Acute Exposure Guideline Levels for Selected Airborne Chemicals: Volume 17 (2014)

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6 Toluene1 Acute Exposure Guideline Levels PREFACE Under the authority of the Federal Advisory Committee Act (FACA) P.L. 92-463 of 1972, the National Advisory Committee for Acute Exposure Guide- line Levels for Hazardous Substances (NAC/AEGL Committee) has been estab- lished to identify, review, and interpret relevant toxicologic and other scientific data and develop AEGLs for high-priority, acutely toxic chemicals. AEGLs represent threshold exposure limits for the general public and are applicable to emergency exposure periods ranging from 10 minutes (min) to 8 hours (h). Three levels—AEGL-1, AEGL-2, and AEGL-3—are developed for each of five exposure periods (10 and 30 min and 1, 4, and 8 h) and are distin- guished by varying degrees of severity of toxic effects. The three AEGLs are defined as follows: AEGL-1 is the airborne concentration (expressed as parts per million or milligrams per cubic meter [ppm or mg/m3]) of a substance above which it is predicted that the general population, including susceptible individuals, could experience notable discomfort, irritation, or certain asymptomatic, nonsensory effects. However, the effects are not disabling and are transient and reversible upon cessation of exposure. 1 This document was prepared by the AEGL Development Team composed of Sylvia Talmage (Oak Ridge National Laboratory), Chemical Manager George Woodall (Na- tional Advisory Committee [NAC] on Acute Exposure Guideline Levels for Hazardous Substances), and Ernest V. Falke (U.S. Environmental Protection Agency). The NAC reviewed and revised the document and AEGLs as deemed necessary. Both the document and the AEGL values were then reviewed by the National Research Council (NRC) Committee on Acute Exposure Guideline Levels. The NRC committee has concluded that the AEGLs developed in this document are scientifically valid conclusions based on the data reviewed by the NRC and are consistent with the NRC guidelines reports (NRC 1993, 2001). 289 

290 Acute Exposure Guideline Levels AEGL-2 is the airborne concentration (expressed as ppm or mg/m3) of a substance above which it is predicted that the general population, including sus- ceptible individuals, could experience irreversible or other serious, long-lasting adverse health effects or an impaired ability to escape. AEGL-3 is the airborne concentration (expressed as ppm or mg/m3) of a substance above which it is predicted that the general population, including sus- ceptible individuals, could experience life-threatening health effects or death. Airborne concentrations below the AEGL-1 represent exposure concentra- tions that could produce mild and progressively increasing but transient and nondisabling odor, taste, and sensory irritation or certain asymptomatic, nonsen- sory effects. With increasing airborne concentrations above each AEGL, there is a progressive increase in the likelihood of occurrence and the severity of effects described for each corresponding AEGL. Although the AEGL values represent threshold concentrations for the general public, including susceptible subpopula- tions, such as infants, children, the elderly, persons with asthma, and those with other illnesses, it is recognized that individuals, subject to idiosyncratic respons- es, could experience the effects described at concentrations below the corre- sponding AEGL. SUMMARY Toluene is a widely used raw material in the chemical manufacturing in- dustry. It is a component of automotive and aviation gasoline and a solvent in lacquers, paint thinners, glue, and other household products. A major concern with the uncontrolled release of toluene is explosion and fire. The odor threshold for toluene ranges from 0.16 to 100 ppm for detection and 1.9 to 69 ppm for recognition; the odor is not unpleasant. Toluene is readily absorbed by the respiratory tract and distributed throughout the body, accumu- lating in tissues with high lipid content. Liquid toluene can be absorbed through intact skin and the alimentary tract. Toluene is a central nervous system (CNS) depressant and is irritating to the eyes at high concentrations. Other effects ob- served in humans after accidental or intentional inhalation of high concentra- tions of toluene include renal toxicity, cardiac arrhythmias, hepatomegaly, and developmental abnormalities. Considerable human and animal toxicity data were available for deriving AEGL values. Clinical, metabolism, and occupational-monitoring studies were available for deriving AEGL-1 values. Many of the studies evaluated sensory irritation and CNS depression. Numerous studies of neurotoxicity have also been conducted in rodents. Lethality data on toluene were available for the mouse and rat. AEGL-1 values were based on the preponderance of data from clinical and occupational studies and from metabolism studies of human subjects that indi- cated that an 8-h exposure to toluene at 200 ppm is near a threshold for AEGL-1 effects (headache), and also near a level for detectable neurologic effects (mod- erate lightheadedness and increased simple reaction time). More than 300 indi- 

Toluene 291 viduals have been evaluated in 20 clinical studies that involved exposures to toluene at 40-700 ppm, and several thousand workers were surveyed in occupa- tional-monitoring studies that involved exposures at up to 1,500 ppm. Those populations are presumed to be composed of healthy individuals, but they repre- sent a broad spectrum of uptake rates (sedentary, working, and exercise condi- tions) and individual differences in metabolism rates (Gamberale and Hulten- gren 1972; Veulemans and Masschelein 1978; Brugnone et al. 1986; Hjelm et al. 1988). Although many clinical studies tested toluene at 100 ppm, the addition of exercise to the protocol in the studies of Astrand et al. (1972), Baelum et al. (1990), and Rahill et al. (1996) more than doubled the toluene blood concentra- tion; concentrations were greater than that from a 200-ppm exposure with the subject at rest (Astrand et al. 1972; Veulemans and Masschelein 1978). The weight of evidence from these studies indicates that an 8-h exposure to toluene at 200 ppm was without adverse health effects in the tested popula- tions, and was an appropriate basis for the AEGL-1 values. At concentrations of 80-200 ppm, toluene approaches a steady-state in the blood within 15-30 min (Astrand et al. 1972; Carlsson 1982). Storage takes place in lipid-rich tissues (including the brain), but elimination is rapid. Toluene reaches a steady-state in the blood and brain fairly rapidly, and no cumulative effects were observed after repeated exposure at 100 ppm for 5 days (Stewart et al. 1975); therefore, 200 ppm was used as the basis for all AEGL-1 durations. Although there was no notable discomfort and only mild irritation (effects expected to be concentration dependent and not subject to changes in activity level), headaches (potentially related to CNS effects), dizziness, and measurable neurologic effects were re- ported after exposure to toluene at 100-200 ppm. Neurologic effects would be expected to be affected by an increase in activity level, leading to higher concen- trations in the brain (target tissue for CNS effects). As noted earlier, physical activity may double the blood concentration of toluene. On the basis of the range of alveolar concentrations among humans exposed to anesthetic gases, an uncer- tainty factor of 3 for human variability was applied to calculate an AEGL-1 val- ue of 67 ppm for all durations. That concentration is deemed protective for all observed effects, including those at 100-200 ppm. The AEGL-2 values for toluene are based on impaired neurologic function that affects the ability to escape. The point of departure was a no-observed- adverse-effect level (NOAEL) of 1,600 ppm for a doubling of the choice reac- tion time in Long-Evans rats exposed for 34 min (Bushnell et al. 2007a). The CNS effects observed during exposure were assumed to be directly related to parent material reaching the brain. Therefore, the toluene concentration in brain (BrTC) after 34 min provides an internal dose measurement correlating with the NOAEL. The physiologically-based pharmacokinetic (PBPK) model of Kenyon et al. (2008) was used to calculate the internal dose or BrTC in the rat. An inter- species uncertainty factor of 1 was applied because pharmacokinetic modeling eliminated the toxicokinetic component of the uncertainty factor, and the phar- macodynamic component was assigned a value of 1 because similar effects (CNS depression) were observed in humans and animals. An intraspecies uncer- 

292 Acute Exposure Guideline Levels tainty factor of 3 was applied because the minimum alveolar concentration for volatile anesthetics should not vary by more than 2- to 3-fold among humans. A human model for toluene (Benignus et al. 2006) was then used to determine the exposure concentrations at each of the AEGL exposure durations that would yield the same brain concentration in humans. Further support for the AEGL-2 values is provided by comparisons with the results obtained from the published model of Benignus et al. (2009, 2011), which calculates the BrTC leading to an effect level comparable to an individual with a blood ethanol level of 0.10% (the legal level of incapacitation in the United States). Although the routes of exposure are different between the two chemicals (inhalation for toluene inhalation and oral for ethanol), the relative effect levels are relevant for comparison purposes. The AEGL-3 values for toluene were based on a NOAEL for lethality in rats. A 2-h exposure to toluene at 6,250 ppm was not lethal but produced pros- tration in rats (Mullin and Krivanek 1982). A 2-h exposure of rats at 10,000 ppm resulted in 20% mortality (Kojima and Kobayashi 1973). The same PBPK mod- els and uncertainty factors that were used to derive the AEGL-2 values were used to calculate the AEGL-3 values. The AEGL values for toluene are presented in Table 6-1. TABLE 6-1 AEGL Values for Toluene End Point Classification 10 min 30 min 1h 4h 8h (Reference) AEGL-1 67 ppm 67 ppm 67 ppm 67 ppm 67 ppm No-effect level for (nondisabling) (250 (250 (250 (250 (250 notable discomfort mg/m3) mg/m3) mg/m3) mg/m3) mg/m3) and neurologic effects in 20 clinical studies.a AEGL-2 1,400 ppmb 760 ppm 560 ppm 310 ppm 250 ppm No-effect level for (disabling) (5,300 (2,900 (2,100 (1,200 (940 impaired ability to mg/m3) mg/m3) mg/m3) mg/m3) mg/m3) escape, decrement in neurological function.c AEGL-3 5,200 ppmb 3,700 ppmb 1,800 ppmb 1,400 ppmb No-effect level for (lethal) (20,000 (14,000 (6,800 (5,300 lethality in rats –d mg/m3) mg/m3) mg/m3) mg/m3) (Mullin and Krivanek 1982) a Clinical studies include Astrand et al. (1972), Gamberale and Hultengren (1972), Stewart et al. (1975), and Baelum et al. (1990). b Concentration is one-tenth or more of the lower explosive limit of 14,000 ppm for toluene in air. Therefore, safety considerations against the hazard of explosion must be taken into account. c No-effect level for a doubling in choice reaction time in rats (Bushnell et al. 2007a). Effect level supported by comparison of toluene inhalation with ethanol consumption in humans (Benignus et al. 2011). d The 10-min AEGL-3 value of 10,000 ppm (38,000 mg/m3) is higher than 50% of the lower explosive limit of 14,000 ppm for toluene in air. Therefore, extreme safety considerations against the hazard of explosion must be taken into account. 

Toluene 293 1. INTRODUCTION Toluene is a colorless, flammable liquid with a pungent floral or aromatic odor similar to that of benzene (Henderson 2001). The odor has also been de- scribed as sour or burnt (Hellman and Small 1974), rubbery, or similar to that of moth balls (Billings and Jonas 1981; Ruth 1986). The chemical and physical properties of toluene are presented in Table 6-2. A major concern with the uncontrolled release of toluene is explosion and fire. The flash point of toluene is 4.4°C and the ignition temperature is 536°C. The saturated vapor is only slightly heavier (about 10% more) than air and may travel a considerable distance in still air to a source of ignition and flash back. Toluene may be rapidly dispersed by normal eddy currents. The vapor may ex- plode if ignited in an enclosed area (Weiss 1980). For example, a fatal explosion occurred when workers were sawing an opening in the side of an empty 10,000- gallon toluene storage tank (NIOSH 1985). TABLE 6-2 Chemical and Physical Properties of Toluene Parameter Value Reference Synonyms Methyl benzene; phenyl methane; ATSDR 2000 methyl benzol; monomethyl benzene; toluol; methacide; tolu-sol; antisal 1a CAS registry no. 108-88-3 O’Neil et al. 2006 Chemical formula C7H8 O’Neil et al. 2006 Molecular weight 92.140 O’Neil et al. 2006 Physical state Clear liquid O’Neil et al. 2006 Boiling point 110.6°C O’Neil et al. 2006 3 Density/specific gravity 0.866 g/cm O’Neil et al. 2006 Solubility in water Slightly soluble, 0.067% O’Neil et al. 2006 Vapor density (air = 1) 3.1 Henderson 2001 Vapor pressure 36.7 mmHg Henderson 2001 Log Kow 2.72 ATSDR 2000 Flash point 4.4°C (closed cup) O’Neil et al. 2006 12.8°C (open cup) Weiss 1980 Flammability limits Henderson 2001 Lower explosive limit 1.4% Upper explosive limit 7.9% Conversion factors in air 1 ppm = 3.77 mg/m3 NIOSH 2011 1 mg/m3 = 0.265 ppm 

294 Acute Exposure Guideline Levels In 1999, world production of toluene was nearly 13,000,000 tons. Approx- imately 79% of total production is from catalytic reforming of refinery streams, an additional 16% is separated from pyrolysis gasoline, and 4% is produced via separation from coal tars. Most of the toluene produced (85-90%) is not isolated but remains as a benzene-toluene-ethylbenzene-xylene (BTEX) mixture for use in gasoline as an octane booster. Of the remaining capacity, the primary use is for chemicals and solvents such as benzene (via dealkylation). In the chemical industry, toluene is used as raw material in the production of benzyl chloride, benzoic acid, phenol, cresols, vinyl toluene, trinitrotoluene (TNT), and toluene diisocyanate. Approximately 14% of toluene is also used as a solvent for paints and coatings and in adhesives, inks, and pharmaceuticals (US Air Force, 1989; EPA, 1990; Ozokwelu 1997; Chemical Week 2000). In the past, commercial toluene contained benzene and xylenes at up to 2-15% and 10%, respectively (NIOSH 1973; Low et al. 1988). Highly purified toluene (benzene at less than 0.01%) began to be produced commercially in 1973. Therefore, greater consideration was given to more recent toxicity studies, in which the toluene is more chemically pure. For both the general population and for occupationally-exposed individu- als, inhalation is the primary route of exposure to toluene. Evaporation of gaso- line and automobile exhaust is the largest source of toluene in the environment, and industries that use toluene as a solvent are the second largest source (EPA 1990). Toluene is also a common indoor-air contaminant due to releases from common household products and from cigarette smoke (ATSDR 2000). 2. HUMAN TOXICITY DATA The typical sequence of events that result from exposure to toluene at con- centrations high enough to produce unconsciousness include euphoria, delu- sions, and sedation (Benignus 1981; Bruckner and Warren 2001). Mood eleva- tion, nausea, and subtle changes in performing intricate tasks have been reported at 200 ppm and higher (ACGIH 2005), although some of the studies are poorly documented (see Table 6-3). Exposure to toluene may be occupational or recrea- tional; in the latter case it is reported to produce a pleasant euphoria with few side effects (Massengale et al. 1963). Its abuse potential may be enhanced by its apparent low irritancy in humans (von Oettingen et al. 1942; Carpenter et al. 1944; Nielsen and Alarie 1982). The major effect of toluene is its narcotic effects, manifested in muscular weakness, incoordination, and mental confusion (NIOSH 1973). Adverse effects on the liver, kidneys, lungs, and heart are limited to acute and chronic exposures at high vapor concentrations. Early reports suggesting deleterious effects on the bone marrow involved the use of toluene contaminated with benzene (NIOSH 1973). The health effects of toluene have been reviewed by Cohr and Stockholm (1979), NRC (1981, 2008), WHO (1985), CIR (1987), Low et al. (1988), EPA (1990), ATSDR (2000), Bruckner and Warren (2001), and Henderson (2001). 

TABLE 6-3 Sensory and Neurobehavioral Effects of Toluene in Controlled Human Studies Concentration (ppm) Duration Subjects/Effects Reference 10, 40, 100 6h 16 males (21-32 y): slight irritation of eyes and nose at 100 ppm; no effect Andersen et al. 1983 on mood, fatigue, or sleepiness; increased frequency of headache, dizziness, and feeling of slight to moderate intoxication; no effect on pulmonary function or nasal mucous flow; no significant effect on performance in eight psychomotor tests. 40 4h 12 males (20-50 y): no effects on measures of motor performance, attention, Lammers et al. 2005a perceptual coding and memory, or mood. 110 Three 30-min peaks over 4 h 50a 3h 10 males, 20 females (19-45 y): no subjective symptoms. Luderer et al. 1999 50 4.5 h 20 males: no increase in sleepiness; increase in scores of unpleasant smell Muttray et al. 2005 and irritation to the throat. 80 4h 8 males (22-50 y): no impairment on neurobehavioral tasks. Cherry et al. 1983 80 4h 16 males (23-38 y): no differences in subjective symptoms compared with Olson et al. 1985 controls; no impairment in tests of simple reaction time, short-term memory, or choice reaction time; no effect on heart rate. 80 4.5 h 12 males (22-44 y): increase in subjective symptoms (nausea, headache, Iregren et al. 1986 irritation), but rated negligible; no impairment in tests of simple and choice reaction time, color-word vigilance, or memory; no effect on heart rate, electroencephalograph results, or sleep latency. 100 3.5 h 18 subjects: no behavioral deficits in psychomotor tests. Winneke 1982 (Continued) 295 

296 TABLE 6-3 Continued Concentration (ppm) Duration Subjects/Effects Reference 100 4h 30 males and females: no serious impairment in neurobehavioral tests Dick et al. 1984 (small impairment in one measure of a visual-vigilance test). 100 6h 6 males and females (27-38 y): No significant effect on pulmonary function Rahill et al. 1996 (subjects exercised for 30 min); slight effect on some multitask and neuropsychologic tests (increased latency but not accuracy on neurobehavioral tasks); no symptoms reported in a double-blind questionnaire. 100 6.5 h 43 male printers and 43 referents (29-50 y). Four groups tested (two exposed Baelum et al. 1985; (printers were previously and two controls): sensory irritation (no annoyance or nausea); sleepiness; Nielsen et al. 1985 exposed for 9 to 25 y) fatigue; slightly decreased performance on four of 10 tests (manual dexterity, color discrimination, visual perception) in one or both exposed groups; no changes in renal function. 100 1, 3, or 7.5 h/d 10 males and 9 females (19-47 y): no decrement in psychomotor-test results Stewart et al. 1975 for 5 d on first day of exposure; slight decrement in performance in tests involving visual vigilance and tone detection on days 2 and 5 in females exposed for 7.5 h; similar subjective symptoms between exposed and control groups. 100 (constant) or 100 7h 32 males and 39 females (31-50 y): sensory irritation of nose and lower Baelum et al. 1990 (TWA; exposure varied (three 15-min airways; increase in dizziness and feeling of intoxication; slight decrement with peaks of 300 ppm exercise periods; in one of four psychomotor-performance tests; no differences in symptoms every 30 min) both exposures) or performance found between constant and varying exposures. 75, 150 7 h/d for 3 d 42 male and female students (18-35 y): 7% (mean) decrement in several Echeverria et al. 1989; 1991 neurobehavioral tests at 150 ppm; slight increases in headache, ocular irritation; sleepiness on first day; CNS effect demonstrated by dose-response in number of times subjects slept. No clear pattern of neurobehavioral effects. Variation in the control data across 3 d was greater than with toluene. 

100, 200a 30, 60 min 11 males and 4 females (18-46 y): no difference in heart rate, pulmonary Astrand et al. 1972; ventilation, oxygen consumption, or blood lactate, either at rest or during a Astrand 1975 work load of 50 W. 100, 200 3 h or 7 h with 23 males (23 y, average age): decrease in pulse rate at 200 ppm for 3 h; Ogata et al. 1970 1-h break tendency to have prolonged reaction time at 200 ppm; no clear concentration-response relationship. 100, 300, 500, 700a Successive 20-min 12 males (20-35 y): no effect on reaction time or perceptual speed at 100 Gamberale and exposure at increasing ppm; increase in simple reaction time at 300 ppm; increase in complex Hultengren 1972 concentrations (one reaction time at 500 ppm; decrease in perceptual speed at end of exposure at 5-min break); total 700 ppm; no effect on heart rate during total exposure; one of 12 subjects 85 min. able to distinguish between control and toluene exposures. 220b 15 min 6/6 subjects willing to work for 8 h; negligible sensory symptoms. Carpenter et al. 1976 427b 15 min 3/6 subjects willing to work for 8 h; 2 subjects reported slight “lightheadness”; 1 reported a “stuffy, drowsy feeling.” 200 6h 5 males, resting: no change in respiration; increased heart rate. Suzuki 1973 240 Three 30-min sessions 11 males (20-21 y): impaired vigilance in third session; decreased fatigue Horvath et al. 1981 during second session. a Subjects exposed via a mouthpiece. b Measured as toluene in “toluene concentrate.” 297 

298 Acute Exposure Guideline Levels 2.1. Acute Lethality Inhalation of “high concentrations” can result in paresthesia, vision dis- turbances, dizziness, nausea, CNS depression, and collapse (Henderson 2001). Most deaths involve solvent abuse or “glue sniffing”, which involves sniffing a mixture of solvents from a plastic bag to concentrate the vapors. Solvents, such as paint thinners, may contain as much as 99% toluene (Donald et al. 1991). Prior to 1975, an estimated 125 deaths involving solvent abuse occurred per year in the United States (Winek and Collom 1975). Few deaths have been attributed solely to the inhalation of pure toluene, but are associated with paint thinners, spray paints, glues, and other products containing toluene. Few data are availa- ble on the concentrations of toluene that caused deaths in these studies. The con- centration of toluene achieved when inhaling directly from a paper bag contain- ing gauze soaked with toluene from a tube of polystyrene cement is estimated to be 10,000 ppm (Press and Done 1967). According to the authors, this concentra- tion causes unconsciousness within a few minutes, which results in cessation of exposure. Bass (1970) reviewed reports of “sudden sniffing death syndrome.” Eye- witness accounts of the events prior to death were similar and included: inhala- tion of volatile hydrocarbons from a bag, panic, physical exertion (usually in- volving running), and sudden collapse and death. This pattern was characterized by the author as being the result of severe cardiac arrhythmia associated with fulminate pulmonary edema, the excitement of a light plane anesthesia, hypera- drenergic crisis, or some combination of these and possibly unknown factors. The author suggests a mechanism of action involving sensitization of the myo- cardium by volatile hydrocarbons and subsequent physical exertion coalescing to produce sudden and severe arrhythmia. No cardiac-sensitization tests of tolu- ene in dogs was found, but dogs exposed to toluene at 30,000 ppm for 9-10 min died of ventricular fibrillation and severe hypoxia (Ikeda et al. 1990; see Section 3.1.1). 2.2. Nonlethal Toxicity The odor threshold of toluene in air ranges between 2 and 40 ppm (Amoore and Hautala 1983; Ruth 1986). According to Hellman and Small (1974), the odor can be detected at 0.17 ppm and recognized at 1.74 ppm. The American Industrial Hygiene Association (AIHA1989) reports the detectable range as 0.16-100 ppm and the odor recognition range as 1.9-69 ppm. According to a literature survey (Ruth 1986), the threshold for “irritation” is 200 ppm. In a more recent series of studies, the odor threshold and thresholds for ocular and nasal irritation were measured using squeeze bottles and a two-alternative, forced-choice procedure with an ascending method of limits (Cometto-Muniz and Cain 1995; Abraham et al. 1996). Thresholds for ocular irritation and nasal pungency were approximately >20,000 and 29,850 ppm, respectively. The nasal 

Toluene 299 pungency threshold was developed with anosmics (subjects who were clinically diagnosed as lacking a sense of smell and were, thus, unbiased by odor sensa- tions). Solvent abusers repeatedly inhale anesthetizing concentrations on a daily basis. Toluene abuse, an extreme form of exposure, has resulted in myocardial infarction and cardiac effects (Cunningham et al. 1987; Wiseman and Banim 1987; Carder and Fuerst 1997), renal toxicity that includes renal tubular acidosis (Taher et al. 1974; Reisin et al. 1975; Patel and Benjamin 1986; Gupta et al. 1991; Kamijima et al. 1994), metabolic acidosis often with “anion gap” (the sum of the cations in the blood minus the sum of the anions in the blood) (Fischman and Oster 1979; Jone and Wu 1988), acute encephalopathy in children (8-14 years old) (King et al. 1981), and cerebellar ataxia (Boor and Hurtig 1977). Streicher et al. (1981) described syndromes of toluene sniffing in adults, which included a pattern of three dominant symptoms: muscle weakness, gastrointesti- nal disorders, and neuropsychiatric disorders. Neuropsychiatric symptoms in- cluded headache, dizziness, syncope, paresthesias or peripheral neuropathy, hal- lucinations, lethargy, and cerebellar ataxia. Some exposures were to mixtures of solvents and ethanol. Exposure concentrations could not be ascertained. In a review of neurologic and psychiatric consequences of toluene abuse, Ron (1986) concluded that evidence for such sequelae remains inconclusive. Distal renal tubular acidosis is an established consequence of toluene abuse and has been reported in numerous studies. This consequence is notable with the extremely high vapor concentrations associated with chronic abuse sit- uations (O’Brien et al. 1971; Taher et al. 1974; Fischman and Oster 1979; Kroeger et al. 1980; Moss et al. 1980; Russ et al. 1981; Streicher et al. 1981; Patel and Benjamin 1986; Marjot and McLeod 1989). In life-threatening cases, patients present with severe generalized muscle weakness, nausea and vomiting, and neuropsychiatric derangements (Streicher et al. 1981; Marjot and McLeod 1989). The mechanism of action for this disorder is discussed in Section 4.2. In spite of reports of hepatic, adrenal, and renal damage, there appears to be a low incidence of these injuries among glue sniffers. Only modest elevations of serum glutamic-oxaloacetic transaminase and alkaline phosphatase and transient ab- normalities in urinalyses were observed among groups of glue sniffers (Press and Done 1967; Litt et al. 1972; Weisenberger 1977). 2.2.1. Occupational Exposures Studies of workers exposed to toluene in occupational settings have fo- cused on functional impairment. These exposures are usually not of a magnitude required to produce serious sustained effects. Even though exact exposure pa- rameters of concentration and duration are usually not determined in these stud- ies, the investigations provide information about the more common effects and at what approximate concentrations these effects are observed. Interpretation of most occupational exposure studies of toluene is confounded by co-exposure to 

300 Acute Exposure Guideline Levels other solvents. Only the study by Wilson (1943) addressed acute effects imme- diately following the work day. Additional occupational monitoring studies are briefly discussed below. Wilson (1943) surveyed the effects of various concentrations of toluene in workers at a large industrial plant. Approximately 1,000 workers were exposed to toluene at concentrations of 50-1,500 ppm for periods of 1-3 weeks. Approx- imately 10% of the employees had symptoms severe enough to require examina- tion at a hospital. The employees were grouped according to the concentration of toluene fumes measured at their job sites; measurements were made with a combustible gas indicator. (Combustible gas indicators measure all combustibles and are generally incapable of measuring concentrations of less than 5%.) In workers exposed to toluene at 200 ppm or lower, the most common complaints were headache, lassitude, and loss of appetite. At 200-500 ppm, complaints in- cluded headache, lassitude, and anorexia that were more pronounced. These subjects also complained of nausea, a bad taste in the mouth, loss of coordina- tion, decreased reaction time, and momentary loss of memory. At concentrations greater than 500 ppm, the major complaints were nausea, headache, dizziness, anorexia, palpitation, and extreme weakness. Physical and laboratory examina- tions of the approximately 60 workers exposed to toluene at 200 ppm or lower were negative. Also, no significant physical or laboratory findings were noted for the 30 workers exposed at 200-500 ppm. Physical examination of the re- maining 10%, exposed at 500 ppm or higher, found loss of coordination, de- creased reaction time, and petechiae under the skin. Laboratory results from these patients revealed low erythrocyte counts and leukopenia, and bone marrow biopsy demonstrated aplastic anemia in two subjects. The hospitalized workers were treated symptomatically. No deaths occurred. The analytic methodology in this study and the confounding issue of benzene exposure precludes the use of this study for developing AEGL values. Monitoring studies show that workers have been routinely exposed to tol- uene at 32 ppm (0.1-457 ppm) (Neubert et al. 2001b); a lifetime weighted aver- age of 45 ppm (Seeber et al. 2005); a time-weighted average (TWA) of 63-118 ppm (5-353 ppm) (Ovrum et al. 1978); a TWA of 88 ppm (49-130 ppm) (Foo et al. 1990); 50-100 ppm (Neubert et al. 2001a); 50-150 ppm (Iregren 1982); ≥100 ppm (Ukai et al. 1993); an estimated average of 117 ppm (Juntunen et al. 1985); 132 ppm (66-250 ppm) (Zavalic et al. 1998); 1->100 ppm (Lee et al. 1988); 100- 440 ppm with peak values of 200-500 ppm (Eller et al. 1999); 9-467 ppm (Des- champs et al. 2001); ≥200 ppm (Forni et al. 1971); 200-800 ppm (Parmeggiani and Sassi 1954); and ≤ 500 ppm (Greenberg et al. 1942). In most cases when psychomotor tests were performed, the tests were administered before the work- day; therefore, acute effects were not measured. In most of these studies, only subtle differences in neurologic parameters, such as alterations in the visual evoked response or small decreases in reaction time, were found compared with control groups (Yin et al. 1987; Foo et al. 1990; Abbate et al. 1993; Murata et al. 1993; Vrca et al. 1995; Boey et al. 1997; Zavalic et al. 1998; Eller et al. 1999). Seeber et al. (2005) found no evidence of neurobehavioral effects following 

Toluene 301 long-term exposure to toluene below 50 ppm. Irritation of the conjunctiva and upper respiratory tract was found in only one of 11 workers exposed at 200-800 ppm (Parmeggiani and Sassi 1954). In some studies, incidences of subjective symptoms, such as sore throat, were greater than in matched control groups (Tanaka et al. 2003). Symptoms correlated with duration and extent of exposure in some studies (Lee et al. 1988) but not in others (Gericke et al. 2001). Chronic exposures of workers to toluene at low concentrations have not resulted in alter- ations in liver-enzyme activity or hormone concentrations (Gericke et al. 2001) or in serious renal damage (ATSDR 2000). An acute exposure of flexoprint workers to toluene at approximately 100 ppm for 6.5 h failed to show significant changes in β-microglobulin or albumin excretion compared with air-exposed controls (Nielsen et al. 1985). Studies of occupationally-exposed workers indicate that chronic exposure to toluene at low concentrations results in hearing loss (Morata et al. 1997; ATSDR 2000). However, in a study of 333 rotogravure printing workers, expo- sure at less than 50 ppm could not be related to ototoxicity (Schaper et al. 2003). In animal models, toluene was shown to damage outer hair cells in rats exposed to toluene at 1,400 ppm for 14 h/day for 8 days (Johnson and Canlon 1994; see also Section 3.2.2). A question of whether toluene impaired color vision in workers was raised by Zavalic et al. (1998). Color vision was impaired in these workers, but alcohol and age were confounding factors. 2.2.2. Accidental Exposures Two men working with toluene to remove excess glue from tiles in a swimming pool were exposed to toluene at concentrations greater than 1,842 ppm for 2 or 3 h (Meulenbelt et al. 1990). The concentration of toluene was measured at the edge of the pool by Drager tube 3 h after the men were rescued. Concentrations were presumed to be higher at the bottom of the pool where the men were found because toluene is heavier than air and would have accumulated at the bottom of the pool. Both men were disoriented when found; one was una- ble to walk or sit, and the other was barely able to walk. Physical examinations carried out 1 h after they were found revealed mucosal irritation of the eyes, slurred speech, headache, paresis, and amnesia. The patient exposed for 3 h had an excessive anion gap and a sinus bradycardia. The second patient, who was exposed for 2 h, complained of headache, and clinical examination revealed a sinus tachycardia and a slightly excessive anion gap. Neither patient showed abnormalities in hepatic function or hematologic parameters. Blood toluene con- centrations taken 2 h after exposure were 4.1 and 2.2 mg/L in the patients ex- posed for 3 and 2 h, respectively. The most striking effect of this acute exposure was the increased anion gap in both patients, which the authors attributed to either the high plasma concentration of toluene metabolites (benzoic acid and/or hippuric acid) or distal tubular acidosis. Recovery for both patients appeared complete at the 1-week postexposure medical examination. 

302 Acute Exposure Guideline Levels Two cases of accidental occupational exposure to toluene at very high concentrations were reported by Longley et al. (1967). One of the case reports involved several men who were exposed to toluene in an enclosed space aboard a commercial ship. Initially, two men were assigned to spray ballast tanks with an “anti-rust” paint containing toluene. One man climbed out of the tank be- cause he felt dizzy, and shortly afterwards noticed that the other man had col- lapsed. Seventeen more men suffered symptoms of exposure during rescue op- erations, which lasted for about 2 h. Symptoms of exposure included unconsciousness, severe mental confusion, amnesia, and illogical behavior. All affected workers recovered fully within 30 min after breathing oxygen. No esti- mate of the toluene concentrations was possible. The second incident also took place on a merchant ship (Longley et al. 1967). An accidental exposure to a concentrated insecticide containing malathi- on (20%), piperonyl butoxide (8%), pyrethrum (1.5%), and toluene (to 100%) occurred. Thirty gallons of this mixture, which contained 21 gallons of toluene, was mistakenly sprayed undiluted into a hold with a volume of about 102,000 cu. ft. within about 75 min. Seven men, including the workers and rescuers, were overcome, and three lost consciousness. The men were taken off the ship and given medical examinations. Because the cholinesterase activity of the men remained at 100%, the authors judged that the malathion was not absorbed ap- preciably. Using a Department of Scientific and Industrial Research (DSIR) pump which was designed by the British Department of Scientific and Industrial Research to make spot determinations of hazardous atmospheres, the toluene concentrations were estimated to be 5,000-10,000 ppm. However, calculations of the distribution of the 21 gallons of toluene in the hold results in approximate concentrations of 10,000-12,000 ppm. Because the vapor rapidly anesthetized the kneeling worker but not for a standing one, the concentrations were probably higher near the floor than at head level. Therefore, some sources estimate or report that the concentrations of toluene were 10,000 ppm at waist level and 30,000 ppm at floor level (NIOSH 1973; NRC 1981). In addition to uncon- sciousness, the men also suffered nausea, incoordination, amnesia, and feelings of intoxication, but they did not complain of ocular or throat irritation. The ex- posed men recovered without persistent effects. 2.2.3. Clinical Studies Numerous clinical studies have been conducted with healthy human sub- jects exposed in controlled settings to monitored concentrations of toluene for varying durations. Studies performed in the 1940s are now considered compro- mised because toluene was less pure at that time and contained other solvents, including benzene, and limited analytic characterization of exposure concentra- tions were available. However, there are recent, well-conducted clinical studies of toluene that are suitable for estimating AEGL values (Table 6-3). 

Toluene 303 More than 300 individuals have been evaluated in clinical studies involv- ing toluene exposures of 40-700 ppm. The subjects are presumed to be healthy individuals, but represent a broad spectrum of uptake rates (sedentary, working, and exercise conditions). Although many clinical studies used a toluene concen- tration of 100 ppm, the addition of exercise to the protocol in the studies of Astrand et al. (1972), Baelum et al. (1990), and Rahill et al. (1996) more than doubled the blood concentration of toluene to a level greater than that from a 200-ppm exposure with the subjects at rest (Astrand et al. (1972; Veulemans and Masschelein 1978). Baelum et al. (1990) investigated peak exposures of 300 ppm (14 times during a 7-h exposure; mean concentration of 100 ppm) with exercise (50-100 W) undertaken for 15 min during three of the peak exposures. Astrand et al. (1972) also incorporated exercise into the 200-ppm exposures. These studies generally addressed the threshold for subjective and CNS ef- fects to set guidelines for chronic exposures. Some of these studies provide in- formation on the threshold for subtle psychomotor dysfunction (impairment of mental traits, abilities, and processes) in humans. Several studies also addressed air quality and odor adaptation or olfactory fatigue. Although slight irritation involving the eyes and nose in humans was reported in several studies of toluene at 80-100 ppm, toluene is not a primary respiratory irritant as evidenced by the high RD50 (concentration the reduces respiratory rate by 50%) of 5,300 ppm (see Section 3.2.3). In general, complaints increased among control subjects, espe- cially in studies with long exposure durations. Other studies reported exposures at 80-100 ppm to be nonirritating (Stewart et al. 1975; Cherry et al. 1983; Olson et al. 1985; Rahill et al. 1996). No CNS effects were reported at 80-100 ppm in studies of Winneke (1982), Cherry et al. (1983), and Stewart et al. (1975); ef- fects were minor in other studies at 100-700 ppm (Gamberale and Hultengren 1972; Dick et al. 1984; Baelum et al. 1990). There were no biologically signifi- cant pulmonary or cardiovascular effects at 100 and 200 ppm for exposures up to 6 h (Astrand et al. 1972; Suzuki 1973) and no indications of renal damage (Nielsen et al. 1985). Exposure to toluene at 100 ppm in the study by Stewart et al. (1975) was repeated for 5 days, with no greater effects over time. Two studies that addressed only sensory effects are summarized here (Astrand et al. 1972; Suzuki 1973). Experimental studies in controlled settings that evaluated neurotoxicity and subjective symptoms are summarized in Sec- tion 2.3 (Neurotoxicity), and a reproductive study that evaluated subjective symptoms (Luderer et al. 1999) is summarized in Section 2.4 (Developmental and Reproductive Toxicity). All of these studies are summarized in Table 6-3, and are arranged in order of increasing concentration. Additional studies of con- trolled human exposures that evaluated the metabolism and disposition of tolu- ene, but that did not address subjective symptoms, are summarized in Section 4.1 (Uptake, Metabolism, and Disposition). Astrand et al. (1972) exposed 15 healthy male and female subjects, ages 18-46 year, to toluene at concentrations of 100 or 200 ppm for 30-min periods at rest and during exercise on a bicycle ergometer at work loads of 50 and 150 W. Some exposure periods were extended to 60 min at 100 ppm and 90 min at 200 

304 Acute Exposure Guideline Levels ppm, the latter with exercise at 75 and 50 W for 30 min each. Toluene concen- trations were measured by gas chromatography. No differences in measurements taken before or during exposure of heart rate, pulmonary ventilation, oxygen consumption, or blood lactate content for the corresponding workloads were found. Differences between males and females were minor. Although individual differences in uptake were noted, childhood asthma and obesity did not appear to affect uptake, and the presence of heart arrhythmias in two individuals, noted preexposure, was unaffected by the exposures. Exercise at 50 W in subjects ex- posed to toluene at 100 ppm more than doubled the subjects’arterial concentra- tion of toluene (Astrand et al. 1972; Astrand 1975). Suzuki (1973) exposed groups of five male students to toluene at 0 or 200 ppm for 6 h and measured several physiologic functions. The students reclined on a bed during the exposure. No significant changes were observed in galvanic skin reflex, vasoconstriction, respiration rate, or activity on electroencephalo- gram (EEG). Only heart rate was significantly increased (by 0.1 second) in the exposed group. 2.3. Neurotoxicity The effects of toluene on nasal mucus flow, pulmonary function (nasal flow resistance, forced vital capacity, and forced expiratory volume), subjective response (headache and dizziness), and psychometric performance (both manual and mental tests) were evaluated several times during 6-h exposures to toluene at 10, 40, or 100 ppm (Andersen et al. 1983). The tests were carried out in an atmosphere-controlled chamber, and toluene concentrations were continuously monitored using gas chromatography and photo-ionization detection. Sixteen healthy male Danish students (average age 24), who were “nose breathers” vol- unteered for the study. Adaptation to the odor occurred, but odor was still no- ticeable at the higher concentrations at the end of the exposures. Three subjects reported that the odor/air quality at 100 ppm was unacceptable (not further ex- plained). There was an increase in slight irritation of the eyes and nose, de- scribed as slight, and a reported decrease in air quality at 100 ppm as well as an increase in the perceived odor levels as test concentrations increased. The aver- age irritation score was 13 on a scale of 100, with one subject reporting a score of 64; six subjects reported no irritation during exposure at 100 ppm. No irrita- tion of the throat or lower airways was reported at any concentration, and there was no effect on mood, fatigue, or sleepiness and no cough or nausea. However, an increase in the occurrence of headache, dizziness, and feeling of intoxication was found during the 100-ppm exposure; effects were described as slight to moderate and involved about half of the subjects (data were not presented). Pulmonary function and nasal mucus flow were unaffected by toluene inhala- tion. Furthermore, toluene had no significant effects on the performance of eight psychometric tasks measuring 20 different parameters, but there was a border- line significant decrease in three tests (multiplication errors, Landolt’s rings, and the screw plate test) at 100 ppm. 

Toluene 305 Twelve healthy, adult men (ages 20-50) inhaled toluene at a constant con- centration of 40 ppm for 4 h or were exposed to three 30-min peaks at 110 ppm over a 4-h period (Lammers et al. 2005a). Neurobehavioral tests were performed repeatedly during and after exposure. No effect on motor performance, attention, perceptual coding and memory, or mood was found with either exposure regi- men. There were no further details in the available abstract. In a study that evaluated acute symptoms and neurotoxicity manifest as sleepiness, Muttray et al. (2005) exposed 20 healthy male subjects to toluene at 0 or 50 ppm for 4.5 h. Toluene was monitored by infrared spectroscopy. Acute symptoms were assessed with a questionnaire, and sleepiness was assessed with the pupillographic sleepiness test, which measures changes in the diameter of the pupil of the eye. The subjects reported a bad smell and irritation of the throat during exposure at 50 ppm, but symptoms of headache, dizziness, nausea, tired- ness, pain or pressure on the chest, coughing spells, shortness of breath, irrita- tion of the eyes, watering eyes, blurred vision, irritation to the nose, running nose, an unpleasant taste, irritation to the skin, and feeling of fainting or vertigo were not significantly increased over control symptoms. Sleepiness was not in- creased. Cherry et al. (1983) assessed the effects of a 4-h exposure to toluene at 80 ppm on four measures of performance and on mood. The subjects were eight male postgraduate students (ages 22-50), who were tested in groups of four in an atmosphere-controlled chamber. Test results of simple reaction time, four-choice reaction time, tracking task, and visual search were compared with those from a control group. Test results were also compared with those following exposure to alcohol (0.4 mL/kg) and following exposure to toluene and alcohol (0.4 mL/kg). In the control exposures, the chamber was primed with toluene to mask the con- trol chamber. In both the control and exposure sessions, peppermint oil was used to mask the odor of toluene. The exposures were controlled by the investigators, but they took no part in the testing which was carried out blind. For the single exposures, the mean alcohol blood level was 49.9 mg%, and the mean blood toluene concentration was 12.7 mmol/L (both taken at the end of 4 h). Toluene alone had no significant effect on any of the behavioral measures, whereas alco- hol caused a significant deterioration on pursuit tracking, visual search tasks, and mood. Although the combination of toluene and alcohol had no significantly greater effect on any of the test results than alcohol alone, there was a nonsignif- icant tendency for performance and mood to deteriorate more than when alcohol was administered alone. In a similar study, 16 healthy adult male volunteers, ages 23 to 38, were exposed to toluene at 0 or 80 ppm for 4 h in a controlled atmosphere chamber (Olson et al. 1985). Three different performance tests were administered: simple reaction time, choice reaction time, and memory. There were no differences in performance of tests involving simple reaction time, short-term memory, or choice reaction time immediately after entering the exposure chamber or after 2 or 4 h of exposure. On a rating scale of no, negligible, slight, and considerable, 

306 Acute Exposure Guideline Levels the subjects rated the discomfort (nausea, headache, or irritation in the eyes, nose, or esophagus) during the exposure as negligible. The same investigators (Iregren et al. 1986) conducted another study in which they compared effects of toluene exposure to alcohol ingestion. Twelve men, ages 22-44 years, were exposed to toluene at 80 ppm toluene for 4.5 h. Results were compared with control exposures, exposure to toluene at 80 ppm accompanied by ingestion of alcohol (15 mmol/kg), and ingestion of alcohol alone. Neurobehavioral tests addressed choice reaction time, simple reaction time, color-word vigilance, and memory. The tests were administered prior to exposure and after 2 and 3.5 h of exposure. Symptoms and mood, including wakefulness, were also surveyed. Exposure to toluene at 80 ppm failed to affect performance on any of the tests, whereas the moderate ethanol intake resulted in gross performance changes in simple reaction time and color-word vigilance. There were no interactive effects of toluene and ethanol. Heart rate, EEG record- ings, and sleep latencies were not affected by toluene treatment, although sub- jective symptoms of nausea, headache, and irritation were increased during the toluene and combined toluene and ethanol exposures. However, mood symp- toms (feeling bored, sleepy, or irritated) were not different between the exposed and control groups. The subjective symptoms were the same as those reported in the earlier study of Olson et al. (1985), which were rated in that study as negli- gible in the control and exposed groups. Winneke (1982) exposed 18 subjects to either air or toluene at 100 ppm in a controlled chamber for a 3.5 h period. Critical flicker frequency (the illusion of motion due to persistent sensory neuron excitation after a stimulus has ended) was measured as a perceptual measure of CNS activation. Sustained attention was measured with a bisensory vigilance task. At the end of the exposure, a comprehensive battery of psychomotor tests (not defined) was administered. None of the measures showed a significant effect from toluene exposure. Dick et al. (1984) exposed 30 male and female subjects to toluene at 100 ppm for 4 h. Subjects were exposed to either a placebo (a 2-min exposure to toluene at 25 ppm followed by air) or toluene at a concentration of 100 ppm in an atmosphere-controlled chamber. Solvent concentration was monitored using an infrared analyzer and analysis was carried out by gas chromatography every 3 min. Compared with the control values, toluene exposure did not adversely af- fect the outcomes on 27 psychomotor tests. A slight, but statistically significant, decrement was observed on the visual-vigilance test; the percentage of correct hits was lower. The authors considered the overall results to demonstrate a fail- ure to induce cognitive effects. A double-blind study by Rahill et al. (1996) tested toluene at 100 ppm or air only for 6 h. Six healthy male and female subjects, ages 27-38, performed complex psychometric and response-time tasks during rest and following exer- cise sessions sufficient to quadruple the resting ventilation rate. Pulmonary func- tion tests, consisting of forced vital capacity and forced expiratory volume in 1 second (sec), were also carried out prior to and after the exposure. The subjects filled out a 56-item questionnaire concerning symptoms and mood before and 

Toluene 307 after the exposure. Each subject served as his or her own control. The exposures were carried out in a large atmosphere-controlled chamber, and the toluene con- centration was monitored by an infrared analyzer. Latency but not accuracy dur- ing neuropsychologic tests proved sensitive to toluene. The latency was greatest following the exercise period. The composite score obtained over time during toluene exposure was lower than that during the air exposure. However, the dif- ferences between the air- and toluene-exposed groups, while in some cases sta- tistically significant, were characterized by the study authors as subtle. For ex- ample, the composite score for an hour-long multitasking test was reduced by 11% following the toluene exposure compared with control score. Pulmonary function was not affected by toluene exposure in this study. No subjective symp- toms were reported, and mood was only slightly changed. The mood change was characterized as a “reduced positive mood.” This experiment demonstrated that physical activity can exacerbate the response to toluene as greater, but subtle, differences in performance were observed after exercise. Baelum et al. (1985) reported that the acute effects of toluene exposure are slightly more pronounced for previously exposed workers (rotogravure printers) compared with previously unexposed controls. The printers had been exposed to solvents for 9-25 years. Male subjects (43 printers and 43 controls), ages 29-50 years, were divided into four matched groups (20 control printers, 19 toluene- exposed printers, 21 unexposed controls, and 15 toluene-exposed controls) and exposed to either air (20 control printers and 21 controls) or toluene at 100 ppm (19 printers and 15 controls) for 6.5 h, preceded by a 1-h acclimatization period. Subjective evaluations concerning the quality of the environment were taken before exposure and at 0.5 and 6.5 h. Nine visuomotor-coordination and percep- tual-speed tests were administered at several hours into the exposure. A color- discrimination test was administered before and during exposure. The effects observed at 100 ppm for both exposed groups compared with control groups included discomfort with complaints of low air quality, strong odor, fatigue, sleepiness, a feeling of intoxication, and irritation of the eyes, nose, and throat. However, complaints increased for all subjects during the test periods (including the controls) and neither annoyance nor nausea was experienced. Toluene- exposed printers were slightly slower than nonexposed printers on one of six manual dexterity tests (p < 0.05), but toluene-exposed controls and nonexposed controls had similar results on this test. The same relationship was found for one visuomotor test (Landolt’s rings); more errors were made by exposed printers compared with unexposed printers and no differences were found between ex- posed and unexposed control groups. Both groups of printers made fewer errors on a color-discrimination test administered before exposure compared with both groups of controls, but the error rate decreased later in the exposure for both groups of controls, whereas there was almost no improvement for either of the toluene-exposed groups. Although no significant differences were observed be- tween the occupationally-exposed group and the naive subjects, the authors stat- ed that there was a tendency toward greater sensitivity among occupationally- exposed subjects. 

308 Acute Exposure Guideline Levels As part of the same study, Nielsen et al. (1985) evaluated the renal func- tion of the printers and control groups. Urinary samples were taken before the exposure, at 3 h after the start of exposure, and again at 6 h after the start of ex- posure. No significant changes in β-microglobulin- or albumin-excretion rates were observed for the exposed subjects compared with the air controls. The au- thors concluded that there is no causal relationship between moderate exposure to organic solvents and renal injury. Stewart et al. (1975) exposed 10 male subjects to toluene at 0, 20, 50, or 100 ppm and nine female subjects at 100 ppm for 1, 3, or 7.5 h over several days. The exposures at 100 ppm were for five consecutive days. Males were also exposed to a fluctuating concentration of 50-150 ppm (mean concentration 100 ppm) for 1, 3, or 7.5 h for 2 days. Ages ranged from 19-47 years old. Subjects were exposed at each concentration in groups of two to four, with never more than eight subjects in the chamber at one time (subjects exposed for 1 h exited the chamber after 1 h and the subjects exposed for the longer durations re- mained). Male subjects exercised for 5-6 min once during the shorter exposures and twice during the 7.5-h exposure. Chamber atmospheres were monitored with gas chromatography and infrared spectroscopy. Each subject was given a com- plete medical examination before and after exposure; additional evaluations in- cluded hematology and clinical chemistry parameters, EEG recordings (with an amplified visual evoked response), pulmonary-function tests, heart rate, equilib- rium tests (modified Romberg), and cognitive tests. Subjective symptoms were also evaluated. Not all evaluations took place every day or with all subjects. There was no decrement in several psychomotor performance tests, except for females exposed at 100 ppm for 7.5 h on the second day. They had fewer correct responses on a dual task involving visual vigilance and tone detection on the second and fifth days of exposure. This decrement was not observed in male subjects or in females during the 3-h exposure. Performance on time estimation, addition, and coordination tests did not change. On the fifth day of exposure at 100 ppm for 7.5 h and during the fluctuating exposure at 100 ppm, one of two male subjects had a slight increase and decrease, respectively, in the visual evoked potential. This change was not observed in female subjects. When tolu- ene was present in the chamber, all subjects noticed a mild to strong odor when entering the chamber; adaptation occurred for most subjects. There was no in- crease in drowsiness, fatigue, sleepiness, or headache or change in appetite or sleep habits. Subjective complaints of irritation were greater during the 3-h ex- posure at 100 ppm than during the 1- or 7.5-h exposure at 100 ppm. Types and incidences of subjective complaints were similar between controls and the ex- posed subjects during the 7.5-h exposure at 100 ppm. An evaluation of human responses to varying concentrations of toluene with a TWA of 100 ppm (with peaks at 300 ppm every 30 min) was conducted by Baelum et al. (1990). The authors compared the symptoms during the varying concentrations to those during exposure to a constant concentration of toluene at 100 ppm and to those reported in a clean air control. Thirty-two males and 39 females comprised a random sample of the population between the ages of 31 

Toluene 309 and 50. The subjects were healthy males and females who did not abuse alcohol or drugs and were able to exercise for 15-min periods with a load of 50, 75, or 100 W during the peak exposures. The clean air, constant exposure, and varying exposure groups comprised 23-24 subjects each. Exposures were carried out for 7 h, and concentrations were measured using a flame ionization detector. Both toluene-exposed groups complained significantly more about the poor air quali- ty, altered temperature perception, feeling of intoxication, and increased irrita- tion in the nose and lower airways (but not of the eyes); there was a tendency toward an increase in dizziness and feeling of intoxication in the exposed groups (controls reported scores similar to those of the constant exposure-group for fatigue, sleepiness, and headache). There was a tendency toward lower scores on vigilance tests for the toluene-exposed groups compared with the control, indi- cating only a minimal effect on psychomotor performance, as scores on other assessment tests were normal. There was not, however, a significant difference between symptoms or performance between the two toluene-exposed groups. Echeverria et al. (1989, 1991) reported on the acute neurobehavioral ef- fects of toluene in tests with 42 healthy male and female college students. The toluene concentrations tested were 0, 75, and 150 ppm over a 3-day period (7 h each day) and were administered in random order. The odor of toluene was masked with menthol (0.078 ppm). Chamber atmospheres were measured with an infrared analyzer and confirmed by gas chromatography. A battery of 12 per- formance tests (verbal, visual, and psychomotor) was administered to each par- ticipant before the exposures and again at 4 and 7 h during the exposures. Test results were averaged over the 3 days. The initial test results served as control values. A mood and fatigue checklist was also incorporated into the protocol. A 5-12% decrement in performance was considered significant if consistent with a linear trend. The results of this study included a significant decrement in per- formance on several tasks when subjects were exposed to toluene at 150 ppm; they included losses of 6.0% for digit span, 12.1 % for pattern recognition (in- crease in latency of 0.3 sec), 5.0% for pattern memory (number correct), 6.5% for one hole, and 3% for critical tracking. These differences, although statistical- ly significant, were small. Although the pattern of moods scale and fatigue symptoms were not affected by toluene, the frequency of headaches and ocular irritation increased slightly in a concentration-dependent manner, as did the number of observations of sleep during exposure. For example, headache was reported by 8, 11, and 14% of individuals exposed at 0, 75, and 150 ppm, re- spectively. Sleep did not confound the behavioral scores. The reports of ocular irritation and headache were greatest on the first day of exposure. Ogata et al. (1970) measured the effects of exposure to toluene at 100 or 200 ppm on blood pressure, pulse rate, flicker value (not defined), and eye-to- hand reaction time in 23 male students. Subjects were exposed for 3 h in the morning and 4 h in the afternoon, the latter following a 1-h break. After a 3-h exposure at 200 ppm, the mean pulse rate was decreased significantly compared with a control group. However, there was no dose-response relationship and all pulse rates were lowered as exposures continued. The authors stated that there 

310 Acute Exposure Guideline Levels was a tendency for reaction time to be prolonged during the 200-ppm exposure, but there was no clear concentration-response relationship. Twelve healthy male subjects, ages 20 to 35, were exposed to successively increasing concentrations of toluene at 100, 300, 500, and 700 ppm, for four successive 20-min periods (Gamberale and Hultengren 1972). There was a 5- min break between the exposure at 300 ppm and at 500 ppm. The air-toluene mixture was administered via a breathing valve and a mouthpiece to the seden- tary subjects. The odor of toluene was masked with menthol-crystals, and only one of the 12 subjects was able to correctly distinguish between conditions with and without toluene. Four performance tests were administered during the last 15 min of each exposure: two perceptual speed tests, simple reaction time, and choice reaction time. Test results were compared with results during air expo- sures. An increase in simple reaction time (to a stimulus) was observed at 300 ppm (controls, 222 msec; 300 ppm, 236 msec), complex reaction time was af- fected at 500 ppm, and a decrease in perceptual speed occurred at 700 ppm. The decrease in perceptual speed was relative to the control values, which decreased with each successive exposure (learning effect); speed also decreased for the exposed group but not as rapidly as for the control group. The authors stated that short-term exposures to toluene below 300 ppm are not associated with psycho- motor dysfunction. Heart rate declined during the 85-min test period in accord- ance with increasing familiarity with the test situation and individual test and was little affected by toluene exposure. Carpenter et al. (1976) reported several human and animal responses to “Toluene Concentrate,” a hydrocarbon mixture produced by a large solvent manu- facturer in the United States. This mixture contained toluene (45.89 %), butanes, pentanes, hexanes, heptanes, octanes, cyclopentanes, cyclohexanes, and benzene. Gas-chromatographic analysis was used to monitor exposure concentrations. Six of six subjects indicated they would be willing to work an 8-h day while being exposed to toluene concentrate at 480 ppm (220 ppm of toluene), a concentration at which some ocular irritation was reported after a 15-min exposure. Three of these six people reported that they would be willing to work an 8-h shift while being exposed to toluene concentrate at 930 ppm (427 ppm of toluene). Horvath et al. (1981) exposed a group of 11 male volunteers, ages 20-21, to three 70-min sessions of toluene at 240 ppm, during which the subjects per- formed a vigilance task. The task consisted of spatial discrimination of low fre- quency acoustical clicks, which contributed to the monotony of the task, and a concurrent visual feedback task. The toluene concentration was increased gradu- ally over the test period, reaching and maintaining a maximum concentration of 240 ppm over the last 30 min of each session. There was a 70-min period be- tween exposures. For comparison purposes, additional subjects ingested 5 or 10 mg of diazepam, a known psychotropic drug, or were exposed to a combination of toluene and diazepam (5 mg). There were no effects from exposure to either toluene or diazepam on vigilance during the first session, and there was no effect of toluene on vigilance during the second session, although vigilance errors were increased in the groups that had ingested diazepam (both doses) and in the group 

Toluene 311 exposed to both toluene and diazepam. During the third session, vigilance errors were increased for all groups except the control group. Errors were highest in the groups exposed to 10 mg of diazepam and both diazepam and toluene. The authors considered the effect an impaired alertness. The subjective symptom survey found decreased fatigue and sleepiness, especially during the second ses- sion. Two older studies are reported here for completeness of the data base (von Oettingen et al. 1942; Carpenter et al. 1944). These studies used outdated analyt- ic techniques and a small number of subjects compared, and are not included in Table 6-3. von Oettingen et al. (1942) conducted a study with three healthy hu- man volunteers, ages 35 to 53; toluene concentrations were controlled and measured by interferometric determinations. Subjects were exposed at 0, 50, 100, 200, 300, 400, or 600 ppm for 8-h sessions (with a 30-min break) over the course of an 8-week period (each exposure was separated by several days). Two of the subjects were also exposed at 800 ppm for 3 h, then for another 2 h fol- lowing a 2-h break. Many of the exposures were repeated, with a break of sever- al days between exposures. Blood pressure, pulse rate, respiratory rate and vol- ume, differential blood cell count, and clinical chemistry parameters were monitored. Subjective symptoms were also evaluated. There were no effects of exposure on cardiac or respiratory parameters or hematology or clinical chemis- try parameters at concentrations up to 500 ppm. Decreased erythrocyte count was observed at 800 ppm. At 50 ppm, one of two subjects had no subjective complaints and the other complained of very mild headache and drowsiness (subjects also complained of moderate tiredness toward the end of the control exposure). At 100 ppm, moderate fatigue and sleepiness were the only com- plaints. Acute exposure to toluene at a concentration of 200 ppm for 8 h pro- duced headache, nausea, muscular weakness, confusion, impaired coordination, and dilated pupils, as well as after-effects including fatigue, general confusion, and moderate insomnia in one, two, or all of the subjects. Higher concentrations produced effects similar to the 200-ppm exposure; however, the effects were more pronounced and the after-effects were prolonged. Olfactory adaptation to the odor was rapid, and other than a slight smarting of the eyes and nose, none of the exposures produced irritation of the mucous membranes. Carpenter et al. (1944) exposed two subjects to toluene at 200, 400, 600, or 800 ppm for 7-8 h. Subjective symptoms ranged from transitory mild throat and ocular irritation and slight exhilaration at 200 ppm to metallic taste, transito- ry headache, extreme lassitude, scotomata, verbosity, inebriation, and slight nau- sea at 800 ppm. After the initial exposure, some adaptation to the subjective symptoms occurred. The threshold for inability to perform a steadiness task (holding a wire in a 0.25-in hole without touching the sides) was 800 ppm. Tol- uene was evaporated from a heated dish; vapor concentrations were measured with an interferometer. In order to simulate an abuse situation, a researcher inhaled toluene from a paper bag containing gauze soaked with toluene (Press and Done 1967). The exposure consisted of “rapid, deep inhalations and exhalations directly into a 

312 Acute Exposure Guideline Levels brown paper bag containing the contents of two tubes of 3/4 oz each of Testor’s cement...”. The estimated concentration of toluene in the paper bag was 10,000 ppm (3.6 mg of toluene/100 mL of air). During the 5-min exposure, the subject had sensations of dizziness, blurred vision, roaring and buzzing in the ears, and slurred speech. An electroencephalogram taken during the exposure was normal. “Blood levels exceeding 1.75 mg/100 mL were reached.” Because the concen- tration was estimated, this study is not included in Table 6-3. 2.4. Developmental and Reproductive Toxicity Reproductive and developmental toxicity of toluene have been reviewed by Donald et al. (1991) and ATSDR (2000). Data regarding human developmen- tal and reproductive toxicity are restricted to chronic exposures and include only continuous occupational or abuse situations. Intrauterine growth retardation, spontaneous abortion, premature delivery, congenital malformations, and post- natal developmental delays are clearly associated with gross toluene exposure; maternal toxicity at very high abuse concentrations includes overt CNS depres- sion, renal tubular acidosis, and fatty liver. Further confounding these report are social and health status variables, as well as the possibility of exposure to other fetotoxic agents (either as impurities or admixtures in toluene-containing prod- ucts) or deliberate or accidental exposures to other chemicals or drugs (especial- ly ethanol), which are not accounted for in these reports. These studies provide little quantitative information regarding dose response and are not considered in the development of AEGL values. Luderer et al. (1999) examined the reproductive endocrine effects of acute exposure on males and females. Women were divided into two groups com- prised of those in the follicular phase and those in the luteal phase of the men- strual cycle. Groups consisted of four or five individuals with a similar number of matched controls. A 3-h exposure at 50 ppm via a mouthpiece did not result in alterations of the serum gonadotrophins comprising luteinizing hormone and follicle-stimulating hormone; however, subtle effects on luteinizing-hormone secretion in men and women, the latter in the luteal phase, were found. There was no effect on blood testosterone concentrations in men. The authors stated that the clinical relevance of the subtle effects on luteinizing-hormone secretion is unclear. Gericke et al. (2001) found no effect of chronic toluene exposure on folli- cle-stimulating hormone, luteinizing hormone, or testosterone of 1,077 male subjects compared with a referent group. There is some indication that lower concentrations of hormones, follicle-stimulating hormone, luteinizing hormone, and serum testosterone (Svensson et al. 1992) and dysmenorrhea (Ng et al. 1992) may be associated with occupational exposure to toluene. However, Ng et al. (1992) state that it is uncertain whether other behavioral and work-related factors may also have contributed to the incidence of dysmenorrhea. 

Toluene 313 2.5. Genotoxicity Several investigators examined the effect of occupational exposures to toluene on peripheral lymphocytes. Chromosome analyses of 24 workers (Forni et al. 1971) and 32 workers (Maki-Paakkanen et al. 1980) at rotogravure plants revealed no significant differences in the frequencies of sister chromatid ex- changes (SCEs) or chromosomal aberrations between the workers and matched controls. Bauchinger et al. (1982) examined peripheral lymphocytes in a group of male rotogravure workers who were occupationally exposed only to toluene for more than 16 years. The ages ranged from 32 to 60; 11 workers were heavy smokers (more than 10 cigarettes per day). A similar number of smokers (eight of 24) comprised the control group. Subjective complaints were similar among both groups and there was no evidence of neurologic damage in either group. The rotogravure workers had significantly more chromatid breaks, chromatid exchanges, and gaps than the control group. The continuously measured toluene concentration in the work room was between 200 and 300 ppm (benzene was <0.3%). Hammer (2002) found a three-fold increase in SCEs in 42 rotogravure printing plant workers exposed to toluene at 38-88 ppm compared with a control group. Ambient air and blood toluene concentrations did not show a relationship to SCEs, but there was a significant relationship with cresol metabolites in the urine. Five adult male volunteers were exposed to toluene at a concentration of 50 ppm for 7 h/day for 3 days; this exposure regimen was repeated three times over 2 weeks (Richer et al. 1993). Peripheral blood lymphocytes were evaluated for SCEs, cell-cycle delay, and cell mortality. Although cell mortality was tem- porarily increased, disappearing after 15 h, there were no changes in cytogenetic parameters. An in vitro experiment conducted by Gerner-Smidt and Friedrich (1978) showed that toluene concentrations up to of 1.52 mg/mL failed to alter the num- ber of SCEs or the number of chromosomal aberrations in human lymphocytes compared with controls. In this study, toluene at 1,520 ppm did, however, pro- duce significant cell-growth inhibition compared with controls. 2.6. Carcinogenicity An epidemiologic study by Carpenter et al. (1988) compared deaths from cancers of the CNS in workers at the Y-12 nuclear facility or Oak Ridge Nation- al Laboratory (Oak Ridge, TN) with a group of controls. The workers had been exposed for a mean period of 5 years to solvents (toluene, xylene, and methyl ethyl ketone). No increased incidence in death from cancers of the CNS was found compared with controls. No estimation of exposure concentrations was made in this study. Another study of occupational exposure to toluene was conducted involv- ing 1,020 rotogravure printers exposed to toluene who were employed for a min- imum of 3 months between 1925 and 1985 (Svensson et al. 1990). Estimated 

314 Acute Exposure Guideline Levels concentrations of toluene ranged from 450 ppm in the 1940s to as low as 30 ppm by the mid-1980s; workers were also exposed to various concentrations of benzene and other hydrocarbons for various durations. When the data were ana- lyzed for workers who had been exposed for at least 5 years and a latency of 10 years, no significant increase in deaths from cancers was observed. The International Agency for Research on Cancer (IARC 1999) has as- sessed the carcinogenicity of toluene. Its evaluation states that there is inade- quate evidence in humans and evidence suggesting lack of carcinogenicity of toluene in experimental animals. IARC (1999) concluded that “toluene is not classifiable as to its carcinogenicity to humans (Group 3).” EPA (2007) has not classified toluene as to its carcinogenicity because there are no human data and animal data are inadequate. 2.7. Summary Toluene is relatively nontoxic (NRC 1981). Effects of acute exposures are limited to CNS depression, cardiac arrhythmias, and renal toxicity (NRC 1981). Even concentrations that produced unconsciousness failed to produce residual organ damage. Deaths have been reported following exposure at “high concen- trations” of toluene and are usually associated with intentional solvent abuse. The “high concentrations” in these abuse situations can seriously inhibit CNS function and predispose subjects to cardiac arrhythmias. Severe renal tubular acidosis has been described in several abuse situations and in some accidental exposures. Effects on the blood and bone marrow were observed during some early studies, when industrial toluene was contaminated with benzene or used in conjunction with benzene (von Oettingen et al. 1942; Massengale et al. 1963), a known bone marrow suppressant. The highest non-fatal vapor exposures oc- curred during accidental exposures and the concentrations were either calculated or measured after the exposure. Concentrations of greater than 5,000 ppm for an undefined time (Longley et al. 1967) and greater than 1,842 ppm for several hours (Meulenbelt et al. 1990) resulted in unconsciousness and disorientation, respectively. Recovery was complete in both cases. Toluene is not a primary irritant. Slight irritation of the eyes and nose has been reported in several controlled clinical studies during 6- to 8-h exposures to at 100 ppm or higher (Carpenter et al. 1944; Andersen et al. 1983; Baelum et al. 1985, 1990; Echeverria et al. 1989), but there was no annoyance or discomfort associated with the exposures (Baelum et al. 1985). Furthermore, irritation was not among the reported symptoms during exposures of up to 800 ppm (von Oettingen et al. 1942) or in many occupational monitoring studies where con- centrations were 100 ppm or higher (Greenberg et al. 1942; Wilson 1943; Foo et al. 1990; Ukai et al. 1993; Neubert et al. 2001a). No dermal, ocular, or throat irritation was reported in painters exposed to toluene at 1,100 ppm (Greenberg et al. 1942) or in accident situations where concentrations may have been greater than 5,000 ppm (Longley et al. 1967). Adaptation occurs to the odor (Stewart et 

Toluene 315 al. 1975; Andersen et al. 1983; Mergler and Beauvais 1992) and potential drying effects on the mucous membranes (Carpenter et al. 1944; Andersen et al. 1983). The primary effect associated with inhalation exposure to toluene is CNS depression. Fifteen of the approximately 20 studies in Table 6-3 addressed neu- robehavioral effects. The neurobehavioral end points in these studies measure very subtle changes in reaction time and cognitive ability. The concentration of 100 ppm was a no-effect level for most neurobehavioral end points, including vigilance and reaction time (Ogata et al. 1970; Gamberale and Hultengren 1972; Stewart et al. 1975; Winneke 1982; Andersen et al. 1983; Dick et al. 1984; Baelum et al. 1990; Rahill et al. 1996). The effect of exercise, which results in increased uptake of toluene, was evaluated in the studies by Baelum et al. (1990) and Rahill et al. (1996). No gross neurobehavioral deficits were observed at 150 ppm (Echeverria et al. 1989), 200 ppm (Ogata et al. 1970), or 100 ppm with peaks to 300 ppm with an exercise protocol (Baelum et al. 1990). No greater effects were observed when exposures were repeated over several days (Stewart et al. 1975; Echeverria et al. 1989), and no significant gender differences were observed (Stewart et al. 1975). No impairment in alertness occurred during two successive 70-min exposures to toluene at 240 ppm (Horvath et al. 1981), alt- hough the results of the study were difficult to interpret. A significant increase in time to complete a complicated task involving perceptual speed (45.5 vs 36.9 sec) was observed at 700 ppm, following 20-min exposures at lower concentra- tions (Gamberale and Hultengren 1972). The studies by von Oettingen et al. (1942) and Carpenter et al. (1944) were reported for completeness of the data base, but were not considered in development of AEGL values because of the impurity of the toluene, the less accurate analytic measurement methods (infer- ometrics is no longer an acceptable analytic method and the reliability of the combustible gas indicator is lower than the range measured), and the small number of subjects compared with later studies. Developmental toxicity data from animal studies show that the developing embryo is no more sensitive to the toxic effects of toluene exposure than is the mother (ACGIH 2005). Developmental delay and congenital malformations resembling those of fetal alcohol syndrome have been observed in pregnant women after intentional inhalation of concentrated toluene vapor. These condi- tions were the result of chronic toluene abuse during pregnancy. There is no evidence that this syndrome occurs in women who are exposed occupationally to toluene. The relationship of toluene exposure to mutagenicity and genotoxicity is unclear as conflicting results were observed in several studies. Examination of peripheral lymphocytes of clinically-exposed volunteers and occupationally- exposed workers were generally negative for chromosome aberrations and SCEs, but were positive or questionable in other studies (Bauchinger et al. 1982; Hammer 2002). The human data are insufficient for cancer classification of tol- uene exposure in humans at this time, but examinations have found no associa- tion between cumulative toluene dose (as ppm-years) and standardized mortality ratios for either all sites or for respiratory tract cancers (Svensson et al. 1990). 

316 Acute Exposure Guideline Levels 3. ANIMAL TOXICITY DATA 3.1. Acute Lethality Acute lethality data were available for the rat and mouse, and lethal con- centration data from these studies are summarized in Table 6-4. Additional data from studies with the dog, cat, and rabbit were available but provide little infor- mation on the threshold for lethality. 3.1.1. Dogs Ikeda et al. (1990) examined the cardiovascular response in 25 dogs dur- ing acute inhalation of toluene at 30,000 ppm for 9-10 min. Electrocardiograms revealed no changes for the first 3-4 min of exposure; tachycardia ensued for several minutes and was followed by a period of bradycardia. In some dogs, ventricular fibrillation ensued shortly thereafter, followed by death. Kobayashi et al. (1989) reported that the threshold concentration of toluene for decreasing left-ventricle contractility in dogs was 3,800 ppm. 3.1.2. Rats Pryor et al. (1978) exposed groups of male Fischer rats to various unde- fined concentrations of toluene. All rats died during a 1-h exposure at 40,000 ppm. The 1-h LC50 (lethal concentration, 50% lethality) was 26,700 ppm. Cameron et al. (1938) in a summary of their earlier work reported that the 6.5-h LC50 for Wistar rats was approximately 12,200 ppm. Mortality was 60% during a 1.5 h exposure at 24,400 ppm and all 10 rats died when this exposure was extended to 6 h. No rats died during a 24-h exposure to toluene at 6,100 ppm. Nine-day old rats were more resistant to toluene than adult rats. Rats sur- vived fourteen 8-h exposures at 1,525 ppm. No details of the exposures were provided. Smyth et al. (1969) reported that one of six rats exposed at 4,000 ppm for 4 h died. No further details were provided in this range-finding study, and the study is not reported in Table 6-4. In a study by Shell Oil Company (1982), groups of three male and three female Wistar rats inhaled measured concentrations of toluene at 18,100-18,800 ppm for up to 7 h. All rats survived the 3-min exposure. After exposures at 18,100 ppm for 1 min, 18,500 ppm for 30 min, and 18,900 ppm for 7 h, mortali- ty was 17, 83, and 100%, respectively. At all concentrations, signs during expo- sure included agitation, salivation, and ocular and nasal discharge. Except for the 3-min exposure, rats became comatose, usually with whole-body tremors. Animals gained consciousness 1h after exposure. DuPont Chemical Company (1966) reported a 4-h LC50 of 18,300 ppm for male CD rats. 

TABLE 6-4 Summary of Acute Lethal and Sublethal Toxicity of Toluene in Laboratory Animals Species Concentration (ppm) Exposure Duration Effects Reference Dog 30,000 10 min Successive tachycardia, bradycardia, and Ikeda et al. 1990 ventricular fibrillation, followed by death. Rat 26,700 1h LC50 Pryor et al. 1978 40,000 1h 100% mortality Rat 24,400 6h 100% mortality Cameron et al. 1938 24,400 1.5 h 60% mortality 12,200 6.5 h 50% mortality 6100 24 h No deaths Rat 20,000 50 min 100% mortality Kojima and Kobayashi 1973 15,000 2.5 h 80% mortality 12,200 2-2.5 h LC50 10,000 2h 20% mortality 5,000 2h No deaths Rat 18,900 7h 100% mortality Shell Oil Company 1982 18,500 30 min 83% mortality 18,100 10 min 17% mortality 18,500 3 min No deaths Rat 18,300 4h LC50 DuPont Company 1966 Rat 15,000 1h No deaths Hinman 1987 Rat 6,000 4h No deaths Wada et al. 1989 7,670 4h 25% mortality Rat 6,250 2h No deaths Mullin and Krivanek 1982 3,100 4h No deaths Rat 2,667 7.5 h No deaths Lammers et al. 2005b (Continued) 317 

318 TABLE 6-4 Continued Species Concentration (ppm) Exposure Duration Effects Reference Mouse 38,465 10 min LC50 Moser and Balster 1981; 1985 21,872 30 min LC50 19,018 60 min LC50 6,000a 30 min No deaths Mouse 24,400 6h 100% mortality Cameron et al. 1938 24,400 1.5 h 10% mortality 12,200 6.5 h 100% mortality 6,100 24 h No deaths Mouse 12,000 20 min No deaths Bruckner and Peterson 1981a,b 8,600 3h LC50 4,000 3h No deaths Mouse 10,000 1h No deaths Bushnell et al. 1985 Mouse 6,940 6h LC50 Bonnet et al. 1979 Mouse 5,320 7h LC50 Svirbely et al. 1943 a Exposures were repeated for 7 weeks. 

Toluene 319 Kojima and Kobayashi (1973) exposed groups of five male albino rats to concentrations of toluene ranging from 5,000 to 25,000 ppm for 2 to 2.5 h. No rats died following exposure at 5,000 ppm for 2 h. One of five rats died during a 2-h exposure at 10,000 ppm. Four of five rats died during a 2.5-h exposure at 15,000 ppm. All rats exposed at 20,000 ppm died within 50 min. Death was at- tributed to CNS depression. As part of a neurotoxicity study (see Section 3.3.2), Mullin and Krivanek (1982) exposed groups of six male CD rats to measured concentrations of tolu- ene at 810, 1,660, and 3,100 ppm for 4 h and at 6,250 ppm for 2 h. No deaths were reported following exposures to toluene, whereas deaths were reported in similar tests with other chemicals. In other neurotoxicity tests described in Sec- tion 3.3, no deaths occurred in adult male Long-Evans rats exposed to toluene at 15,000 ppm for 1 h (Hinman 1987) or in male WAG/RijCrlBR rats exposed at 2,667 ppm for 7.5 h (Lammers et al. 2005b). No deaths occurred in adult male Wistar rats exposed at 6,000 ppm for 4 h, but two of eight rats exposed at 7,670 ppm for 4 h died (Wada et al. 1989). The fact that some of these animals were performing learned tasks increased the rate of uptake of toluene. 3.1.3. Mice Moser and Balster (1985) exposed groups of 12 male CD-1-mice to sever- al undefined toluene concentrations for 10, 30, or 60 min. Animals were exam- ined for lethality and behavioral toxicity (inverted screen test). The LC50 values for the 10-, 30-, and 60-min durations were 38,465 ppm (confidence limit: 36,067-41,023 ppm), 21,872 ppm (confidence limit: 20,731-23,076 ppm), and 19,018 ppm (confidence limit: 17,350-20,846 ppm), respectively. The authors noted that as solvent concentrations were increased for the lethality studies, mice displayed a progression of clinical signs from excitability and hyperactivity to lethargy and hypoactivity. Shallow, rapid respiration ensued and was followed by death within 1 h after the exposure. All mice survived repeated exposure at 6,000 ppm for 30 min/day, 5 days/week for 7 weeks and appeared in good health (Moser and Balster 1981). Cameron et al. (1938) exposed groups of 10 mice (strain not specified) to concentrations of toluene at 6,100-24,400 ppm for various durations. Mortalities were 10% and 100% following 1.5- and 6-h exposures, respectively, at 24,400 ppm. Mortality was also 100% after a 6.5-h exposure at 12,200 ppm. All mice survived a 24-h exposure at 6,100 ppm. No further details of the exposures were given. As part of a study on the pharmacology and pharmacodynamics of tolu- ene, Bruckner and Peterson (1981a) exposed groups of up to 14 male ICR mice to toluene at concentrations ranging from 2,600 to 12,000 ppm. The 3-h LC50 was 8,600 ppm (95% confidence interval: 8,000-9,200 ppm). No further details were provided. In a companion paper, no deaths were reported in mice exposed at 4,000 ppm for 3 h/day, 5 times weekly for 8 weeks (Bruckner and Peterson et al. 1981b). Similarly, no deaths were reported in adult C57BL/6J male mice 

320 Acute Exposure Guideline Levels exposed at 10,000 ppm for approximately 1 h or in mice exposed at 3,000 ppm for 5 h/day for 90 days (Bushnell et al. 1985). In a study by Bonnet et al. (1979), the 6-h LC50 was 6,940 in mice. No further details were available. Acute toxicity studies in Swiss mice conducted by Svirbely et al. (1943) revealed progressive symptoms prior to death, including restlessness, muscular twitching, an S-shaped curve in the tail, dyspnea, incoordination, and evidence of a narcotic effect. Toluene concentrations ranged from 3,660-8,520 ppm, as measured by a refractometer. The 7-h LC50 was 5,320 ppm (confidence limits: 4,960 and 5,710 ppm). Microscopic examination of the major organs failed to identify lesions with the exception of casts and debris in the renal tubules of some mice. 3.2. Nonlethal Toxicity 3.2.1. Dogs von Oettingen et al. (1942) exposed six dogs to toluene at 850 ppm for 1 h. This exposure resulted in an increase in respiratory rate and a decrease in res- piratory volume. No further details were provided. 3.2.2. Rats Most studies of acute exposures of rats to sublethal concentrations of tolu- ene evaluated neurotoxicity and are summarized in Section 3.3.2. Subacute ex- posures to toluene have produced hearing loss in rats. Hearing loss was found in young rats after exposure to toluene at 2,000 ppm for 8 h/day for 3 days or at 1,500 ppm for 14 h (Pryor et al. 1984). Permanent loss of hearing in the high frequency range was found when young rats were exposed at 1,200 ppm for 14 h/day, 7 days/week for 5 weeks. Morphologic examinations revealed loss of, or damage to, hair cells in the basal turn of the cochlea. Toluene was also tested for its influence on ventricular arrhythmias in the rat. Previous inhalation of toluene by Wistar rats, at up to 7,387 ppm for 15 min, reduced the ectopic ventricular activity caused by coronary ligation or admin- istration of aconitine (Magos et al. 1990). These results contrast with those with benzene, in which arrhythmias were increased in the 30 min following induction of arrhythmia. Following a near-lethal exposure to toluene at 66,000 ppm for 30 min, injection of epinephrine did not induce arrhythmia or ectopic beats in anes- thetized rats (Vidrio et al. 1986). 3.2.3. Mice Exposure of eight male ICR mice to toluene at 4,000 ppm for 3 h resulted in elevated blood SGOT 24 h after (Bruckner and Peterson 1981b). Repeated expo- sures for 3 and 5 days also resulted in elevated SGOT, with the increase significant after the 1- and 3-day exposures. When the exposures were extended to five times 

Toluene 321 weekly for 8 weeks, transient body weight depression and increased hepatic weights were observed. Histopathologic findings in the heart, lungs, kidneys, brain, and liver were generally unremarkable. The sensory irritation response in groups of four male Swiss-Webster mice during a 30-min exposure at several concentrations of toluene (900, 1,700, 2,600, 3,500, 4,100, 5,050, 6,400, or 7,800 ppm) was evaluated by Nielsen and Alarie (1982). From these data, the RD50, the concentration that depresses the respiratory rate by 50%, was calculated. At 900 ppm, there was no effect on the respiratory rate. Toluene concentrations of 2,600 ppm and higher produced a rapid, brief (approximately 1 min) decrease in the respiratory rate, followed by an increase above the control level within the next 6 min (stimulatory effect). At 7,800 ppm, the initial depressive effect on the respiratory rate was modified by the stimulatory effect at this concentration; after the brief depression, the in- creased respiratory rate, up to 140% of the control value, was sustained over the 30-min exposure. The RD50, calculated on the basis of the initial depression, was 5,300 ppm. In cannulated mice, only a small decrease in respiratory rate oc- curred at the beginning of exposures, even at the higher concentrations. The authors discuss the fading of the sensory-irritation response or desensitization that occurs with some chemicals including some alkylbenzenes, making meas- urement of an RD50 difficult. The stimulatory effect was attributed to systemic absorption of toluene from the lung. In a similar study and using OF1 mice, De Ceaurriz et al. (1981) reported a RD50 of 3,373 ppm. 3.3. Neurotoxicity 3.3.1. Non-human Primates Weiss et al. (1979) evaluated the capacity of squirrel monkeys to self- administer toluene as a model of abuse. Monkeys (number not specified) previ- ously trained to self-administer drugs on a single response were tested with tolu- ene at concentrations of 0, 560, 1,000, 3,000, or 10,000 ppm during 1-h sessions. Pushing a button delivered a 15-sec exposure to toluene vapor. The highest rate of response (141 responses/h) was at 1,000 ppm. This rate decreased thereafter with increasing concentrations. Taylor and Evans (1985) subjected adult female cynomolgus macaques to 50-min head-only exposures to toluene at 0, 100, 200, 500, 1,000, 2,000, 3,000, or 4,500 ppm and simultaneously tested for delayed matching-to-sample behav- ior as a measure of cognitive function. Monkeys were exposed singly to each concentration, with each monkey tested twice at each concentration. This proce- dure took 6 weeks. Previously, two of the monkeys had been exposed at 100 ppm and one monkey had been exposed at 1,000 ppm toluene for 6 h/day, 5 days/week for 90 days. Toluene concentration was monitored continuously with an infrared gas analyzer. Responses at 100 and 200 ppm were similar to those during the control sessions. The responses at 500 and 1,000 ppm were lower than, but not significantly different from the control responses. Cognitive func- 

322 Acute Exposure Guideline Levels tion was impaired at 2,000 ppm or higher, as indicated by an increase in re- sponse time and a decrease in accuracy of matching. Response time at 4,500 ppm increased by 0.26 sec over the control response time, and monkeys failed to respond during the second half of the session. Most monkeys remained awake at 4,500 ppm, but failed to respond. The effect was characterized as an attention deficit with no specific memory effect. Expired carbon dioxide, measured during the exposures, displayed an inverted U-shaped concentration-effect curve. 3.3.2. Rats Many of the nonlethal studies in rats evaluated the acute CNS effects of tol- uene exposure (Table 6-5). Most of these studies demonstrated the biphasic nature of reaction to toluene: an initial stimulatory effect followed by CNS depression. The effects of 0.5-, 1-, 2-, and 4-h exposures at 150 ppm in male Sprague-Dawley rats were evaluated using a multiple fixed ratio-fixed interval (FR-FI) schedule of reinforcement (Geller et al. 1979). Both schedules of reinforcement were increased during the shorter exposures (0.5 and 1 h), showing a stimulatory effect, and de- creased during the longer exposures (2 and 4 h) compared with controls. Values for individual rats were presented graphically; aside from the general pattern of an increase followed by a decrease in rate of responding over time, there were con- siderable individual differences among toluene-exposed rats. Furthermore, the pattern of the control session over time was not provided. The acute effects of toluene inhalation on the detection of auditory signals (sensitivity index and response index) were evaluated by Bushnell et al. (1994). Male Long-Evans rats were exposed to toluene at 0, 1,000, 1,500, or 2,000 ppm for 1 h. Toluene eliminated the normal increase in sensitivity index that occurred over a session. The responsitivity index was decreased by toluene at the begin- ning of each session but returned to control levels during exposure at 1,000 or 1,500 ppm, and within 40 min of exposure at 2,000 ppm was increased above control levels. Increases in latency were concentration- and time-dependent. Groups of four adult male Long-Evans rats weighing 350 g were exposed to toluene at 0, 1,200, 1,600, 2,000, or 2,400 ppm for up to 70 min (Bushnell et al. 2007a), and responses were measured at five 12-min intervals from 22-70 min (22, 34, 46, 58 and 70 min). Each rat inhaled one concentration/day and each rat was exposed at each concentration over the course of 5 days; concentra- tions were administered in counter-balanced order. Rats were previously trained in a signal-detection task consisting of a food reward in response to a visual sig- nal. There was a concentration-related change in a signal-detection task, with accuracy (attention to the signal) decreased and response time increased, but the number of false hits was not affected. Mean response times in the control group (exposure to air) and the group exposed to toluene at 2,400 ppm were approxi- mately 0.4 sec and 1.9 sec, respectively. A doubling in the response time (from 

TABLE 6-5 Neurbehavioral Effects of Acute Inhalation Exposure to Toluene in Rats Concentration (ppm) Duration Effects Reference 150 0.5, 1 h Stimulatory effect, multiple schedule performance. Geller et al. 1979 2, 4 h Reduced performance. 0, 1,200, 1,600, 22, 34, 46, 58, Signal detection task: concentration- and duration-related reduction in attention and Bushnell et al. 2007a 2,000, 2,400 70 min increase in response time; no effect on false hits; NOAEL for doubling of reaction time was 1,600 ppm for 34 min and 2,000 ppm for 46 min. 178, 300, 560 2h Increased activity (for reward). Wood and Cox 1995 1,000, 1,780 2h Increased activity, then return to control rate. 3,000 2h Increased activity, then decrease below control rate. 810 4h Threshold, decreased unconditioned reflex. Mullin and Krivanek 1982 1,660 4h Increased number of failures. 3,100 2h Decreased conditioned avoidance response. 6,500 1h Rats failed numerous test; prostrate after 2 h. 1,340 1-4 h EC50: most sensitive unconditioned reflex (calculated). 125, 250, 500 4h Decreased conditioned avoidance responses. Kishi et al. 1988 1,000, 2,000 4, 2 h Increased incorrect responses and reaction time. 4,000 4h Excitation, increased response rate, ataxia. 1,000, 1,500, 2,000 1h Initial decrease in detection of auditory signals at all concentrations followed by Bushnell et al. 1994 return to control levels. 1,000 4h Little effect on avoidance responses. Shigeta et al. 1978 3,000 4h Changes in response pattern. (Continued) 323 

324 TABLE 6-5 Continued Concentration (ppm) Duration Effects Reference 1,000, 1,780, 3,000 2h 1,780 and 3,000 ppm: concentration-dependent increase in response rates to food Wood et al. 1984 reward despite electric shock punishment. 3,000 4h Ataxia. 1,000 4h No change in behavior (number of rearings). Takeuchi and Hisanaga 1977 2,000 4h Increased rearings and seizures. 4,000 4h Excitation followed by narcosis. 2,000 4h Increased number of lever presses to avoid shock; no change in avoidance behavior. Harabuchi et al. 1993 4,000 4h Increased number of lever presses to avoid shock; decrease in avoidance response. 1,700 4h No decrease in activity following exposure. Miyagawa et al. 1986 3,400 4h Activity decreased by 31% followed by recovery. 5,100 4h Inactivity followed by partial recovery. 2,000 4h Increased locomotor activity. Wada et al. 1989 4,000 4h Decreased conditioned avoidance responses. 6,000, 8,000 4h Decreased conditioned avoidance responses, ataxia, narcosis. 1,333, 2,667 or 8,000 7.5 h Effects on visual discrimination; increased motor activity; return to baseline on van Asperen et al. 2003 (five 15-30 min peaks) following day. 2,500 1h No effect on motor activity during exposure. Hinman 1987 5,000 1h Increased locomotor activity. 10,000 1h Increased activity followed by slight decrease. 15,000 1h Increased activity followed by cessation of activity. 

Toluene 325 0.4 sec to 0.8 sec) compared with controls was evident at exposures of 2,000 and 2,400 ppm for 34 min and at 1,600 ppm for 46 min. The NOAEL for this effect was noted at 1,600 ppm for 34 min and at 2,000 ppm for 46 min. A doubling in reaction time was identified by Dr. Bushnell as a relatively unambiguous demar- cation of a clear effect on CNS function and comparable to the effect level ob- served in ethanol intoxication (P.J. Bushnell, personal communication, 2013; Appendix D). A physiologically-based toxicokinetic (PBPK) model quantita- tively predicted behavioral effects based on concentrations in the brain (Bush- nell et al. 2007a), but not predictions based on the area under the curve (AUC) of exposure or on the AUC of brain toluene concentration. Rats developed a tolerance to toluene after repeated exposure at high concentrations during per- formance testing (Oshiro et al. 2007), and performance was also affected by motivation as reward or punishment (Bushnell et al. 2009). Wood and Cox (1995) exposed 12 male Long-Evans rats to toluene at 178, 300, 560, 1,000, 1,780, or 3,000 ppm for 2 h and performed behavioral evalua- tions of nose-poking on a probabilistic schedule of food delivery. Animals served as their own controls, and exposures were repeated. Exposure sessions were 3-4 days apart. The general pattern of responses over the 2-h session for all exposures including the control was an initial high response rate during the first 20 min of the exposure followed by a tapering-off effect; the number of re- sponses also generally increased with increasing concentration. This pattern of concentration-dependent increased numbers of responses over the control value was observed except at the higher doses. During the 3,000-ppm session, the ini- tial high rates of responding fell below the control rates after 50 min of expo- sure. During the session at 1,780 ppm, the rates returned to control values by the end of the exposure. Between 178 and 560 ppm, responses over the number of control responses increased in both concentration-dependent and time-dependent manners. The authors described these results as biphasic concentration-effect, time-effect, and concentration-response functions at the higher concentrations. A weighted regression analysis determined that a response increase of 10% in all animals would be achieved at a concentration of 182 ppm, with a lower 95% confidence limit of 157 ppm. In an earlier study with a similar protocol (Wood et al. 1984), rats trained to press a lever for a food reward increased their rate of response following 2-h exposures to toluene at 1,780 and 3,000 ppm in spite of electric shock punishment. Mullin and Krivanek (1982) tested unconditioned reflexes and conditioned reflex tasks in groups of six 5-week-old male Charles River CD rats exposed to toluene at 0, 800, 1,600, 3,200, or 6,400 ppm for 4 h. Measured concentrations were 810, 1,660, 3,100, and 6,250 ppm. Unconditioned reflex tests consisted of locomotor activity, coordination, corneal reflex, and righting reflex. The condi- tioned reflex involved shock avoidance. A few rats began to fail unconditioned reflex tests at 800 ppm (one of 16 tests); the 1-, 2-, and 4-h EC50 for the most sensitive test were all 1,340 ppm. Rats showed excitement during the first half- hour of exposure at 3,100 ppm, decrements in conditioned avoidance were ob- served after exposure for 1 h, and rats began to show prostration by 2 h. At 

326 Acute Exposure Guideline Levels 6,250 ppm, rats failed numerous tests at 1 h and were prostrate at 2 h, at which time the test was terminated. There were no mortalities. A group of eight male Wistar rats were exposed to toluene at 0, 125, 250, 500, 1,000, 2,000, and 4,000 ppm for 4 h on separate days (Kishi et al. 1988). Exposures were in ascending order of concentration. Chamber concentrations were measured with a gas chromatograph. This experiment incorporated shock- avoidance behavioral observations during toluene exposure. Rats were exposed to toluene or air for 4 h a day, and the interval between exposures was 10-20 days to avoid lingering effects. Rats were tested continuously for 2 h after each exposure. When rats were exposed at 125, 250, or 500 ppm, a decline in condi- tioned avoidance responses 20 min into the exposure were found compared with baseline. However, results were variable, and there was no concentration- response relationship. Rats almost completely recovered during the postexpo- sure period. After exposure at 1,000 ppm for 4 h or 2,000 ppm for 2 h, there were concentration-related increases in lever presses and incorrect responses (implying excitation), acceleration of the reaction time, and decreases in the effective avoidance response rate. At the beginning of the 4,000 ppm exposure, the response rate increased and then gradually decreased until slight ataxia was observed in six of eight rats. A similar study by Wada et al. (1989) also used shock-avoidance training as a measure of CNS impairment when male Wistar rats (in groups of eight) were exposed to measured concentrations of toluene at 2,000, 4,000, 6,000, or 8,000 ppm for 4 h. Shock avoidance, motor activity, and latency of response were measured immediately after the exposure and at 3 and 6 h and 1, 2, and 3 days postexposure. As in the previous experiment, shock-avoidance responses were decreased in pretrained rats at 4,000 ppm and higher, but recovery was achieved within 3-6 h postexposure. Also, at 2,000 and at 4,000 ppm, locomotor activity was transiently increased. Paradoxically, at 4,000 ppm, response laten- cies were increased. The authors also reported a failure in rats exposed at 6,000 or 8,000 ppm to avoid electrical shock, and the percentage of escape responses was decreased as well. Locomotor activity was dramatically decreased at those concentrations; therefore, ataxia and narcosis could have contributed to impaired avoidance performance. Takeuchi and Hisanaga (1977) exposed groups of male Wistar rats to tolu- ene at 1,000, 2,000, or 4,000 ppm for 4 h. They observed general activity and measured EEG activity. No change in general activity was found at 1,000 ppm, as evidenced by the number of rearings, and there were few changes in the EEG results. At 2,000 ppm, there were increased rearings and occasional myoclonic seizures; these changes were accompanied by increases of high-frequency EEG activity and a disturbance of the sleep pattern. At 4000 ppm there was increased activity followed by narcosis. These rats also experienced myoclonic seizures. All sleep pattern activity was disturbed. Shigeta et al. (1978) also found that a 4- h exposure at 1,000 ppm had little effect on avoidance responses (to an electrical shock) of adult male Wistar rats. At 3,000 ppm, the responses increased, but the inter-response pattern shortened, with the result that there was no significant 

Toluene 327 difference in shock counts. Behavior recovered in an hour. Harabuchi et al. (1993) also found little difference in shock avoidance in male Wistar rats ex- posed to toluene at 2,000 ppm for 4 h. At 4,000 ppm, lever presses in response to a warning sound were increased, but shock avoidance responses were signifi- cantly decreased soon after exposure began. A biphasic recovery curve for a variable interval response schedule (lever press for food reward) was observed by Miyagawa et al. (1986). Young male Sprague-Dawley rats were exposed in groups of four to toluene at 1,700, 3,400, or 5,100 ppm for 4 h. The response rate was then assessed at recovery intervals of 0-30, 30-60, 60-90, and 90-120 min. The same rats were used at each concen- tration for the four response times (four different days). At 1,700 ppm, the be- havioral response rate was increased by about 40% compared with baseline lev- els, and duration of recovery period did not influence activity. A decrease in responses compared with baseline was observed in the 3,400-ppm group imme- diately after exposure, but the response rate increased to greater than baseline during the next 30-120 min. Immediately after exposure at 5,100 ppm, an almost total decrease in responses was observed, followed by a linear increase with respect to duration of recovery period. The authors also noted that at low brain toluene concentrations an increase in response rate occurs, which is reversed at higher concentrations so that an inverted U-shaped curve is obtained for the rela- tionship between lever-pressing behavior and toluene concentration in the brain. A biphasic concentration-effect relationship was observed in male Long- Evans rats exposed to increasing concentrations of toluene by Hinman (1987). Six rats were used with some rats being exposed at several concentrations. Spontaneous locomotor activity was monitored continuously during inhalation of toluene at 0, 2,500, 5,000, 10,000, or 15,000 ppm for 60 min. Sham exposures resulted in a period of activity for approximately 30 min followed by period of low activity (habituation). Before each toluene exposure, the rats were allowed a 30-min habituation period during which they adjusted to their surroundings. The pattern of activity during the exposure at 2,500 ppm was the same as that of the control exposure (no increased activity during the exposure). At 5,000 ppm, locomotor activity increased monophasically during exposure and subsequently decreased in the same manner during recovery. At higher concentrations (10,000 and 15,000 ppm), locomotor activity initially increased, but decreased with con- tinued exposure and eventually ceased. The highest concentrations of toluene produced a biphasic recovery, with time to maximum activity and time to recov- ery dependent on the concentration of toluene during exposure. No rats died during the exposures. Lammers et al. (2005b) and van Asperen et al. (2002) exposed groups of eight male WAG/RijCrlBR rats to toluene at constant concentrations of 1,333 or 2,667 ppm for 7.5 h or to five peaks of 8,000 ppm for 15 or 30 min, alternating with toluene-free intervals, so that total exposures were to averages of 1,333 or 2,667 ppm, respectively. Visual discrimination was tested prior to, immediately after, and 24 h postexposure. Spontaneous motor activity was monitored contin- uously from preexposure to postexposure. All exposure scenarios resulted in 

328 Acute Exposure Guideline Levels changes in visual discrimination performance, defined as a slowing of response speed and disinhibition of responding (results presented graphically). Short-term fluctuating exposure scenarios resulted in greater effects on behavior than expo- sures at a constant concentration. But effects were time- and exposure-scenario- related. Visual discrimination and activity returned to preexposure levels by 24 h postexposure. 3.3.3. Mice Several studies in mice have focused on the neurobehavioral effects of acute toluene exposure (Table 6-6). Bushnell et al. (1985) exposed groups of eight male C57BL/6 mice to toluene at 0, 100, 1,000, 3,000, or 10,000 ppm for 72 min on successive days to measure effects on motor activity and carbon- dioxide production. The same group was used for all exposures (the first expo- sure was at 10,000 ppm). For the 10,000-ppm exposure, mice were placed into the chamber before the vapor was generated for safety reasons. The concentra- tion climbed exponentially from 0 to 10,500 ppm over the course of the expo- sure (t 1/2 = 15 min). Activity was measured by interruptions of a photobeam in a computerized system. Activity was similar during exposure to air and to toluene at 100 ppm (activity followed by a gradual decline). At 1,000 ppm, motor activi- ty began to increase above that of the control after 60 min. At 3,000 ppm, the increased activity began 12 min after the start of the experiment and continued throughout the exposure. At 10,000 ppm, activity was similar to that of the con- trol group during the first 24 min, after which it declined precipitously, reaching 0 by 48 min. In the latter experiment, toluene was allowed to decline in the chamber over a period of 72 min. Carbon-dioxide production was initially sup- pressed at 1,000 ppm and higher. At vapor concentration below 6,000 ppm, an- imals recovered and became hyperactive 24 min post-exposure. In a second ex- periment, exposure of a separate group of mice to toluene at 3,000 ppm for 5 h/day for 5 days had no effect on activity when measured 30-90 min after expo- sure. In a third experiment, groups of eight mice were exposed to toluene at 0, 100, 1,000, or 3,000 ppm for 5 h/day, 5 days/week for 12 weeks. These expo- sures affected minute volume of expired carbon dioxide, but did not decrease postexposure locomotor activity or have an effect on body weight. Using the inverted screen test to measure motor performance, Moser and Balster (1985) exposed groups of 12 CD-1 mice to several undefined concentra- tions of toluene for periods of 10, 30, or 60 min. Within 60 sec after exposure, mice were tested for the ability to hold onto or climb to the top of a screen rotat- ed 180°. Significantly lower EC50 values were obtained at each increase in expo- sure duration; for the 10-, 30-, and 60-min durations, the EC50 values were 2,959, 2,012, and 1,445 ppm, respectively. The authors also presented recovery 

TABLE 6-6 Neurobehavioral Effects of Acute Inhalation Exposure to Toluene in Mice Concentration (ppm) Duration Effects Reference 100 72 min No effect on locomotor activity. Bushnell et al. 1985 1,000 72 min Increased activity after 60 min. 3,000 72 min Increased, sustained activity after 12 min. 10,000 72 min No effect on activity for 24 min followed by narcosis. 200, 400, 800 30 min No change in operant behavior. Moser and Balster 1981 1,600 30 min Increased responding, decreased reinforcement. 3,200 30 min Ataxia. 6,400 30 min Narcosis. 2,959 10 min EC50 for inverted screen test. Moser and Balster 1985 2,012 30 min EC50 for inverted screen test. 1,445 60 min EC50 for inverted screen test. ≥2,000 20 min Functional observational battery changes: abnormal posture, abnormal gait, decreased Tegeris and Balster 1994 arousal, decreased rearing. 100, 250, 500, 1,000, 2,000 30 min Concentration-related increase in locomotor activity. 4,000, 6,000, 8,000 30 min Biphasic locomotor activity; overall concentration-related decrease in locomotor activity; Bowen and Balster 1998 narcosis at end of 30-min session at 8,000 ppm; slight concentration-related decrease in schedule-controlled behavior at 100-8,000 ppm. 500 4h No effect on schedule-controlled behavior. Glowa 1981 1,000 4h Increased rate of responding. 2,000 4h Decreased rate of responding. 1,657 30 min EC50 for decreased responding for schedule-controlled behavior. Glowa et al. 1986 722-1193 4h 31-74% decrease in immobility in “behavioral despair” swimming test. De Ceaurriz et al. 1983 2,600 1-1.5 h Ataxia, no loss of consciousness at 3 h. Bruckner and Peterson 1981a, b 5,200 45 min Immobility, loss of consciousness at 1.5 h. 12,000 10 min Loss of consciousness. 329 

330 Acute Exposure Guideline Levels times with respect to maximum concentrations tested for each duration. There was only a 5-min recovery time for a 10-min exposure at 5,000 ppm. However, for the 30- and 60-min exposures at 3,000 and 2,000 ppm, respectively, there was a 30-min recovery period. In a study of operant behavior, Moser and Balster (1981) exposed groups of eight CD-1 mice individually to toluene at 200, 400, 800, 1,600, 3,200, or 6,400 ppm for 30 min in a static-exposure chamber. Tests were performed dur- ing a 15-min postexposure period. Diet was maintained at 80% free feeding weight, and sweetened milk was used as a reinforcer for lever-press responding. Reinforcement occurred only with a 10-sec pause between responses. Response rates increased in the groups exposed at up to 3,200 ppm; at 6,400 ppm, the re- sponse rate decreased below that of the control group. At concentrations above 800 ppm, the rate of reinforcement decreased in a concentration-dependent manner. When removed from the exposure cage, mice exposed at 1,600 ppm were excitable and mice exposed at 3,200 ppm had marked ataxia. Animals ex- posed at 6,400 ppm were almost anesthetized. As part of the same report, mice were exposed to toluene at 6,000 ppm for 30 min/day, 5 days/week for 7 weeks; operant testing was carried out with the acute exposures. All mice survived the repeated exposures and appeared in good health. Response rate was variable, and the reinforcement rate remained low. Operant behavior returned to baseline 3 days after exposure ended. Tegeris and Balster (1994) exposed groups of eight male Swiss mice to toluene at 0, 2,000, 4,000, or 8,000 ppm for 20 min. During the last 2 min of the exposure, the mice were observed for neurologic deficits. A functional observa- tional battery (FOB) was administered following exposures. Effects occurred both during and after exposures and were concentration-related. Motor incoordi- nation (abnormal gait), decreased muscle tone, and equilibrium changes, as well as decreased rearing occurred at 2,000 ppm and higher. Loss of the righting re- flex occurred at 4,000 ppm. By the end of the exposure at 8,000 ppm, all mice were essentially anesthetized. However, recovery was rapid when the rats were removed from exposure. Lacrimation was observed only after exposure at 8,000 ppm. The profile of neurobehavioral effects was similar to that induced by phe- nobarbital (intraperitoneal injections of 0-40 mg/kg). In another study, Bowen and Balster (1998) tested locomotor activity and schedule-controlled behavior in adult male CFW mice exposed to toluene at 100, 250, 500, 100, and 2,000 ppm. During 30-min exposures, locomotor activi- ty increased with increasing concentrations; the increases were significant dif- ferent from controls at concentrations of 500 ppm and higher. Total locomotor activity decreased with increasing concentrations of 4,000, 6,000, and 8,000 ppm. Activity became biphasic, increasing and then decreasing at both 6,000 and 8,000 ppm. By the end of the 8,000-ppm exposure session, the mice were essentially immobile and appeared anesthetized. The effect of these concentra- tions on schedule-controlled behavior (pressing a lever for a liquid reward) was also studied. According to the authors, the expected increases in rates of behav- 

Toluene 331 ior were not observed. Instead, there was a concentration-related decrease in responding, which was most obvious at 4,000 ppm and higher. The behavioral effects of toluene exposure in CD-1 mice were also evalu- ated by Glowa et al. (1986). A fixed-interval (FI) 60-sec schedule of milk deliv- ery after a response (breaking a beam of light), with an alternating series of eight consecutive rewards followed by an inter-series time out of 30 min, was devel- oped as a means of behavioral assessment. Nominal concentrations of toluene at 250 to 4,000 ppm were added to the sealed exposure chambers. Concentration- effect curves were constructed by exposing mice to incremental additions of toluene at 30-min intervals. The rate of responding in mice exposed to toluene at 700 ppm was increased, and higher concentrations progressively reduced re- sponding. The calculated EC50 for a reduced rate of responses was 1,657 ppm. In a similar study by Glowa (1981), the effects of 4-h exposures to toluene for 5 consecutive days produced similar results. The FI 60-sec milk presentation was again used, with 10-min sessions of milk availability followed by 25-min periods were responding had no consequences. The effects of toluene on sched- ule-controlled behavior were concentration-dependent: 500 ppm had little effect on the rate of responding, 1,000 ppm consistently increased rates of responding, and 2,000 ppm consistently decreased rates of responding. Four different toluene exposure concentrations of 722, 785, 977, or 1,193 ppm were evaluated in a “behavioral despair” swimming test with male Swiss OF1 mice (De Ceaurriz et al. 1983). Rodents forced to swim in a restricted space became immobile after approximately 3 min. After 4-h exposures, all four tolu- ene concentrations significantly decreased the mean duration of immobility (in- creased activity during a 3-min test period). A concentration-effect relationship was observed at the lowest concentration of toluene producing a 31% decrease in immobility and successively increasing concentrations producing 36, 54, and 74% decreases for exposures at 722, 785, 977, or 1,193 ppm, respectively. The concentration associated with a 50% decrease in immobility was 915 ppm. Bruckner and Peterson (1981a) exposed groups of four male ICR mice to toluene at concentrations of 2,600, 5,200, or 12,000 ppm for up to 3 h to evalu- ate the narcotic potency, speed of onset, and duration of CNS-depressant effects of inhaled toluene. Five reflex tests (balance, visual placing, grip strength, tail pinch, and righting reflex) were used to evaluate the onset of loss of reflex- es/narcosis during the exposures. Tests were performed at 5- to 15-min intervals. Seven-week-old mice exposed to toluene at 12,000 ppm became depressed very rapidly and were unconscious within 15 min. Mice exposed to toluene at 5,200 ppm became immobile within 45 min and unconscious after approximately 1.5 h. At 2,600 ppm, mice became ataxic in 1-1.5 h and, within 2 h, were immobile in the absence of stimulation, although consciousness was not lost within the 3-h test period. Four-week-old mice tested separately were slightly more sensitive to toluene-induced narcosis than were 8- and 12-week old mice. Rats, tested at the same concentrations, were slightly less sensitive than mice. Specific data were not provided for rats. 

332 Acute Exposure Guideline Levels Bruckner and Peterson (1981a) also reported on recovery times of mice following 5-, 10-, or 20-min exposures to toluene at concentrations of 4,000, 8,000, or 12,000 ppm. Seven-week old mice were tested in groups of five. Min- imal decrements in reflexes were observed at 4,000 ppm for up to 20 min. Re- covery to preexposure performance and reflexes took 10 min or less. Depression was greater and recovery took longer with increasing concentrations and in- creasing exposure durations. For example, animals became immobile and lost consciousness during the 10- and 20-min exposures at 12,000 ppm, but full re- covery took place after breathing fresh air for 12 and 37 min, respectively. In exposures designed to approximate human solvent abuse episodes, mice and rats were subjected to seven consecutive inhalation cycles consisting of 10- min exposures to toluene at 12,000 ppm followed by a 20-min solvent-free re- covery period (Bruckner and Peterson 1981b). Signs of ataxia characteristic of inebriation in humans were observed after 2-3 min and unconsciousness was observed in about 10 min. Although mice were depressed to a greater degree than rats, the mice exhibited marked recovery during each fresh air interval. Re- coveries in rats progressively declined over the 190-min regimen. No deaths were reported. 3.3.4. Rabbits Kobayashi (1985) described effects of toluene in rats exposed at 4,000 ppm either in air or mixed with oxygen. For both mixtures, the observed re- sponses included Cheyne-Stokes respiration (within 2-3 min), arrhythmia, in- creased blood pressure, and slow wave hypersynchronies in cortical and subcor- tical EEGs which progressed to pH decreases and arousal reaction disappearance in the sensorimotor cortex and hippocampus. Most of these effects occurred within 15-20 min. Finally, grand mal seizures were followed by postictal de- pression. The respiratory acidosis shifted gradually to metabolic acidosis with continued exposure. 3.4. Developmental and Reproductive Toxicity In a review of reproductive and developmental toxicity in animal models, reduction of late fetal body weight and retardation of skeletal development were the most consistent fetotoxic effects (Donald et al. 1991). Although intrauterine growth was retarded, there is little evidence that exposure to toluene causes tera- togenic effects. Selected studies identified in the literature are summarized in Table 6-7. Although continuous exposures and 12-h exposures to toluene at low con- centrations appeared to be maternally toxic in rat and mice (Hudak and Ungvary 1978; Tatrai et al. 1980; Ungvary and Tatrai 1985), the lowest concentration that retarded fetal growth in the rat was 1,200 ppm when administered under devel- opmental and reproductive guidelines suggested by EPA (6 h/day during organ- ogenesis) (Thiel and Chahoud 1997). 

TABLE 6-7 Reproductive and Developmental Toxicity of Toluene in Animals Concentration (ppm) Duration Effects Reference Rat 266 GD 1-21 (8 h/d) Slightly reduced fetal body weights. Hudak and Ungvary 1978 399 GD 1-8 (24 h/d) Reduced fetal body weight; maternal deaths. 399 GD 9-14 (24 h/d) No effect on fetuses; maternal deaths. 100, 400 GD 6-15 (6 h/d) No effect at either concentration. LBI 1978 266 GD 7-14 (24 h/d) Delayed skeletal development. Tatrai et al. 1980 100, 500, 2,000 80 d before mating and Reduced fetal body weight (by 8%) at 2,000 ppm; no evidence of API 1985 through lactation maternal toxicity. 750, 1,500, 3,000 GD 6-15 (6 h/d) No fetal effects at 750 ppm; minimal reduction in mean litter and fetal API 1993 weights and unossified sternebrae at 1,500 and 3,000 ppm; maternal clinical signs at all concentrations. 300, 600, 1,000, 1,200 GD 9-21 (6 h/d) Reduced fetal and dam body weights at 1,200 ppm; higher mortality Thiel and Chahoud 1997 up to weaning at 1,200 ppm; no neurobehavioral or reproductive deficits in F1 generation. 1,200, 1,800 GD 7-19 No effects on reproductive parameters. Dalgaard et al. 2001 1,800 GD 7-20 (6 h/d) Increased neuronal apoptosis in brain of male offspring at PND 21. 1,500 GD 7-20 (6 h/d) Reduced fetal birth weight and lower maternal weight gain. Hougaard et al. 2003 600, 2,000 GD 7-17 (6 h/d) No effect at 600 ppm. At 2,000 ppm: fetal and dam body weight Ono et al. 1995 decreases; no anomalies or neurobehavioral effects on offspring but fetal mortality and growth retardation. 600, 2,000 6 h/d; Fertility and mating performance unaffected at both concentrations. Ono et al. 1996 females: 14 d pre-gestation to Males: no clinical signs at 2,000 ppm. Females: salivation, lacrimation, GD 7; males: 90 d (60 d prior and decreases in body weight and food consumption; increased fetal to mating) mortality at 2,000 ppm. 333 (Continued) 

334 TABLE 6-7 Continued Concentration (ppm) Duration Effects Reference Mouse 133, 399 GD 6-13 (24 h/d) Significantly reduced fetal weight at 133 ppm; all dams died Hudak and Ungvary 1978 at 399 ppm. 133, 266, 399 GD 6-15 (12 h/d) Significantly reduced fetal weight at 266 ppm; all dams died Ungvary and Tatrai 1985 at 399 ppm. 200, 400 GD 7-16 (7 h/d) Increased incidence of dilated renal pelves at 200 ppm but not at 400 Courtney et al. 1986 ppm; increase in fetuses with 13 ribs (normal number for mice). 100, 1,000 GD 1-17 (6 h/d) Significant increase in incidence of fetuses with extra (14) ribs Shigeta et al. 1981 at 1,000 ppm. Rabbit 30, 100, 300, 500 GD 6-18 (6 h/d) No effects on dams or fetuses at any concentration. Klimisch et al. 1992 Abbreviations: GD, gestation day; PND, postnatal day. 

Toluene 335 Male F344/N rats and B6C3F1 mice evaluated in the toluene inhalation studies of NTP (1990) exhibited no compound-related effects on sperm count or motility when exposed at concentrations of 3,000 ppm or less for 14 weeks or at 1,200 ppm or less for 2 years. No histopathologic lesions were observed in the epididymis, prostate, or testes either species. Male SD rats exposed to toluene at 600 ppm for 6 h/day for 90 days exhib- ited a decrease in sperm count of 13%; when exposed at 2,000 ppm under the same protocol, the sperm count decreased significantly by 26% and the weight of the epididymis declined by 15% (Ono et al. 1996); these changes had no ef- fect on mating performance or fertility as characterized by fertility and copula- tion indices. 3.5. Genotoxicity Toluene has been extensively studied for genetic toxicity both in vitro and in vivo. There is an overwhelming body of evidence that indicates that toluene is not genotoxicy. Very few positive studies exist, and the few that are positive have confounding factors which limit their reliability and relevance (NTP 1990). These confounding factors include the purity of the toluene in the case of in vitro studies and the presence of other chemicals and smoking habits in studies of workers. The metabolites of toluene, such as benzyl alcohol, are also nongenotoxic (NTP 1990). While the majority of toluene metabolites are nongenotoxic, the minor metabolite, o-cresol, has shown genotoxicity in some in vitro tests with Chinese hamster ovary cells (ATSDR 2008). Selected studies reviewed in CIR (1987), NTP (1990), IARC (1999), and ATSDR (2000) are discussed below. Toluene was assayed for mutagenicity using the Ames Salmonel- la/microsome assay by Bos et al. (1981). In this study, toluene was unable to re- vert Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 either with or without metabolic activation by S9 mix derived from livers of rats either untreated or induced with Aroclor 1254. Several other studies in S. typhimurium were also negative for gene mutation and growth inhibition due to DNA damage (Mortelmans and Riccio 1980; Anderson and Styles 1978; LBI 1978; Florin et al. 1980; Nestmann et al. 1980; Snow et al. 1981; Spanggord et al. 1982; Haworth et al. 1983). Studies in Bacillus subtilis (McCarroll et al. 1981a), Escherichia coli (Fluck et al. 1976; McCarroll et al. 1981b; Mortelmans and Ric- cio 1980), and Saccharomyces cerevisiae (LBI 1978; Mortelmans and Riccio 1980) were also negative for genotoxic effects. In vitro studies using mouse lymphoma L5178Y cells (LBI 1978; McGregor et al. 1988) or Chinese hamster ovary cells (Evans and Mitchell 1980) were also negative. Mammalian in vivo studies in rats have produced some positive, yet ques- tionable, results. In one inhalation study, rats that were exposed to toluene at 80 ppm for 4 h/day for 4 months (Dobrokhotov and Enikeev 1975) had an in- creased incidence of chromosomal aberrations. Three oral studies in rats (Do- brokhotov 1972; Lyapkalo 1973; and Sina et al. 1983) also observed chromoso- 

336 Acute Exposure Guideline Levels mal aberrations or DNA single-strand breaks. However, three of these studies used toluene preparations of unspecified purity and could have been contaminat- ed with benzene (a known clastogen). The results observed in the study by Sina et al. (1983) were probably due to cell lysis, because the single-strand breaks were only observed when cytotoxicity was greater than 30% (NTP 1990). In vivo studies in the mouse have produced negative results. Several stud- ies in the mouse were negative for genotoxic effects when several different pa- rameters were evaluated. These included micronucleus induction (Kirkhart 1980; Gad-El-Karim et al. 1984), sperm-head abnormalities (Topham 1980), dominant-lethal mutations (LBI 1981), sister-chromatid exchange (Tice et al. 1982), and chromosomal aberrations (Gad-El-Karim et al. 1984). 3.6. Subchronic and Chronic Toxicity and Carcinogenicity Jenkins et al. (1970) exposed Sprague-Dawley rats to repeated or continu- ous concentrations of toluene to study long-term effects on mortality, body weight, and hematology parameters. A group of 15 male and female rats was exposed to toluene at 1,085 ppm for 8 h/day, 5 days/week for 6 weeks, and a group of 13 male and female rats exposed continuously at 107 ppm for 90 days. A control group was composed of 14 rats. No rats died in the repeated-exposure study. Two rats continuously exposed to toluene died, one on day 28 and the second on day 56. Rats in both treatment groups gained more weight than the control group; however, rats in the treatment groups were heavier than the con- trols at the start of the study. There were no apparent effects on hemoglobin, hematocrit, or leucocyte count. No further details were reported. Poon et al. (1994) exposed groups of 10 male and 10 female Sprague- Dawley rats to toluene at 30 or 300 ppm for 6 h/day, 5 days/week for 4 weeks. The higher concentration caused mild biochemical changes (increased serum alkaline phosphatase activity in males), an occasional moderate reduction in follicle size of thyroid gland cells, and subepithelial nonsuppurative inflamma- tion of the nasal passages. There is no evidence to indicate toluene produces increased incidences of tumors in rats or mice. Gibson and Hardisty (1983) conducted a chronic toxicity and carcinogenicity inhalation study in male and female F344 rats. Groups of 120 animals were exposed to toluene at 0, 30, 100, or 300 ppm for 6 h/day for 5 days/week for up to 2 years. Subgroups of animals were killed after 6, 12, and 18 months for interim evaluations. There were no treatment-related effects on hematology or clinical-chemistry parameters and no tissue or organ lesions at- tributable to treatment. No increases in the incidences of neoplasms in treated rats compared with controls were found. Because IARC (1989) judged that the test concentrations may have been low, NTP (1990) conducted a second series of oncogenic bioassays in rats and mice. In those studies, groups of 60 male and 60 female F344/N rats and 60 male and 60 female B6C3F1 mice were exposed to toluene at 120, 600, or 1,200 ppm for up to 2 years (6.5 h/day, 5 days/week). 

Toluene 337 The results revealed no evidence of carcinogenicity in treated animals compared with concurrent controls. Mild degeneration of the nasal cavity olfactory and respiratory epithelium was observed at 600 and 1,200 ppm. Because these ef- fects on the respiratory system were not observed at a higher concentration (3,000 ppm) in a 15-week study, also conducted by NTP (1990), they can be attributed to the repeated nature of the chronic exposure regime. The NOAELs for carcinogenicity and survival were both 1,200 ppm in rats and mice. In addi- tion, no clinical signs were observed in rats or mice. In 1999, IARC concluded that there is evidence suggesting a lack of carcinogenicity in experimental ani- mals treated with toluene. 3.7. Summary Lethality data on toluene were available for only the rat and mouse. Based on LC50 values, the mouse is slightly more sensitive to toluene than the rat. Mouse LC50 values ranged from 38,465 ppm for 10 min to 5,320 ppm for 7 h. The highest nonlethal concentrations were 12,000 ppm for 20 min (Bruckner and Peterson 1981a), 5,000 and 6,250 ppm for 2 h (Kojima and Kobayashi 1973; Mullin and Krivanek 1982), and 6,000 ppm for 4 h (Wada et al. 1989). Toluene, like all CNS depressants and anesthetics, produces an initial ex- citatory stage followed by narcosis. Except for increased activity, concentrations below 1,000 ppm have little or no effect on gross manifestations of animal be- havior (NRC 1981; WHO 1987). At approximately 2,000 ppm, increased motor activity and an increased rate of responding in neurobehavioral tests occur. Higher concentrations suppress activity. In neurotoxicity tests, increased motor activity and response rates (excitation) at low concentrations and decreased ac- tivity and responses at higher concentrations are the result of CNS depression (Moser and Balster 1981, 1985; Wood et al. 1983). Increases in activity with no or minor decrements in accuracy on tasks occurred in rats and mice exposed to toluene at 1,000-2,000 ppm (Mullin and Krivanek 1982; Kishi et al. 1988; Wood and Cox 1995). Mice exposed at approximately 2,000 ppm for short periods of time began to fail equilibrium tests in some studies (Moser and Balster 1985; Tegeris and Balster 1994), but had increased activity in others (Kishi et al. 1988; Wada et al. 1989). Mice exposed at 5,200 ppm became immobile after 45 min and lost consciousness after 1.5 h (Bruckner and Peterson 1981a). The neuro- logic deficits are similar to those observed in humans. Unfortunately, the onset of neurobehavioral deficits is not readily observable in rodents, so extrapolation to humans is difficult. Furthermore, the increased activity of rodents at low con- centrations is not clearly observable in humans. A number of developmental studies have reported fetotoxicity, including reduced fetal weight and retarded skeletal development, but no evidence of tera- togenicity. Continuous exposures and 12-h exposures to relatively low concen- trations of toluene appeared to be more toxic to pregnant rat and mice (Hudak and Ungvary 1978; Tatrai et al. 1980; Ungvary and Tatrai 1985) than 6- to 8-h 

338 Acute Exposure Guideline Levels exposures at higher concentrations. The lowest concentration that retarded fetal growth of the rat was 1,200 ppm when administered under developmental and reproductive guidelines suggested by EPA (6 h/day during organogenesis) (Thiel and Chahoud 1997). These rodent studies duplicate the developmental delays associated with gross toluene exposures in humans. Studies of acute exposures to toluene at high concentrations and of repeat- ed and chronic exposures show that toluene is relatively nontoxic. Toluene at concentrations that produced unconsciousness failed to produce residual organ damage (Svirbely et al. 1943; Bruckner and Peterson 1981b; NTP 1990). Re- peated and chronic exposures at moderately high concentrations (e.g., 1,200 ppm) also failed to produce organ damage (Gibson and Hardisty 1983; NTP 1990). Effects appeared to be limited to reversible liver enzyme-activity changes (Bruckner and Peterson 1981b). In concordance with the available human epi- demiologic data, evidence of carcinogenicity from inhalation exposure to tolu- ene has not been substantiated in well-conducted rodent studies. IARC (1999) concluded that there is evidence suggesting lack of carcinogenicity of toluene in experimental animals. 4. SPECIAL CONSIDERATIONS 4.1. Uptake, Metabolism, and Disposition As shown in controlled-exposure studies with humans and animals, tolu- ene is readily absorbed through the respiratory tract. Uptake is proportional to the concentration in the inspired air, length of exposure, and pulmonary ventila- tion (Astrand et al. 1972; Astrand 1975; Veulemans and Masschelein 1978; Bruckner and Peterson 1981a). Blood:air partition coefficients of 15-21, meas- ured both in vitro at 37°C and in vivo in humans and laboratory animals, indi- cate that toluene is readily absorbed into the blood (Astrand et al. 1972; Sher- wood 1976; Sato and Nakajima 1979a; Gargas et al. 1989; Pierce et al. 1996a). The pulmonary retention percentage of toluene (measured by concentrations in inspired and expired air) was 50% in healthy male subjects exposed at 50 ppm with a workload of 50 W or at 80 ppm under sedentary conditions (Lof et al. 1990, 1993). Because uptake depends on respiratory rate and cardiac output, both of which increase during exercise, uptake increases during exercise com- pared with that at rest (Astrand et al. 1972; Carlsson and Lindqvist 1977; Veulemans and Masschelein 1978; Carlsson 1982; Nadeau et al. 2006). In hu- mans, dermal absorption, measured during atmospheric exposures, is about 1% of that measured after respiratory absorption (Kezic et al. 2000). Toluene can be detected in human blood within 10-15 sec of initiation of an exposure, and reaches 60% of maximum arterial concentrations within 10-15 min at concentrations as low as 100-200 ppm (Astrand et al. 1972; Benignus 1981). During a 4-h exposure to toluene at 80 ppm under sedentary conditions, approximately 90% of the 4-h blood value was attained at 2 h; steady-state was reached more rapidly under exercise conditions (Hjelm et al. 1988; Lof et al. 

Toluene 339 1990, 1993). In a study of mice exposed to toluene at 4,000 ppm, arterial blood concentrations did not reach maximum values until about 2 h after the onset of exposure (Bruckner and Peterson 1981a). After the first 20-30 min of inhalation, the ratio of toluene concentration in the brain and blood is constant (Benignus et al. 1981). Distribution of toluene to body tissues depends on blood flow to the tissue or organ, lipid content of the tissue, rate of metabolism, and duration of expo- sure. After absorption, toluene is rapidly distributed to highly vascularized tis- sues, such as the liver, kidneys, and brain. It rapidly accumulates and affects the brain due to that organ’s high lipid content (Bruckner and Warren 2001). It is eventually absorbed and stored in adipose tissue; consequently, obese people tend to accumulate more toluene than do lean people (Carlsson and Lindqvist 1977). Male Sprague-Dawley rats exposed to toluene at 550 ppm for 1 h had the following concentrations of toluene in their tissues immediately after exposure: adipose tissue, 87 µg/g; adrenal glands, 56 µg/g; kidneys, 55 µg/g; liver 21 µg/g; and brain, 15 µg/g (Carlsson and Lindqvist 1977). Absorbed toluene undergoes rapid, extensive metabolism, primarily in the liver (Low et al. 1988; ATSDR 2000). The major pathway of toluene detoxifica- tion and elimination is methyl hydroxylation to form benzoic acid followed by dehydrogenation to benzoic acid and conjugation with glycine to form hippuric acid which is excreted in the urine (Figure 6-1). In humans, oxidation of the methyl group by the hepatic cytochrome isozyme CYP2E1 yields benzyl alcohol (Liira et al. 1991; Nakajima et al. 1997). Benzyl alcohol is rapidly oxidized by alcohol dehydrogenase to benzaldehyde, which in turn is converted to benzoic acid by aldehyde dehydrogenases. About 75-80% of the absorbed dose of tolu- ene is metabolized to benzoic acid, which is also a common food constituent. Following conjugation with glycine, benzoic acid is excreted in the urine as hip- puric acid (75-80% of the absorbed dose in humans); smaller amounts of benzo- ic acid are excreted as the sulfate or glucuronide conjugate. Toluene can also be hydroxylated to form o-, m-, or p-cresol, which are conjugated with sulfate or glucuronide and excreted in the urine. The cresols are minor urinary metabolites (less than 1% of the absorbed dose). The remainder of the absorbed dose, about 18%, is eliminated via the lungs as unchanged toluene. Studies with rat liver microsomes indicate that pathways are the same in humans and rats. Pierce et al. (1999, 2002) measured exhaled toluene and excreted metabo- lites in 25 men, ages 20-62, who were exposed to 2H-toluene at 50 ppm and un- labeled toluene at 50 ppm through a gated mouthpiece for 2 h while at rest. Me- tabolites were evaluated for 4 days, and the disposition was as follows: exhaled toluene, 13 ± 6.2%; hippuric acid, 75 ± 6.4%; o-cresol, 0.31 ± 0.22%; m-cresol, 0.53 ± 0.44%; and p-cresol, 11 ± 3.8%. The exhalation rate of toluene was ex- ponentially triphasic, whereas metabolite excretion rates were biphasic. 2H- toluene was cleared slightly faster than unlabeled toluene, but the difference was small compared with individual differences in toluene kinetics. Body weight, adipose tissue fraction, and blood:air partition coefficient were correlated with 

340 FIGURE 6-1 Metabolism of to oluene in mammalian n systems (heavy arro ows indicate major pathways of metabolissm). Source: ATSDR R 2000. 

Toluene 341 terminal half-life, steady-state volume of distribution, and terminal volume of distribution (Pierce et al. 1996a). During the 2-h exposure, a 10-fold interindi- vidual range in venous blood concentrations of toluene was found between the highest and lowest values (ventilation rate differed between these two subjects by a factor of two). Benoit et al. (1985) reported that following a 90-min exposure of seven human subjects to toluene at 50 ppm, retention at steady-state (measured by elimination in exhaled air) was 83%. In the postexposure period, toluene was eliminated occurred by first order kinetics, with a half-life of 25 min. Lof et al. (1993) found that toluene elimination from the blood of nine human volunteers after exposure at 53 ppm for 2 h was triphasic, with half-lives of 3, 40, and 738 min. The longer half-life is presumed to be representative of the mobilization of toluene from adipose tissue, given the chemical’s high lipophilicity. Brugnone (1985) reported half-lives of 1.8 and 29 min in vessel-rich tissue and muscle of humans, respectively, and 36 and 2.7 h in fat and vessel-poor tissues. Metabolism and excretion of toluene are rapid, as indicated by recovery from behavioral deficits in rodents and rapid elimination from the brain follow- ing exposure (Gospe and Calaban 1988). 14C-Labeled toluene decayed in an exponential manner from brain compartments of male Long-Evans rats, with negligible amounts detected at 4 h. At 15 min postexposure, concentrations were half of those at the end of exposure. Nonvolatile metabolites were not detected in the brain, and the CNS-depressant activity was attributed to toluene. Initial activity was highest in the lipid-rich medulla/pons followed by the mid-brain. However, elimination coefficients were not correlated with regional lipid con- tent. The atmospheric concentration during the 10-min exposure was not speci- fied. Radiolabeled toluene, injected into the antecubital vein, was cleared from the brain of baboons with a half-life of 10-20 min (Gerasimov et al. 2002). Several factors influence rate and pathway of metabolism in humans (Nakajima et al. 1992; ATSDR 2000). The cytochrome P-450 isozymes respon- sible for toluene metabolism influence the relative production of benzyl alcohol, o-cresol, and p-cresol. There are two cytochrome P-450 isozymes that play ma- jor roles in the metabolism of toluene in rats: CYP2C11 and CYP2E1. The for- mer has a high Km (metabolic rate constant) and becomes increasingly im- portant with increasing exposure concentrations of toluene. The latter has a low Km and is induced by fasting and ethanol. CYP2B1 is induced by toluene con- centrations as low as 500 ppm and is important for the formation of the genotox- ic o-cresol (Wang et al. 1993). The action of these isozymes is influenced by factors, such as age, sex, and pregnancy (Nakajima eta l. 1992). Therefore, ex- trinsic and intrinsic factors can influence the relative amounts of the various urinary metabolites that are produced after toluene exposure. Several studies indicated that measurement of hippuric acid in the urine could be used as an indicator of toluene exposure (Ogata et al. 1970; Nomiyama 

342 Acute Exposure Guideline Levels and Nomiyama 1978; Hasegawa et al. 1983; Ogata 1984). However, measure- ment of hippuric acid may not be a good indicator of toluene exposure, as me- tabolism of other substances to hippuric acid may override the levels from tolu- ene alone (NIOSH 1973; Stewart et al. 1975). When volunteers were exposed to deuterium-labeled toluene at 50 ppm and nonlabeled toluene at 50 ppm, there was little variation in labeled hippuric acid excretion among individuals (78% excreted after 20 h) (Lof et al. 1993). However, unlabeled hippuric-acid excre- tion varied widely among subjects and was about four times greater than what would have been generated by toluene exposure alone. In humans there are large variations in the amounts of toluene metabolites; therefore, monitoring of uri- nary metabolites can serve only as a qualitative marker for toluene exposure (Andersen et al. 1983; Hasegawa et al. 1983; Baelum et al. 1987). The concentration of toluene in blood and tissues is proportional to the concentration in alveolar air, which in turn is proportional to the atmospheric concentration. Numerous investigators have reported on the exposure and alveo- lar concentration of toluene in relation to tissue content during controlled expo- sures in human subjects (Astrand et al. 1972; Gamberale and Hultengren 1972; Veulemans and Masschelein 1978; Carlsson 1982; Hjelm et al. 1988; Tardif et al. 1991) and animal models (Benignus et al. 1981, 1984; Bruckner and Peterson 1981a; Tardif et al. 1992; van Asperen et al. 2002). Nadeau et al. (2006) report- ed that toluene concentrations in alveolar air of human subjects exercising at 50- 100 W were 1.4- to 2.0-fold greater than from when exposures administered at rest. Several of the studies also measured toluene concentrations in the brain of rodents. With the exception of concentrations above 5,200 ppm in a study with the mouse (Bruckner and Peterson 1981a), all of the studies demonstrated a lin- ear relationship between concentrations in both atmospheric and alveolar air and blood concentrations. At concentrations above 5,200 ppm, the metabolism of toluene may be saturated in the mouse. Although values appear comparable among some of the studies, differences in analytic techniques and the use of arterial rather than venous blood samples allow only general comparisons to be made. Selected studies of toluene in human subjects and animal models are pre- sented in Table 6-8 and presented graphically in Figure 6-2 (human studies) and Figure 6-3 (animal studies). The clinical studies represent different exposure situations, ranging from sedentary subjects to exercising subjects (Astrand et al. 1972; Lof et al. 1990; 1993) and to routine working conditions (von Oettingen et al. 1942). Steady-state may not have been reached for blood concentrations dur- ing the successive 20-min exposures in the study of Gamberale and Hultengren (1972). Where several studies involving the same species are available, there is relatively good agreement among peak blood concentrations. In general, for sim- ilar air concentrations, peak blood concentrations are inversely related to body size, being greatest in the mouse, followed by the rat and then the dog. 

TABLE 6-8 Relationship between Ambient Air and Blood Concentrations of Toluene Species Exposure Duration Air Concentration (ppm) (level of work) Blood Concentration (mg/L) Reference Human – Background, general population Up to 0.015 ACGIH 2001 Human 8h 40-54 0.41 Brugnone et al. 1986 (occupational) Human 7h 33 0.20 Tardif et al. 1997 Human 6.5 h 50 0.77 Tardif et al. 1991 3.5 h 95 1.36 Human 4.5 h 0 (control) 0.01 Muttray et al. 2005 50 0.50 Human 2h 80 (rest) 0.6 (mg/L, arterial)a Carlsson 1982 0.4 (mg/L, venous) b 80 (50 W) 2.1 (mg/L, arterial) 1.2 (mg/L, venous) Human 4h 53 0.28c; 0.52-0.64 Wallen et al. 1985 Human 4h 80 0.47 Hjelm et al. 1988 Human 4h 80 (no exercise) 0.52 Lof et al. 1990 Human 2h 50 (exercise, 50 W) 0.92 Lof et al. 1993 Human Cherry et al. 1983; 8 men 4h 80 1.17 Waldron et al. 1983 7 men 4h 80 0.98 Human 4 h (four 50-min exposures) 50 0.2 Veulemans and Masschelein 1978 100 0.4 150 0.6 Human 8h 100 1.3 Angerer et al. 1980 8h 200 3.4 (Continued) 343 

344 TABLE 6-8 Continued Species Exposure Duration Air Concentration (ppm) (level of work) Blood Concentration (mg/L) Reference Human 6 h (occupational) 50-100 0.85-1.70 Neubert et al. 2001a Human 20 mind 100 0.6 Gamberale and Hultengren 1972 20 min 300 1.8 20 min 500 3.0 20 min 700 4.5 Human 30, 60 min 100 (no exercise) 1.0 Astrand et al. 1972 30 min 200 (no exercise) 2.0 30, 60 min 100 (exercise, 50 W) 2.3 30 min 200 (exercise, 50 W) 4.8 Human 8 h (occupational) Nonexposed 0.004 Foo et al. 1988 32 (50 W) 0.56 65 (50 W) 0.99 86 (50 W) 1.3 115 (50 W) 1.5 132 (50 W) 1.9 196 (50 W) 2.6 Dog 1h 700 27 Hobara et al. 2000 1h 1,500 56 1h 2,000 67 Rat 1h 2,000 100b Oshiro et al. 2007 Rat 1h 2,000 (sedentary) 31.5-42.8 Bushnell et al. 2007b; 2,000 (active) 36.5-50.4 Kenyon et al. 2008 2,000 (free fed) 25-31.7 2,000 (weight maintained) 16.7-28.1 Rat 7h 100 1.3 Korsak et al. 1991 Rat 5h 75 0.7 Tardif et al. 1992 5h 150 2.7 5h 225 5.1 

Rat 4h 50 0.5 Haddad et al. 1999a 100 1.3 200 5.0 Rat 3h 50 0.45 Benignus et al. 1984 3h 100 0.89 3h 500 10.2 3h 1,000 30.6 Rat 2h 1,000 58 Rees et al. 1985 2h 1,780 94 2h 3,000 120 Rat 4h 125 2.5 Kishi et al. 1988 4h 250 7 4h 500 17 4h 1,000 27 4h 2,000 70 4h 4,000 120 Rat 7.5 h 1,333 45 van Asperen et al. 2003; 7.5 h 2,667 79 Lammers et al. 2005b Mouse 2h 1,300 97 Bruckner and Peterson 1981a 2h 2,600 147 3h 4,000 200 2h 5,200 242 2h 10,400 315 a Blood concentrations were reported in mg/kg or µg/g. b W, watts exercise (1 W = 4.2 calories/h). c Concentrations reached a peak at 2.5 h and then decreased during the last 1.5 h. d These were successive exposures of 20 min each; steady-state may not have been reached at the higher concentrations. 345 

346 25 Gamberale & Hultengren, 1972 20 Blood Concentration (mg/L) 15 Astrand et al., 1972 (No Exercise) 10 Astrand et al., 1972 (With 5 exercise) 0 0 100 200 300 400 500 600 700 800 900 von Oettingen et al., 1942 Air Concentration (ppm) FIGURE 6-2 Relationship between air and blood concentrations of toluene in humans under different exposure conditions. 

Toluene 347   350 300 Blood Concentration (mg/L) 250 Dog 200 Rat Mouse 150 100 50 0 0 2000 4000 6000 8000 10000 12000 Air Concentration (ppm) FIGURE 6-3 Relationship between air and blood concentrations of toluene in different animal species. Data for the dog, rat, and mouse are from studies by Hobara et al. (2000), Rees et al. (1985), and Bruckner and Peterson (1981a), respectively. Using a single exposure concentration, Benignus et al. (1981) showed that blood and brain concentrations of toluene reach an asymptote fairly rapidly (Figure 6-4). The authors measured venous blood and brain concentrations of Long-Evans rats (gender unspecified) exposed to toluene at 575 ppm for 240 min. Animals were killed serially at 15, 30, 60, 120, and 240 min both during exposure and during a 240-min post-exposure period. The investigators fit the raw data to one-compartment, three-parameter models as shown in Figure 6-4. Blood and brain toluene concentrations achieved 95% of estimated asymptotes in 53 and 58 min, respectively. Estimated asymptotes were 10.5 ppm (mg/L) for venous blood and 18.0 ppm (mg/L) for brain. Blood and brain toluene concen- trationss rose and fell at similar rates, although toluene in the brain fell slightly more rapidly than in the blood. The above value for brain concentration follow- ing exposure to toluene at 575 ppm is essentially the same as that of Carlsson and Lindqvist (1977), who measured a brain concentration of 15 µg/g (mg/L) in rats exposed at 550 ppm for 1 h. In studies of higher concentrations, 1,700, 3,400, and 5,100 ppm, Miyagawa et al. (1986) measured brain concentrations of approximately 120, 267, and 400 μg/g (mg/L), respectively (numbers read from graph) in young male Sprague-Dawley rats. Concentrations were measured im- mediately after 4-h exposures. 

348 Acute Exposure Guideline Levels 100 End of Exposure Brain ppm 10 Blood 1 0 60 120 180 240 300 360 420 480 Time (minutes) FIGURE 6-4 Toluene concentrations in the brain and venous blood of Long-Evans rats during and after exposure to toluene at 575 ppm for 4 h. (Blood concentrations of toluene given in ppm = mg/L.) Source: Adapted from Benignus et al. 1981. A few studies reported blood toluene concentrations associated with coma in humans. Two workers admitted to the hospital in comas due to an accidental occupational exposure to mixed solvents had blood toluene concentrations of 823 and 1,122 µg/L at 36 h after the end of exposure (Brugnone et al. 1983). Respective alveolar air toluene concentrations were 53 and 38 µg/L. Calculated blood:air partition coefficients were 23 and 28, respectively. The decline in both compartments over the ensuing 5-day period allowed calculation of blood half- lives of 27.1 and 17.1 h in the respective workers and alveolar half-lives of 20.8 and 17.5 h. The authors noted that these half-lives were for the decline of tolu- ene in fat and other poorly perfused tissues, as the half-life from well-perfused tissues would be on the order of 2 min. Extrapolation to the end of exposure gave blood concentration of 6.2 and 8.4 mg/L, respectively. Toluene concentrations have also been measured in fatalities, but meas- urements were taken several hours after exposure in most cases. In 1983, Pater- son and Sarvesvaran reported a fatality involving a 16-year-old Caucasian male who was found dead with a plastic bag over his head. The trachea and bronchi of the deceased contained inhaled stomach contents. His toluene blood concentra- tion was 20.6 µg/mL, and the brain and liver concentrations were 297 and 89 µg/mg of tissue, respectively. The authors determined the cause of death to be 

Toluene 349 toluene poisoning; they also stated that using a plastic bag to “concentrate” the vapors and direct inhalation from the bag contributed to the fatality. A painter who died following a fall had tissue concentrations of toluene in blood, lungs, liver, and brain of 48, 35, 65, and 80 μg/g, respectively (Takeichi et al. 1986). The authors stated that these concentrations were not definitely lethal, but were high enough to anesthetize the CNS. Kojima and Kobayashi (1973) measured toluene in blood of rats that died during 30-50 min exposures at 20,000 ppm. Average toluene concentrations were 890 µg/g in the brain, 700 µg/g in the liver, and 330 µg/g in the blood. During additional exposures, no rats died when brain concentrations were less than 760 µg/g. No deaths occurred during a 120-min exposure at 5,000 ppm; average toluene concentrations in blood, liver, and brain during exposure were 220, 510, and 480 µg/g. Miyagawa et al. (1984) observed increased response rates of rats to a variable interval schedule-controlled operant task when the brain toluene concentrations were less than 200 µg/g and decreased response rates when brain toluene concentrations were between 200 and 350 µg/g. How- ever, van Asperen et al. (2003) found that repeated high-dose spikes producing peak brain concentrations in rats up to 248 mg/L did not predict the level of be- havioral impairment observed across different exposure scenarios. Bruckner and Peterson (1981a) related the following blood concentrations in mice to states of CNS depression: 40-75 µg/g, ataxia; 75-125 µg/g, immobili- ty in the absence of stimulation; 125-150 µg/g, hypnosis with arousal difficult; and >150 µg/g, unconsciousness. A good correlation between brain toluene con- centrations and the extent of CNS depression was also found. Exposures were to toluene at 4,000 ppm for 3 h or 10,600 ppm for 10 min. 4.2. Mechanism of Toxicity The most common consequence associated with toluene exposure at con- centrations historically found in the workplace is CNS depression. CNS depres- sion and narcosis are thought to involve the reversible interaction of toluene (not its metabolites) and lipid or protein components of nervous system membranes. Bruckner and Warren (2001) summarized present theories on mechanisms of action. These involve (1) a change in membrane fluidity, thereby altering inter- cellular communication and normal ion movements, (2) interaction with hydro- phobic regions of proteins, thereby altering membrane-bound enzyme activity or receptor specificity, (3) enhancement of the neurotransmitter gamma- aminobutyric acid (GABAA) receptor function, and (4) activation of the dopa- minergic system. Two mechanisms of toxicity have been proposed for CNS ef- fects due to repeated exposure: 1) interaction of toluene with membrane proteins and/or phospholipids in brain cells changes the activities of enzymes involved in the synthesis or degradation of neurotransmitters, which in turn may produce subtle neurologic effects, and (2) toluene may change the binding of neuro- transmitters to membrane receptors (ATSDR 2000). 

350 Acute Exposure Guideline Levels Intentional exposure to excessively high concentrations of toluene results in feelings of euphoria, which progress to lethargy and neurobehavioral deficits; these effects resemble those produced by anesthetics. Toluene is highly lipo- philic and, as a nonpolar, planar molecule, can behave as an anesthetic by dis- solving in the interior lipid matrix of a membrane. Increasing toluene concentra- tion produces membrane expansion as well as changes in membrane structure and fluidity. Following an acute exposure, toluene diffuses out of the membrane, original integrity is regained, and functional characteristics can be restored (ATSDR 2000). Renal toxicity with metabolic acidosis may be experienced at high concen- trations of toluene. Distal renal tubular acidosis is an established consequence of toluene abuse and has been reported in numerous studies. In life-threatening cases, patients present with severe generalized muscle weakness, nausea and vomiting, and neuropsychiatric derangements (Streicher et al. 1981; Batlle et al. 1988; Marjot and McLeod 1989). The disorder results from the inability of the distal tubule of the nephron to secrete hydrogen ions through the active transport pathway of the collecting tubule of the kidney, resulting in metabolic acidosis with respiratory compensation and production of alkaline urine and hyper- chloremia. The high anion gap of the blood may be due to the accumulation of the acidic metabolites of toluene, namely benzoic acid and hippuric acid. The mechanism(s) of action at the molecular level for hearing loss and impairment of color vision are poorly understood (ATSDR 2000). In hearing loss, toluene exposure leads to a loss of outer hair cells in the ear. There may also be an effect on neural cell membranes. The postulated mechanism of action for color vision impairment involves toluene interference with dopaminergic mechanisms of retinal cells or demyelinization of optic nerve fibers. 4.3. Structure-Activity Relationships No structure-activity issues have been identified with regard to toluene toxicity. In a summary statement based on the studies of benzene, toluene, and mixed xylenes, NTP (1990) states that methyl and dimethyl substitution on the benzene ring eliminates the carcinogenic activity in rodents. The toxicity of tol- uene resembles that of benzene except that toluene is devoid of benzene’s chron- ic hematopoietic toxicity (Henderson 2001). Because many alkylbenzenes have the same CNS depressant effect, similar to that of CNS-depressant drugs and ethanol, their relative potency as CNS de- pressants might be of relevance. In their study of the effects of toluene on the functional observational battery in mice, Tegeris and Balster (1994) compared the effects of benzene and five alkylbenzenes with that of phenobarbital (5-40 mg/kg, intraperitoneal). The alkylbenzenes were toluene, ethylbenzene, propylbenzene, xylenes, and cumene. All exposures were to concentrations of 2,000, 4,000, or 8,000 ppm for 20 min. All agents decreased arousal, increased the ease of han- dling, decreased muscle tone, produced psychomotor impairment, and reduced reactivity to stimuli. At the two highest concentrations, the alkylbenzenes pro- 

Toluene 351 duced an anesthesia-like effect with loss of righting reflex (for phenobarbital, the loss of righting reflex occurred at 30 mg/kg). Both propylene and m-xylene re- quired slightly higher concentrations to produce effects comparable to those of benzene, toluene, ethylbenzene, and cumene, although insufficient concentrations were studied to allow precise comparisons. The converse appears to be true for lethality. Where data on toluene and xylenes were available for the same species, LC50 values were slightly lower for xylene. For the mouse, the 6-h LC50 values for xylene isomers qwew 3,907-5,267 ppm, whereas the 6-h LC50 for toluene was 6,940 ppm (Bonnet et al. 1979). 4.4. Other Relevant Information 4.4.1. Interspecies Variability Uptake of toluene by dogs (Hobara et al. 2000) appears to be more rapid and the blood concentrations attained slightly higher than those in several hu- man studies (Figures 6-2 and 6-3). Uptake is somewhat greater in the rat than in humans, on the basis of blood: air partition coefficients and higher respiratory rate and cardiac output. The blood:air partition coefficient of 21.0 in rats is higher than that of humans (13.9) (Thrall et al. 2002). Physiologically-based pharmacokinetic (PBPK) modeling was done in rats and humans (see Appendix C). PBPK modeling allows a comparison of the in- ternal dose that is received in both species receiving identical external expo- sures. As can be seen in Figures 6-2 and 6-3, rats achieve higher blood toluene concentrations than humans. This is primarily due to the higher respiration rate and cardiac output, as well as a slightly higher blood:air partition coefficient in rats (13-21) (Sato and Nakajima 1979a; Gargas et al. 1989; Thrall et al. 2002; van Asperen et al. 2003) compared to humans (10-18; Thrall et al. 2002; Sato and Nakajima 1979a; Fiserova-Bergerova and Diaz 1986; Pierce et al. 1996a). The interspecies factor is comprised of a pharmacodynamic component as well. A review of the data indicate little difference in interspecies sensitivity to toluene. Comparison of LC50 values for the rat and mouse shows that the mouse is slightly more sensitive than the rat. The 1-h LC50 in rats was 26,700 ppm (Pryor et al. 1978). In the mouse the 1-h LC50 was 19,018 ppm (confidence lim- its: 17,350-20,846 ppm) (Moser and Balster 1985). The sequelae of death was similar in both species with observations of lacrimation, hyperactivity, hypoac- tivity, lethargy, and shallow respiration followed by death. The greater uptake of toluene in mice compared with rats, on the basis of respiratory rate and cardiac output, is offset to some degree by more rapid toluene metabolism by the mouse. Neurotoxicity end points at the same concentration of toluene were also similar for rats and mice. For both species, 1,200 ppm was a chronic no-adverse- effect level (NTP 1990); for short-term exposures, motor activity was increased in rodents exposed at 1,000 ppm (see Tables 6-5 and 6-6). For rats and mice, 2,000 ppm may be the threshold for CNS depression, but a 50-min exposure at 2,000 ppm failed to produce an attention deficit in monkeys (Taylor and Evans 

352 Acute Exposure Guideline Levels 1985). At the same target tissue dose, humans may experience effects similar to those observed in animals, although it would take longer to attain the effects in humans due to a lower breathing rate per kilogram of body weight. In animals, death is preceded by increased activity, followed by narcosis, and is likely the result of depression of the CNS resulting in respiratory arrest. CNS depression with narcosis has also been observed in several accidental and intentional human exposures. Thus, nonlethal effects in both humans and animals are similar in nature and consist primarily of irritation and CNS effects at high concentrations of toluene. Benignus et al. (2007) compared the acute neurotoxicity of toluene in rats and humans. A meta-analysis of dose-effect functions for variables such as choice reaction time in humans and reaction time in rats suggested that sensitivi- ty to toluene is equivalent in humans and rats if both species performed behav- iors that were controlled to the same extent. 4.4.2. Intraspecies Variability Toluene produces CNS dysfunctions that are similar to those produced by other anesthetics (Bruckner and Warren 2001). Studies indicate that children, and particularly infants, are more resistant than adults to the effects of various volatile anesthetics (Gregory et al. 1969; Stevens et al. 1975; Lerman et al. 1983; LeDez and Lerman 1987; Katoh and Ikeda 1992; Chan et al. 1996). The susceptibility of individuals of different ages has been extensively studied in the anesthesia literature where the concentrations of various anesthetic gases in the lungs which produce “anesthesia” (lack of movement) have been measured. Values are usually reported as the minimum alveolar concentration which pro- duces lack of movement in 50% of persons exposed at that concentration. Min- imum alveolar concentrations for several anesthetic gases have been measured as a function of age. The results consistently show a pattern with maximal sensi- tivity (lowest minimum alveolar concentration) in newborns, particularly prema- ture infants, pregnant women, and the elderly. The least sensitive (highest mini- mum alveolar concentration values) occur in older infants, toddlers, and children compared with normal adults. The total range of sensitivity is 2-3 fold. On the basis of this knowledge, it is not unreasonable to assume that the same 2-3 fold difference in sensitivity among individuals would apply for toluene. In healthy adult male volunteers exposed to toluene at 40 ppm for 2 h (number of subjects not specified), blood toluene concentration differed by no more than 2-fold (Bessems et al. 2004). Small standard deviations in additional studies also indicate little inter-individual variation for a constant level of activi- ty (Astrand et al. 1972; Carlsson 1982; Rahill et al. 1996). In one study, Pierce et al. (1999) reported an approximate 10-fold interindividual range in venous concentrations in two individuals exposed to toluene at 100 ppm for 2 h. The ventilation rate of these two subjects differed by a factor of two. A PBPK model indicates that there are no adult-children differences in tissue dose during inhala- tion (Pelekis et al. 2001). 

Toluene 353 4.4.3. Concentration-Response Relationship The two primary effects of toluene exposure are irritation and CNS de- pression. Irritation is considered a threshold (concentration related) effect and therefore should not vary over time. CNS depression is also a concentration- related effect, with duration of exposure also a factor at higher exposures and short durations (less than 8 h). At low concentrations of 80 to 200 ppm, toluene approaches steady-state in the blood within 15-30 min (Astrand et al. 1972; Carlsson 1982). At higher concentrations, steady-state is approached in a con- centration-related manner within one to several hours (Benignus et al. 1981; Gospe and Al-Bayati 1994). Storage would take place in lipid-rich tissues, but elimination is rapid. Once steady-state is reached, concentration is the prime determinant of toluene-induced CNS effects. Thus, concentrations that are not irritating or narcotic to humans within 30 min are assumed to have no greater effect with prolonged exposure. Although blood concentrations can be used as a surrogate for brain con- centrations, the target tissue for toluene, steady-state is not reached and there is much variability in the brain:blood ratio across durations at higher concentra- tions and the short durations relevant to AEGL derivation. As noted by Bushnell et al. (2007a), momentary brain concentration is most predictive of CNS effects. For higher exposure concentrations, PBPK modeling was used to determine hu- man equivalent AEGL values from the animal data. PBPK modeling was used to calculate the internal dose (BrTC) in several studies with the rat. The general physiological and toxicokinetic (GPAT) human model for toluene was then used to determine the exposure concentrations in humans that yields the same con- centration (Benignus et al. 2006). PBPK modeling allows a determination of the internal dose for the experimental species, and a determination of the external exposure which would lead to the same internal dose in the human. As can be seen in Figures 6-2 and 6-3, rats achieve higher blood toluene concentrations than humans. This is primarily due to the higher respiration rate and cardiac output, as well as a slightly higher blood:air partition coefficient in rodents. 4.4.4. Concurrent Exposure Issues Toluene and a number of other volatile organic compounds are competi- tive metabolic inhibitors, as they are oxidized by some of the same P450 iso- zymes. The net effect is an increase in the blood and tissue (for example, brain) concentrations of each parent compound (despite some increase in exhalation) and an increase in the degree and duration of CNS depression. This may occur at “high concentrations,” but at lower concentrations in both man (100 ppm) and rats (25 ppm), no significant interaction occurred (Ikeda 1974; Sato and Nakaji- ma 1979b). Tardif et al. (1992) reported that, compared with single 5-h exposures to toluene or xylene, the simultaneous exposure of adult male Sprague-Dawley rats 

354 Acute Exposure Guideline Levels to toluene (75, 150, and 225 ppm) and xylene (225, 150, and 75 ppm) resulted in interactive effects on metabolism. The interaction was evidenced by higher tolu- ene concentrations in the blood and lower amounts of excreted metabolites over 24 h. There was some dependence of this effect on the ratio of the two chemi- cals. Furthermore, the effect was not observed at 50 ppm of toluene and 40 ppm xylene (Tardif et al. 1991). Studies of toluene exposure (100 ppm) in combination with alcohol inges- tion report delayed metabolism of toluene (Dossing et al. 1984), but there were no additive effects in neurobehavioral tests (4-h exposure at 80 ppm) (Cherry et al. 1983). 5. DATA ANALYSIS FOR AEGL-1 5.1. Human Data Relevant to AEGL-1 Twenty controlled human studies that describe the threshold for irritation and CNS effects from acute exposures to toluene or toluene concentrate have been conducted (Table 6-3). The effects in these studies include slight sensory irritation, accompanied by headache in some cases, subtle impairment on some sensitive cognitive-function tasks, and occasional slight dizziness. The more recent studies involved exposure durations of up to 4 h at 80 ppm, 7.5 h at 100 ppm, and 7 h at 200 ppm. Although slight sensory irritation was reported in sub- jects exposed to toluene at 80-200 ppm in some studies (Andersen et al. 1983; Baelum et al. 1985, 1990; Echeverria et al. 1989, 1991), these concentrations were not irritating in other studies (Stewart et al. 1975; Cherry et al. 1983; Olson et al. 1985; Rahill et al. 1996) and the exposures were generally considered ac- ceptable (no annoyance). True sensory irritation from toluene was reported at concentrations of 20,000 ppm or higher in human studies (Cometto-Muniz and Cain 1995; Abraham et al. 1996), but could not be determined in the mouse (Nielsen and Alarie 1982). No differences in performance on neurobehavioral tests were found in some studies, and others found only subtle differences that indicated slight reductions in alertness (Ogata et al. 1970; Gamberale and Hul- tengren 1972; Stewart et al. 1975; Winneke 1982; Andersen et al. 1983; Cherry et al. 1983; Dick et al. 1984; Olson et al. 1985; Baelum et al. 1985, 1990; Eche- verria et al. 1989, 1991; Rahill et al. 1996). There were no biologically signifi- cant pulmonary or cardiovascular effects (Astrand et al. 1972; Gamberale and Hultengren 1972; Suzuki 1973) and no indications of renal damage (Nielsen et al. 1985). The studies by Rahill et al. (1996), Astrand et al. (1972), and Baelum et al. (1990) found that exercise doubled the uptake of toluene by blood. The study by Stewart et al. (1975) involved repeated exposure to toluene, and found no greater effects over time. Baelum et al. (1990) also tested concentrations that ranged between 50 and 300 ppm (repeated 14 times during a 7-h exposure) with a TWA of 100 ppm; the subjects exercised during the peak exposures at 300 ppm. Gamberale and Hultengren (1972) tested higher concentrations of toluene, but the exposure durations were relatively short and toluene was delivered via 

Toluene 355 breathing tube. Concentrations of toluene above 300 ppm were associated with subtle differences in the speed of reacting to a stimulus or completing a task. For example, during a 20-min exposure at 700 ppm, it took subjects approximately 30 sec longer than controls to match three-digit numbers in 60 columns with numbers appearing at the top of the column (the test was two pages in length). In metabolism studies, subjects were routinely exposed to toluene at 78- 200 ppm for several hours, with and without exercise (Nomiyama and Nomiya- ma 1978; Veulemans and Masschelein 1978; Carlsson 1982; Dossing et al. 1984; Wallen et al. 1985; Hjelm et al. 1988). These studies did not evaluate sub- jective effects, because they were undertaken to study the toxicodynamics and toxicokinetics of toluene in people exposed in the workplace. Occupational-monitoring studies indicate that workers presumed to be healthy have been exposed to toluene at a TWA of 100 ppm or higher without adverse effects. In a comprehensive, well-conducted occupational-monitoring study involving more than 1,000 workers, Neubert et al. (2001a) failed to find alterations in motor performance or increased subjective complaints in workers exposed to toluene at TWA concentrations of 50-100 ppm compared with matched control groups. Workers exposed chronically to toluene at concentra- tions greater than 100 ppm (66-800 ppm) did not have greater attention deficits or sensory symptoms in neurologic tests than those exposed acutely (Foo et al. 1990; Zavalic et al. 1998; Neubert et al. 2001a). Irritation of the conjunctiva and upper respiratory tract was found in only one of 11 workers exposed at 200-800 ppm (Parmeggiani and Sassi 1954). Occupational exposures to toluene at con- centrations up to 300 ppm (Bauchinger et al. 1982), 350 ppm (Ovrum et al. 1978), and 467 ppm (Deschamps et al. 2001) have also been reported. 5.2. Animal Data Relevant to AEGL-1 Few animal studies of end points relevant to AEGL-1 values for toluene were found. Toluene at 1,200 ppm was a NOAEL for overt behavior changes in rodents exposed chronically (NTP 1990). Subtle changes in rodent behavior are not possible to observe, and the effects on rat and mouse behavior described in Tables 6-5 and 6-6, respectively, are sometimes conflicting. Motor activity ei- ther remained unchanged or increased at concentrations below 1,000 ppm; this stimulatory effect was accompanied by either an increase or decrease in positive responses in subtle neurobehavioral tests. 5.3. Derivation of AEGL-1 Values Multiple clinical, occupational-monitoring, and metabolism studies found effects below the definition of the AEGL-1 (notable discomfort). It is not feasi- ble to model notable discomfort, so AEGL-1 values were based on the prepon- derance of data from clinical and occupational studies and from metabolism studies with human subjects that indicated that an 8-h exposure to toluene at 200 

356 Acute Exposure Guideline Levels ppm is a NOAEL for notable discomfort, but elicited subjective, low-severity, nonsensory effects in some studies. More than 300 individuals were involved in the clinical studies summarized in Table 6-3. Several thousand workers were surveyed in the occupational-monitoring studies. Although these populations are presumed to be composed of healthy individuals, they represent a broad spec- trum of uptake rates (sedentary, working, and exercise conditions) and individu- al differences in metabolism rates (Gamberale and Hultengren 1972; Veulemans and Masschelein 1978; Brugnone et al. 1986; Hjelm et al. 1988). Although slight respiratory irritation was reported at 100 and 200 ppm in several studies, toluene is not a respiratory irritant as evidenced by the high RD50 value of 5,300 ppm (Nielsen and Alarie 1982). In addition, the severity of the irritation and the neurobehavioral effects reported in these studies was generally below the defini- tion of AEGL-1. The highest NOAELs for notable discomfort were 200 ppm for 60 min (Astrand et al. 1972), 300 ppm for 15 min (Baelum et al. 1990), and 700 ppm for 20 min (Gamberale and Hultengren 1972). NOAELs for nonsensory (neurobe- havioral) effects were 100 ppm for 4 h (Dick et al. 1984), 6 h (Rahill et al. 1996), and 6.5 h (Baelum et al. 1985, 1990; Nielsen et al. 1985); 150 ppm for 7 h (Echeverria et al. 1989, 1991); and 200 ppm for 3 h (Ogata et al. 1970). Ex- ercise during the studies of Astrand et al. (1972) and Baelum et al. (1990) takes into account the increased uptake that may occur during an emergency situation. Although a steady-state in blood and brain toluene concentrations would be ap- proached but not attained during the 15-and 20-min exposures, the blood con- centration of toluene has been shown to increase approximately two-fold with moderate exercise of 50 W (Astrand et al. 1972). Therefore, the preponderance of data indicates that an 8-h exposure to toluene at 200 ppm is a NOAEL for AEGL-1 effects; however, some subjective neurobehavioral effects have been reported. Although there was no notable discomfort and only mild irritation (ef- fects expected to be concentration dependent and not subject to changes in activ- ity level), headaches (potentially related to CNS effects), reports of dizziness, and nonmeasurable neurologic effects have been reported at 100-200 ppm in controlled human studies (von Oettingen et al. 1942; Gamberale and Hultengren 1972; Andersen et al. 1983). Neurologic effects would be expected to be affect- ed by an increase in activity level, leading to higher concentrations of toluene in the brain. As noted earlier, moderate physical activity may double the blood concentration of toluene. Empirical data as well as pharmacokinetic modeling in humans and ro- dents indicate that venous-blood and brain concentrations of toluene rapidly increase during the first 15-20 min of exposure, followed by relatively modest increases in blood concentrations with continuing exposure (Gamberale and Hultengren 1972; Tardif et al. 1993, 1995). At low concentrations, toluene reaches an asymptote in the blood within 20-30 min (Gamberale and Hultengren 1972; Carlsson 1982). Although storage of toluene would take place in lipid-rich tissues (including those of the brain), elimination is also rapid. On the basis of the range of alveolar concentrations among humans exposed to anesthetic gases 

Toluene 357 (see Section 4.4.2), an uncertainty factor of 3 for intraspecies variability was applied to derive a value of 67 ppm. That concentration was used for all of the AEGL durations, because a steady-state of at 67 ppm is reach fairly rapidly. The AEGL-1 values for toluene are presented in Table 6-9, and the calculations in Appendix A. The relationship between the AEGL-1 values and human expo- sures is shown in Appendix B. 6. DATA ANALYSIS FOR AEGL-2 6.1. Human Data Relevant to AEGL-2 In humans and animals, the primary effect associated with inhalation of “high concentrations” is CNS depression. Few studies of controlled or acci- dental exposures to toluene evaluated CNS depression of a severity that would inhibit the ability to escape. Gamberale and Hultengren (1972) exposed volun- teers to toluene at 700 ppm for 20 min, following successive 20-min exposures at 100, 300, and 500 ppm (total exposure duration 85 min). The work by Benig- nus et al. (2011) shows a useful comparative analysis equating decrements in neurologic functioning associated with blood ethanol levels across exposures to a number of volatile solvents, including toluene. There was one case report of accidental human exposure to toluene, which was appropriate as a basis for deriving AEGL-2 values (Meulenbelt et al. 1990). Two men used toluene to remove excess glue from tiles in the bottom of a swimming pool. The men were barely conscious when they were found; they were confused and unable to walk. They also suffered paresis, ocular irritation, amnesia, and had increased anion gaps, presumably from the onset of distal re- nal tubular acidosis from the metabolites, hippuric and benzoic acid. The toluene concentration measured above the site several hours later was greater than 1,842 ppm, although the victims were likely exposed at higher concentrations at the bottom of the pool. The duration of exposure was approximately 2.5 h. 6.2. Animal Data Relevant to AEGL-2 Except for increased locomotor activity, toluene concentrations below 1,000 ppm have little or no effect on gross manifestations of animal behavior (NRC 1981; WHO 1987). At concentration of 2,000 ppm lower, increased motor activity and an increased rate of responding in neurobehavioral tests were found in rodents (Glowa 1981; De Ceaurriz et al. 1983; Bushnell et al. 1985; Miyaga- wa et al. 1986; Hinman 1987; Kishi et al. 1988; Wada et al. 1989; Wood and TABLE 6-9 AEGL-1 Values for Toluene 10 min 30 min 1h 4h 8h 67 ppm 67 ppm 67 ppm 67 ppm 67 ppm (250 mg/m3) 250 mg/m3) (250 mg/m3) (250 mg/m3) (250 mg/m3) 

358 Acute Exposure Guideline Levels Cox 1995) and monkeys (Taylor and Evans 1985). Higher concentrations of toluene suppress activity. Rats exposed to toluene at 2,400 ppm for 70 min per- formed more poorly on a signal detection task than controls (Oshiro and Bush- nell 2004). The rats were described as sleepy but arousable. Adult female ma- caque monkeys were affected little by a 50-min exposure to toluene at 2,000 ppm or lower; an attention deficit was found during a 50-min exposure at 3,000 ppm, and failure to respond occurred during the second half of a 50-min expo- sure at 4,500 ppm (presumably indicating narcosis) (Taylor and Evans 1985). The NOAEL for clinical signs, survival, and carcinogenicity in mice and rats after chronic inhalation exposure to toluene is 1,200 ppm (NTP 1990). Expo- sures in the NTP study were for 6.5 h/day, 5 days/week for 2 years. 6.3. Derivation of AEGL-2 Values AEGL-2 values for toluene are based on a notable increase in reaction time. The point of departure was a NOAEL of 1,600 ppm based on doubling of reaction time in Long-Evans rats exposed for 34 min (Bushnell et al, 2007a). This point of departure is supported by modeling that shows that a similar mag- nitude of behavioral impairment is caused by oral ingestion of ethanol to the point of legal intoxication (Benignus et al. 2007, 2011). The CNS effects ob- served following toluene exposure are assumed to be directly related to concen- trations of the parent chemical in the brain at the time the effects were measured. Therefore, the BrTC at the 34-min exposure would be expected to provide an internal dose correlating with the NOAEL. The PBPK model of Kenyon et al. (2008) was used to calculate the inter- nal dose (BrTC) in the rat. An interspecies uncertainty factor of 1 was applied because pharmacokinetic modeling eliminated the toxicokinetic component of the uncertainty factor, and the pharmacodynamic component was assigned a value of 1 because similar effects (CNS depression) were observed in humans and animals. An intraspecies uncertainty factor of 3 was applied because the minimum alveolar concentration (lowest concentration causing an anesthetic effect) for volatile anesthetics should not vary by more than 2- to 3-fold among humans (see Section 4.4.2). The GPAT model developed by Benignus et al. (2006) is a human whole- body differential equation-based model for simulating the function of the vari- ous organs in response to realistic situations, such as exercise, environment, and diet. The environmental aspects include temperature, humidity, and, in this ap- plication, toluene uptake and elimination for each organ. Thus, toluene uptake and elimination under various exposures was estimated for each organ, most notably the brain, and for various exercise levels. In addition, estimates were made of the exposure concentrations that would lead to specific organ concen- trations (BrTC) in humans based on output from the Kenyon et al. (2008) rat PBPK model. The target internal dose metric in humans (human BrTC) was calculated by dividing the rat BrTC by the total uncertainty factor of 3. The hu- 

Toluene 359 man GPAT model (Benignus et al. 2006) was then used to determine the equiva- lent exposure concentration that yields the human BrTC at each of the AEGL exposure durations. Similar types of neurobehavioral effects are caused by ethanol intoxication and have been well documented. Comparisons of the effects caused by the mod- eled levels of toluene and similar effects caused by ethanol intoxication (using blood ethanol concentration as the indicator) have been included in Appendix C (Table C-4), Appendix D, and in the paper by Benignus et al. (2011) to further support the approach taken in deriving the AEGL-2 values. Time-scaling calculations are presented in Appendix A and the PBPK modeling methods are explained in Appendix C. AEGL-2 values for toluene are presented in Table 6-10, and are shown graphically in Appendix B. 7. DATA ANALYSIS FOR AEGL-3 7.1. Human Data Relevant to AEGL-3 Human data on toluene concentrations that are the threshold for lethality are sparse. In a shipboard accident, workers lost consciousness after being ex- posed to estimated concentrations of toluene that ranged from 5,000 to 30,000 ppm (Longley et al. 1967). The exposure was at least 75 min in duration. Be- cause toluene is heavier than air, the higher concentration was most likely near the floor where a kneeling worker rapidly lost consciousness. All workers re- covered. Press and Done (1967) estimate the concentration of toluene achieved when inhaling directly from a paper bag containing gauze soaked with toluene (abuse situation) at 10,000 ppm. One of the authors inhaled from the bag for 5 min, and reported dizziness, blurred vision, roaring and buzzing in the ears, and slurred speech. 7.2. Animal Data Relevant to AEGL-3 On the basis of LC50 values, the mouse is slightly more sensitive to the ef- fects of toluene than the rat. LC50 values for the mouse for 10, 30, and 60 min are 38,465, 21,872, and 19,018 ppm, respectively (Moser and Balster 1985). The 3-h LC50 was 8,600 ppm (Bruckner and Peterson 1981a). A 6-h LC50 of 6,940 ppm was calculated by Bonnet et al. (1979) and a 7-h LC50 of 5,320 was calcu- lated by Svirbely et al. (1943). For the rat, LC50 values ranged from 26,700 ppm for 1 h to 12,500 ppm for 2-2.5 h (Pryor et al. 1978; Kojima and Kobayashi 1973). Death was caused by severe CNS depression and respiratory failure. Data were also available on the highest concentrations of toluene that did not result in lethality. No deaths occurred in mice exposed to toluene for 20 min at 12,000 ppm (Bruckner and Peterson 1981a), in rats exposed for 2 h at 5,000 or 6,250 ppm (Kojima and Kobayashi 1973; Mullin and Krivanek 1982), or in rats exposed for 4 h at 6,000 ppm (Wada et al. 1989). Mice exposed at 6,000 

360 Acute Exposure Guideline Levels ppm for 30 min/day, 5 days/week for 7 weeks and that were deprived of food survived the exposures in apparent good health (Moser and Balster 1981). Addi- tionally, mice and rats tolerated toluene at 1,200 ppm for 2 years without an effect on mortality (NTP 1990). 7.3. Derivation of AEGL-3 Values The AEGL-3 values are based on a NOAEL for lethality in the rat. A 2-h exposure at 6,250 ppm was not lethal but produced prostration in rats (Mullin and Krivanek 1982). CNS effects observed following toluene exposure were assumed to be directly related to concentration of parent chemical in the brain at the time effects were observed. Therefore, the PBPK model of Kenyon et al. (2008) was used to calculate the BrTC in the rat. An interspecies uncertainty factor of 1 was applied because pharmacokinetic modeling eliminated the toxi- cokinetic component of the uncertainty factor, and the pharmacodynamic com- ponent was assigned a factor of 1 because similar effects (CNS depression) were observed in humans and animals. An intraspecies uncertainty factor of 3 was applied to account for intraindividual variability, as was done for the AEGL-2. A human GPAT model (Benignus et al. 2006) was used to determine the equiva- lent exposure concentration in humans for each of the AEGL exposure dura- tions. Time-scaling calculations are presented in Appendix A, and the PBPK modeling methods are explained in Appendix C. AEGL-3 values are presented in Table 6-11, and shown graphically in Appendix B. TABLE 6-10 AEGL-2 Values for Toluene 10 min 30 min 1h 4h 8h 1,400 ppma 760 ppm 560 ppm 310 ppm 250 ppm (5,300 mg/m3) (2,900 mg/m3) (2,100 mg/m3) (1,200 mg/m3) (940 mg/m3) a Concentration is one-tenth of the lower explosive limit of 14,000 ppm for toluene in air. Therefore, safety considerations against the hazard of explosion must be taken into ac- count. TABLE 6-11 AELG-3 Values for Toluene 10 min 30 min 1h 4h 8h –a 5,200 ppmb 3,700 ppmb 1,800 ppmb 1,400 ppmb (20,000 mg/m3) (14,000 mg/m3) (6800 mg/m3) (5300 mg/m3) a The 10-min AEGL-3 value of 10,000 ppm (38,000 mg/m3) is higher than 50% of the lower explosive limit of 14,000 ppm for toluene in air. Therefore, extreme safety consid- erations against the hazard of explosion must be taken into account. b Concentration is one-tenth or more of the lower explosive limit of 14,000 ppm for tolu- ene in air. Therefore, safety considerations against the hazard of explosion must be taken into account. 

Toluene 361 Workers lost consciousness after being accidentally exposed to toluene at an estimated concentration of 5,000 ppm or greater for an undefined period of time (Longley et al. 1967). In rodent studies, concentrations of 5,000 ppm for 2 h (Kojima and Kobayashi 1973), 12,000 ppm for 20 min (Bruckner and Peterson 1981a), and 6,000 ppm for 30 min/day, 5 day/week for 7 weeks (Moser and Bal- ster 1981) were all nonlethal. 8. SUMMARY OF AEGLs 8.1. AEGL Values and Toxicity End Points The AEGL values for toluene are presented in Table 6-12. 8.2. Comparison with Other Standards and Criteria Standards and guidance levels for workplace and community exposures to toluene are presented in Table 6-13. The Occupational Health and Safety Ad- ministration recommends a 10-min maximum exposure of 500 ppm. The Na- tional Institute for Occupational Safety and Health’s immediately dangerous to life or health (IDLH) value is also 500 ppm. The IDLH is based on the human data of Gamberale and Hultengren (1972), von Oettingen et al. (1942), and Wil- son (1943), and was established at a concentration lower than both the 30-min AEGL-2 and AEGL-3 values. The IDLH is based on a weight-of-evidence ap- proach from human data only, with the final value based on a consensus from a panel of experts. No quantitative analysis or application of PBPK models to ap- proximate the human exposure which would lead to a NOAEL in the rat was used to determine the IDLH concentration whereas a quantitative approach was used in the development of the AEGL-2 and AEGL-3 values. The emergency response planning guideline (ERPG) values of the American Industrial Hygiene Association (AIHA 2013) are lower than the AEGL values. The ERPG-1 is based on controlled human studies (von Oettingen et al. 1942; Gamberale and Hultengren 1972; Andersen et al. 1983) in which exposure at 100 ppm produced mild symptoms, such as fatigue, drowsiness, headache, dizziness, and feeling of intoxication without measurable neurotoxic effects; these studies were consid- ered in the derivation of the AEGL-1 values in the context of 20 relevant human studies. The final resulting AEGL-1 value of 67 ppm was deemed protective for these low-level, subjective effects. The ERPG-2 was based on controlled human studies in which exposure at 300 ppm for 8 h did not result in muscular weak- ness or incoordination (von Oettingen et al. 1942). This is an old study and was not considered for AEGL development. The ERPG-3 was based on the 1-h LC50 of 26,700 ppm in rats (Pryor et al. 1978), divided by approximately 20 and the reported loss of consciousness in humans exposed at 5,000 ppm for a few minutes (Longley et al. 1967). The National Research Council (NRC) 1-h emer- gency exposure guidance level for toluene is 200 ppm for workplace conditions (NRC 2008). 

362 Acute Exposure Guideline Levels TABLE 6-12 AEGL Values for Toluene Classification 10 min 30 min 1h 4h 8h AEGL-1 67 ppm 67 ppm 67 ppm 67 ppm 67 ppm (nondisabling) (250 mg/m3) (250 mg/m3) (250 mg/m3) (250 mg/m3) (250 mg/m3) AEGL-2 1,400 ppm a 760 ppm 560 ppm 310 ppm 250 ppm (disabling) (5,300 mg/m3) (2,900 mg/m3) (2,100 mg/m3) (1,200 mg/m3) (940 mg/m3) AEGL-3 –b 5,200 ppma 3,700 ppma 1,800 ppma 1,400 ppma (lethal) 20,000 mg/m3) (14,000 mg/m3) (6,800 mg/m3) (5300 mg/m3) a Concentration is one-tenth or more of the lower explosive limit of 14,000 ppm for tolu- ene in air. Therefore, safety considerations against the hazard of explosion must be taken into account. b The 10-min AEGL-3 value of 10,000 ppm (38,000 mg/m3) is higher than 50% of the lower explosive limit of 14,000 ppm for toluene in air. Therefore, extreme safety consid- erations against the hazard of explosion must be taken into account. TABLE 6-13 Standards and Guidelines for Toluene Exposure Duration Guideline 10 min 15 min 30 min 1h 4h 8h AEGL-1 67 ppm – 67 ppm 67 ppm 67 ppm 67 ppm AEGL-2 1,400 ppma – 760 ppm 560 ppm 310 ppm 250 ppm AEGL-3 –b – 5,200 ppma 3,700 ppma 1,800 ppma 1,400 ppma ERPG-1 (AIHA)c – – – 50 ppm – – ERPG-2 (AIHA) – – – 300 ppm – – ERPG-3 (AIHA) – – – 1,000 ppm – – EEGL (NRC)d – – – 200 ppm – – SMAC (NRC)e – – – 16 ppm – – IDLH (NIOSH)f – – 500 ppm – – – TLV-TWA (ACGIH)g – – – – – 50 ppm h PEL-TWA (OSHA) – – – – – 200 ppm REL-TWA (NIOSH)i – – – – – 100 ppm REL-STEL (NIOSH)j – 150 ppm – – – – PEL-C (OSHA)k 300 ppm 300 ppm 300 ppm 300 ppm 300 ppm 300 ppm PEL-peak (OSHA)l 500 ppm – – – – – MAK (Germany)m – – – – – 50 ppm MAK peak exposure – – 250 ppm – – – (Germany)n MAC – – – – – 40 ppm (The Netherlands)o a Concentration is one-tenth or more of the lower explosive limit of 14,000 ppm for tolu- ene in air. Therefore, safety considerations against the hazard of explosion must be taken into account. b The 10-min AEGL-3 value of 10,000 ppm (38,000 mg/m3) is higher than 50% of the lower explosive limit of 14,000 ppm for toluene in air. Therefore, extreme safety consid- erations against the hazard of explosion must be taken into account. 

Toluene 363 c ERPG (emergency response planning guidelines, American Industrial Hygiene Associa- tion) (AIHA 2013). ERPG-1 is the maximum airborne concentration below which it is believed nearly all individuals could be exposed for up to 1 h without experiencing other than mild, transient adverse health effects or without perceiving a clearly defined objectionable odor. The ERPG-1 for toluene is based on controlled human studies (von Oettingen et al. 1942; Gamberale and Hultengren 1972; Andersen et al. 1983) in which exposure at 100 ppm produced mild symptoms, such as fatigue, drowsiness, headache, dizziness, and feeling of intoxication without neurotoxic effects. ERPG-2 is the maximum airborne concentration below which it is believed nearly all individuals could be exposed for up to 1 h without experiencing or developing irreversi- ble or other serious health effects or symptoms that could impair an individual’s ability to take protective action. The ERPG-2 for toluene was based on controlled human studies in which exposure at 300 ppm for 8 h did not result in muscular weakness or incoordination (von Oettingen et al. 1942). ERPG-3 is the maximum airborne concentration below which it is believed nearly all individuals could be exposed for up to 1 h without experiencing or developing life- threatening health effects. The ERPG-3 for toluene was based on the LC50 in rats (Pryor et al. 1978), divided by approximately 20, and the reported loss of consciousness in hu- mans exposed at 5,000 ppm for a few minutes (Longley et al. 1967). d EEGL (emergency exposure guidance levels, National Research Council) (NRC 2008). The EEGL is the concentration of a contaminant that can cause discomfort or other evi- dence of irritation or intoxication in or around the workplace, but avoids death, other severe acute effects, and long-term or chronic injury. e SMAC (spacecraft maximum allowable concentration, National Research Council) (NRC 2008). SMACs are intended to provide guidance on chemical exposures during normal operations of spacecraft, as well as emergency situations. The 1-h SMAC is a concentration of an airborne substance that will not compromise the performance of spe- cific tasks by astronauts during emergency conditions or cause serious or permanent toxic effects. Such exposures may cause reversible effects, such as dermal or ocular irritation, but they are not expected to impair judgment or interfere with proper responses to emer- gencies. f IDLH (immediately dangerous to life or health, National Institute for Occupational Safe- ty and Health) (NIOSH 1994) represents the maximum concentration from which one could escape within 30 min without any escape-impairing symptoms, or any irreversible health effects. The IDLH for toluene is based on the human studies of Gamberale and Hultengren (1972), von Oettingen et al. (1942) and Wilson (1943). g TLV-TWA (threshold limit value – time-weighted average, American Conference of Governmental Industrial Hygienists) (ACGIH 2005) is the time-weighted average con- centration for a normal 8-h workday and a 40-h workweek, to which nearly all workers may be repeatedly exposed, day after day, without adverse effect. There is a skin notation for the TLV for toluene. h PEL-TWA (permissible exposure limit – time-weighted average, Occupational Health and Safety Administration) (29 CFR 1910.1000 [2006]) is a time-weighted average con- centration that must not be exceeded during any 8-h workshift of a 40-h workweek. i REL-TWA (recommended exposure limit – time-weighted average, National Institute for Occupational Safety and Health) (NIOSH 2011) is a time-weighted average concentration for up to a 10-h workday during a 40-h workweek. 

364 Acute Exposure Guideline Levels h REL-STEL (recommended exposure limit – short-term exposure limit, National Institute for Occupational Safety and Health) (NIOSH 2011) is a 15-min time-weighted average exposure that should not be exceeded at any time during a workday. j PEL-C (permissible exposure limit – ceiling, Occupational Health and Safety Admin- istration) (29 CFR 1910.1000 [2006]) is a ceiling value that should not be exceeded dur- ing any part of the workday. If instantaneous monitoring is not feasible, the ceiling must be assessed as a 15-min time-weighted average exposure. l PEL-peak (permissible exposure limit – peak, Occupational Health and Safety Admin- istration) (29 CFR 1910.1000 [2006]) is the acceptable 10-min maximum peak above the acceptable ceiling value. m MAK (maximale arbeitsplatzkonzentration [maximum workplace concentration]) (Deutsche Forschungsgemeinschaft [German Research Association] (DFG 1999 ) is de- fined analogous to the ACGIH TLV-TWA. n MAK spitzenbegrenzung (peak limit [Category II, 2], Deutsche Forschungsgemeinschaft [German Research Association]) (DFG 1999) is the maximum average concentration to which workers can be exposed for a period up to 30 min with no more than two excur- sions per work shift; total exposure may not exceed 8-h MAK. o MAC (maximaal aanvaarde concentratie [maximal accepted concentration], Dutch Ex- pert Committee for Occupational Standards, The Netherlands (MSZW 2004) is defined analogous to the ACGIH TLV-TWA. Exposure limits for chronic exposures are lower than for emergency guidelines. The American Conference of Governmental Industrial Hygienists (ACGIH 2005) occupational exposure limit for toluene is 50 ppm, as is the German MAK (German Research Association (DFG 1999). The Dutch MAC is 40 ppm (MSZW 2004). The 8-h AEGL-1 for toluene is 200 ppm. The previous ACGIH TWA value of 200 ppm was lowered in 1991 on the basis of slight irri- tation at 100 ppm, described in the studies of Andersen et al. (1983), Baelum et al (1985), Wilson (1943), and Echeverria et al. (1989). The AEGL-1 value is higher than the TLV because slight irritation is acceptable under acute (once-in- a-lifetime) conditions. Furthermore, it is unlikely that an intolerable level of annoyance of sensory irritation was reached in the Andersen et al. (1983) study, given the inability to attain an RD50 (Nielsen and Alarie 1982). The NRC (2008) spacecraft maximum allowable concentration for toluene is 16 ppm for 1 and 24 h. The SMAC values were based on a single study (An- dersen et al. 1983), rather than the preponderance of the evidence from 20 pub- lished studies. 8.3. Data Adequacy and Research Needs Because toluene is a commonly used solvent, its effects on humans have been extensively studied. Numerous controlled studies with human subjects that addressed effects meeting the definition of the AEGL-1 were available. The data base of neurotoxicity studies with animals is extensive, and the supporting ani- mal data were in good agreement with the AEGL values based on the human studies. Toluene is fatal to humans only after exposure at extremely high con- 

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382 Acute Exposure Guideline Levels APPENDIX A DERIVATION OF AEGL VALUES FOR TOLUENE Derivation of AEGL-1 Values Key studies: Astrand, I., H. Ehrner-Samuel, A. Kilbom, and P. Ovrum. 1972. Toluene exposure. I. Concentration in alveolar air and blood at rest and during exercise. Scand. Work Environ. Health 9:119-130. Baelum J., I. Andersen, G.R. Lundqvist, L. Mohave, O. F. Pedersen, M. Vaeth, and D.P. Wyon. 1985. Response of solvent-exposed printers and unexposed controls to six-hour toluene exposure. Scand. J. Work Environ. Health 11(4):271-280. Baelum, J., G.R. Lundqvist, L. Molhave, and N.T. Andersen. 1990. Human response to varying concentrations of toluene. Int. Arch. Occup. Environ. Health 62(1):65-72. Dick, R.B., J.V. Setzer, R. Wait, R., M.B. Hayden, B.J. Taylor, B. Tolos, and V. Putz-Anderson. 1984. Effects of acute exposure of toluene and methyl ethyl ketone on psychomotor performance. Int. Arch. Occup. Environ. Health 54(2):91-109. Echeverria, D., L. Fine, G. Langolf, A. Schork, and C. Sampaio. 1989. Acute neurobehavioral effects of toluene. Br.. J Ind. Med. 46(7):483-495. Echeverria, D., L. Fine, G. Langolf, T. Schork, and C. Sampaio. 1991. Acute behavioral comparisons of toluene and ethanol in human subjects. Br.. J. Ind. Med. 48(11):750-761. Gamberale, F., and M. Hultengren. 1972. Toluene exposure. II. Psychophysiological functions. Work Environ. Health 9(3):131-139. Nielsen, H.K., L. Krusell, J. Baelum, G. Lundqvist, G. O. Omland, M. Vaeth, M., S.E. Husted, C.E. Mogensen, and E. Geday. 1985. Renal effects of acute exposure to toluene. Acta Med. Scand. 218(3):317-321. 

Toluene 383 Ogata, M., K. Tomokuni, and Y. Takatsuka. 1970. Urinary excretion of hippuric acid and m- and p- methylhippuric acid in the urine of persons exposed to vapours of toluene and m- and p-xylene as a test of exposure. Br. J. Ind. Med. 27(1):43-50. Stewart, R.D., C.L. Hake, H.V. Forster, and A.J. Lebrun. 1975. Toluene: development of a biologic standard for the industrial worker by breath analysis. NIOSH-00080663; National Institute for OccupationalSafety and Health, Cincinnati, OH. Toxicity end point: 200 ppm for short- and long-exposure durations; effects would not exceed the definition of an AEGL-1 Time scaling: None applied; toluene rapidly approaches steady-state in the blood at low concentrations. Uncertainty factors: 3 for intraspecies variability; the minimum alveolar concentration for volatile anesthetics differs by no more than 2- to 3-fold in the human population. 10-min AEGL-1: 200 ppm ÷ 3 = 67 ppm 30 min AEGL-1: 200 ppm ÷ 3 = 67 ppm 1-h AEGL-1: 200 ppm ÷ 3 = 67 ppm 4-h AEGL-1: 200 ppm ÷ 3 = 67 ppm 8-h AEGL-1: 200 ppm ÷ 3 = 67 ppm Derivation of AEGL-2 Values Key studies: Bushnell, P.J., W.M. Oshiro, T.E. Samsam, V.A. Benignus, Q.T. Krantz, and E.M. Kenyon. 2007a. A dosimetric analysis of the acute behavioral effects of inhaled toluene in rats. Toxicol. Sci. 99(1):181-189. Toxicity end point: No-observed-adverse-effect level of 1,600 ppm for 34 min based on doubling of reaction time in rats; concentration-related decrease in accuracy and increase in response time in a signal detection task with food reward. 

384 Acute Exposure Guideline Levels Time scaling: CNS effects observed after toluene exposure were assumed to be directly related to parent material reaching the brain. Therefore, a physiologically-based pharmacokinetic (PBPK) model for the rat (see Appendix C) was used to calculate the internal dose (BrTC) correlating with the exposure. A human PBPK model was then used determine the equivalent exposure concentration that would produce the target BrTC in humans at each AEGL duration. Uncertainty factors: 1 for interspecies differences; PBPK modeling allows a comparison of the internal dose in both rats and humans from identical external exposures, and similar CNS effects were observed in rodents and humans. 3 for intraspecies variability; the minimum alveolar concentration for volatile anesthetics differs by no more than 2- to 3-fold in the human population. 10-min AEGL-2: 1,400 ppm (based on results of PBPK model) 30-min AEGL-2: 760 ppm (based on results of PBPK model) 1-h AEGL-2: 560 ppm (based on results of PBPK model) 4-h AEGL-2: 310 ppm (based on results of PBPK model) 8-h AEGL-2: 250 ppm (based on results of PBPK model) Derivation of AEGL-3 Values Key study: Mullin, L.S. and N.D. Krivanek. 1982. Comparison of unconditioned reflex and conditioned avoidance tests in rats exposure by inhalation to carbon monoxide, 1,1,1- trichloroethane, toluene or ethanol. Neurotoxicity 3(1):126-137. Toxicity end point: Threshold for lethality in rats of 6,250 ppm for 2 h Time scaling: CNS effects observed after toluene exposure were assumed to be directly related to parent material reaching the brain. Therefore, a PBPK model for the rat (see Appendix C) was used to calculate the internal dose (BrTC) correlating with the exposure. A human PBPK model was then used determine the equivalent exposure concentration that would produce the target BrTC in humans at each AEGL duration. 

Toluene 385 Uncertainty factors: 1 for interspecies differences; PBPK modeling allows a comparison of the internal dose in both rats and humans from identical external exposures, and similar CNS effects were observed in rodents and humans. 3 for intraspecies variability; the minimum alveolar concentration for volatile anesthetics differs by no more than 2- to 3-fold in the human population. 10-min AEGL-3: 10,000 ppm (based on results of PBPK model) 30-min AEGL-3: 5,200 ppm (based on results of PBPK model) 1-h AEGL-3: 3,700 ppm (based on results of PBPK model) 4-h AEGL-3: 1,800 ppm (based on results of PBPK model) 8-h AEGL-3: 1,400 ppm (based on results of PBPK model) 

386 APPENDIX B CATEGORY PLOT FOR TOLUENE 100000 Human ‐ No  Effect Human ‐ Discomfort/  Mild Human ‐ Disabling 10000 Animal ‐ No  Effect Animal ‐ AEGL‐3 Discomfort ppm 1000 Animal ‐ Disabling Animal ‐ Some  AEGL‐2 Lethality Animal ‐ Lethal 100 AEGL‐1 AEGL‐3 AEGL‐2 AEGL‐1 10 0 60 120 180 240 300 360 420 480 Minutes FIGURE B-1 Category plot of animal toxicity data and AEGL values for toluene. 

TABLE B-1 Data Used in Category Plot for Toluene Source Species ppm Minutes Category AEGL-1 67 10 AEGL AEGL-1 67 30 AEGL AEGL-1 67 60 AEGL AEGL-1 67 240 AEGL AEGL-1 67 480 AEGL AEGL-2 1,400 10 AEGL AEGL-2 760 30 AEGL AEGL-2 560 60 AEGL AEGL-2 310 240 AEGL AEGL-2 250 480 AEGL AEGL-3 10,000 10 AEGL AEGL-3 5,200 30 AEGL AEGL-3 3,700 60 AEGL AEGL-3 1,800 240 AEGL AEGL-3 1,400 480 AEGL Andersen et al. 1983 Human 40 360 0, no neurobehavioral effects Andersen et al. 1983 Human 100 360 1, slight ocular and nasal irritation; no neurobehavioral effects Cherry et al. 1983; Olson et al. 1985 Human 80 240 0, no neurobehavioral effects Iregren et al. 1986 Human 80 270 1, subjective symptoms (Continued) 387 

388 TABLE B-1 Continued Source Species ppm Minutes Category Winneke 1982 Human 100 210 0, no behavioral deficits in psychomotor tests Dick et al. 1984 Human 100 240 0, no neurobehavioral deficits Rahill et al. 1996 Human 100 360 0, slight latency, neurobehavioral tests Baelum et al. 1985 Human 100 390 1, sensory irritation, sleepiness, behavioral deficits Stewart et al. 1975 Human 100 450 0, no neurobehavioral deficits Baelum et al. 1990 Human 100 420 1, sensory irritation Echeverria et al. 1989 Human 75 420 0, no neurobehavioral effects Echeverria et al. 1989 Human 150 420 1, ocular irritation, headache Astrand et al. 1972 Human 100 30 0, no neurobehavioral effects Astrand et al. 1972 Human 200 30 0, no neurobehavioral effects with exercise Ogata et al. 1970 Human 100 180 0, no effects Ogata et al. 1970 Human 200 180 0, no effects von Oettingen et al. 1942 Human 100 480 1, moderate fatigue von Oettingen et al. 1942 Human 300 480 2, increasing fatigue symptoms von Oettingen et al. 1942 Human 400 480 2, increasing fatigue symptoms von Oettingen et al. 1942 Human 600 480 2, increasing fatigue symptoms von Oettingen et al. 1942 Human 800 180 2, incoordination Gamberale and Hultengren 1972 Human 100 20 0, no effect on reaction time 

Gamberale and Hultengren 1972 Human 300 20 0, increase in reaction time Gamberale and Hultengren 1972 Human 500 20 0, increase in complex reaction time Gamberale and Hultengren 1972 Human 700 20 0, decrease in perceptual speed at end of exposure Carpenter et al. 1944 Human 200 420 1, mild irritation Carpenter et al. 1944 Human 400 420 1, mild ocular irritation Carpenter et al. 1944 Human 600 480 1, lassitude Carpenter et al. 1944 Human 800 480 2, inebriation Suzuki 1973 Human 200 360 0, no effect Horvath et al. 1981 Human 240 30 0, no effect von Oettingen et al. Human 200 480 1, mild discomfort Luderer et al 1999 Human 50 180 0, no symptoms Pryor et al. 1978 Rat 26,700 60 SL, LC50 Cameron et al. 1938 Rat 24,400 90 SL, 60% mortality Cameron et al. 1938 Rat 12,200 390 SL, LC50 Kojima and Kobayashi 1973 Rat 15,000 150 SL, 80% mortality Kojima and Kobayashi 1973 Rat 12,200 120 SL, LC50 Kojima and Kobayashi 1973 Rat 5,000 120 2, no mortality Mullin and Krivanek 1982 Rat 6,250 120 2, no mortality Moser and Balster 1985 Mouse 38,465 10 SL, LC50 Moser and Balster 1985 Mouse 21,872 30 SL, LC50 389 

390 TABLE B-1 Continued Source Species ppm Minutes Category Moser and Balster 1985 Mouse 19,018 60 SL, LC50 Bruckner and Peterson 1981a Mouse 12,000 20 2, no mortality Bruckner and Peterson 1981a Mouse 8,600 180 SL, LC50 Bonnet et al. 1979 Mouse 6,940 360 SL, LC50 Svirbely et al. 1943 Mouse 5,320 420 SL, LC50 Bruckner and Peterson 1981a Mouse 2,600 180 2, immobility in absence of stimulus NTP 1990 Rat, mouse 1,200 390 0, no clinical signs Moser and Balster 1985 Mouse 2,959 10 2, EC50, inverted screen test Moser and Balster 1985 Mouse 2,012 30 2, EC50, inverted screen test Moser and Balster 1985 Mouse 1,445 60 2, EC50, inverted screen test Moser and Balster 1981 Mouse 3,200 30 2, ataxia Bruckner and Peterson 1981a Mouse 2,600 60 2, ataxia Glowa 1981 Mouse 500 240 0, no effects, scheduled controlled behavior Glowa 1981; Takeuchi and Hisanga 1977 Mouse 2,000 240 2, changes in behavior Shigeta et al. 1978; Takeuchi and Hisanaga 1977 Rat 1,000 240 1, no changes in behavior Shigata et al. 1978 Rat 3,000 240 2, changes in avoidance response, ataxia Hinman 1987 Mouse 2,500 60 1, no effect on motor activity Mullin and Krivanek 1982 Mouse 800 240 0, threshold, unconditioned reflex change Miyagawa et al. 1986 Rat 3,400 240 2, 31% activity decrease Geller et al. 1979 Rat 150 120 1, reduced neurobehavioral performance 

Hinman 1987 Rat 15,000 60 2, no mortality Nielsen and Alarie 1982 Mouse 7,800 30 2, no mortality Nielsen and Alarie 1982 Mouse 5,300 30 2, RD50 Kojima and Kobayashi 1973 Rat 10,000 120 SL, 20% mortality Pryor et al. 1978 Rat 40,000 60 3, lethal Ikeda et al 1990 Dog 30,000 10 3, lethal Kishi et al. 1988 Rat 4,000 240 2, ataxia Taylor and Evans 1985 Monkey 1,000 50 0, no effect Taylor and Evans 1985 Monkey 2,000 50 1, impairment, cognitive function Taylor and Evans 1985 Monkey 4 500 50 2, failure to respond in cognitive function test Wood and Cox 1995 Rat 3,000 120 1, increased activity Wood et al. 1984 Rat 3,000 240 2, ataxia Bushnell et al. 1985 Mouse 10,000 72 2, decrease in activity Moser and Balster 1981 Mouse 800 30 0, no change in operant behavior Wada et a. 1989 Rat 6,030 240 2, no mortality For category: 0 = no effect, 1 = discomfort, 2 = disabling, SL = some lethality, 3 = lethal 391 

392 Acute Exposure Guideline Levels APPENDIX C PHYSIOLOGICALLY-BASED PHARMACOKINETIC MODELING OF TOLUENE Summary The method used in this appendix to determine human equivalent AEGL values is similar to that previously reported (Bruckner et al. 2004; Krewski et al. 2004) and follows the methodology described in the PBPK Modeling White Paper, Addressing the Use of PBPK Models to Support Derivation of Acute Exposure Guideline Levels (AEGLs), (Dennison et al. 2010). The method reduces the uncertainty inherent in ex- trapolating rat toxicity data to humans and extrapolating toxicity data across time scales, by using validated PBPK models to perform the extrapolation based on an in- ternal measure of dose. This reduces the uncertainty in the pharmacokinetic component of the extrapolation. Uncertainty in the pharmacodynamic component of the rat-to- human extrapolation is handled with standard uncertainty factors. The end points found in the critical studies for AEGL 2 and AEGL 3 values can be reasonably associated with the blood concentration of toluene. The blood concentration is a superior measure of dose than the applied concentration (exposure concentration) because, as an internal measure of dose, pharmacokinetic alterations in tissue dosimetry are addressed in extrapolations by explicit quantification. In an extrapolation, for example, of a 1-h AEGL to an 8-h AEGL, the increase in blood concentration over time is explicitly compensated for by reducing the 8-h AEGL to the point where blood concentrations are equivalent. This obviates the need for the use of algorithms such as the ten Berge et al. (1986) equation, which can result in corresponding errors when the empirical parameters are unknown. Fundamentally, the PBPK-based AEGL values are based on the same critical studies as the AEGL values established in the technical support document; only the method of extrapolating from rat to human (dosimetry replaces pharmacokinetic uncer- tainty factors) and over time (dosimetry replaces empirical formulas) differs. When the PBPK-based approach replaces pharmacokinetic uncertainty factors, the resulting AEGL value may be higher due to the reduction in the total uncertainty factor. The AEGL 1 value for toluene was based on effects that could not feasibly be modeled, so modeling was not applied. Modeling was performed in deriving the AEGL-2 and AEGL-3 values. The PBPK assessment method involves the following specific steps: Step 1) Determine an appropriate dose metric, the pharmacokinetic measure of internal dose that correlates with the critical effect. For toluene, the critical effect is CNS depression, and a dose metric of peak concentration of tolu- ene in brain (BrTC) was used. Step 2) Develop a PBPK model for that chemical that adequately describes the pharmacokinetics of toluene in rats and humans with respect to CV. De- scribe model equations, parameter value development, and evaluation us- ing experimental data from the literature. 

Toluene 393 Step 3) Calculate the dose metric amount under conditions that correspond to the critical study. For example, for toluene’s AEGL 3 values, the critical study point of departure was 6,250 ppm for 2 h in rats. Based on the PBPK mod- el, the CV in rats is 165 mg/L (at 2 h and 6,250 ppm). Step 4) Apply the uncertainty factors determined in the technical support document to the dose metric. For AEGL-3 values, the total uncertainty factor was 3, for intraspecies variability. As described in the technical support document, the interspecies factor was set at 1, for a total uncertainty factor of 3. This total uncertainty factor may have been higher if PBPK modeling was not used. After application of the uncertainty factor of 3, the target internal dose of CV becomes 55 mg/L for AEGL 3. The uncertainty factor can also be applied after derivation of the human equivalent concentration (HEC) as an alternative, and in the derivation section of this appendix, both ap- proaches are provided. Step 5) Scale the model to humans by changing body weight and other parameter values to human values. Step 6) Run the PBPK model to determine the HEC that corresponds to the target internal dose of 55 mg/L for each AEGL. This appendix has three parts. First, the structure and parameterization of the toluene PBPK model is described. Second, validation of the model is provided by showing model performance against rat and human data sets obtained from the lit- erature. Third, derivation of recommended AEGL values is performed. Introduction The critical studies that provide the NOAEL used in this analysis (Table C-1) are the same as those used in the technical support document to calculate AEGL values. For the AEGL-2 values, both a clinical study and a study with rats were con- sidered. The study with rats was chosen as the basis for the AEGL-2 values, because the effect in this study, threshold for narcosis, more closely meets the definition of the AEGL-2. Supporting studies were not used in any of the AEGL calculations. TABLE C-1 Critical Studies for Deriving AEGL Values for Toluene Level Study Species NOAEL Duration AEGL-2 Gamberale et al. 1972 Human 700 ppma 20 min AEGL-2 Bushnell et al. 2007a Rat 1,600 ppm 34 min AEGL-3 Mullin and Krivanek 1982 Rat 6,250 2h a After initial exposures at 100-500 ppm. 

394 Acute Exposure Guideline Levels Additional information and justification of these choices of critical studies is available in the technical support document. The target tissue dose (venous blood concentration) was determined from these studies. Selection of the Dose Metric The dose metric used for the PBPK-based assessment is the concentration of toluene in the brain (BrTC). The critical effect of toluene for the setting of AEGLs has been determined to be CNS depression, on the basis of toxicity studies presented in the technical support document. It has been generally suggested that CNS depres- sion caused by organic solvents, such as toluene, is mediated by the action of the parent chemical and not metabolites (Bruckner and Warren 2001). The concentration of toluene in that target brain tissue is proportional to the concentration in venous blood (van Asperen et al. 2003) once steady state has been achieved. The PBPK models have been parameterized to provide CV as model output under the exposure conditions indicated for this assessment. Model Selection The present approach requires a validated PBPK model for rats and humans. Three options exist for developing or selecting a model to use: 1) develop a wholly new model, 2) modify an existing model, or 3) select an existing model and use in its present form. If an existing model would serve the needs of this assessment, option #3 is the preferred choice and was the first approach to be used. Ultimately, an exist- ing model was used with minor modifications. An evaluation of all existing models can in principle be performed to deter- mine the best available model. However, this process is time consuming and can be arbitrary to some extent. Therefore, the method of selecting a model was to screen models for good candidates using specific criteria, and evaluate models one by one until an acceptable model was identified. The criteria used to screen models includ- ed: 1) the model should include the inhalation route of exposure (primarily), 2) de- velopment of the model should incorporate validation against venous blood data, 3) the model should be reported in the peer reviewed literature, and 4) the model should have as a primary purpose the goal of rat-to-human extrapolation. A number of PBPK models have published for inhalation of toluene (Purcell et al. 1990; Tardif et al. 1993; Pierce et al. 1996a; Tardif et al. 1997; Benignus et al. 1998; Pierce et al. 1998; Ali and Tardif 1999; Haddad et al. 1999a; Pierce et al. 1999; Vicini et al. 1999; Jonsson et al. 2001; Tardif et al. 2002; van Asperen et al. 2003; Benignus et al. 2006; Kenyon et al. 2008). Several of these are variations of each other. Some were developed for rats, others for humans, and some for both (with modification of appropriate parameter values). The purpose for some of the models was evaluation of mixture interactions, although in each of these cases, a model was first developed for toluene as a single chemical. The two most recently published models that met the criteria described above are those of Kenyon et al. (2008) for the rat and that of Benignus et al. (2006) for the human. 

Toluene 395 Model Structure The model structure is shown in Figure C-1. The tissue groups/compartments specified are consistent with models for other inhaled volatile organic solvents (Reddy et al. 2005). The individual compartments (lungs, brain, liver, gastrointesti- nal tissue, fat, and slowly and rapidly perfused tissue groups) are connected by the systemic circulation. The model has distinct arterial and venous blood compartments and tissues are described as homogeneous well-mixed compartments. Metabolism is described by a single metabolic pathway following saturable (Michaelis-Menten) kinetics in the liver. Brain is the target tissue of interest and fat is a major site for sequestration. Lung Brain Arterial Blood Venous Blood RPTG SPTG Fat Liver GI Tract Vmax, KM metabolism FIGURE C-1 Schematic diagram of the toluene PBPK model. Source: Kenyon et al. 2008. Reprinted with permission; copyright 2008, Journal of Toxicology and Environ- mental Health, Part A: Current Issues. 

396 Acute Exposure Guideline Levels TABLE C-2 Physiologic Parameters for the Toluene Rat Model Parameter, units Symbol Value Footnote 1 Body weight, kg BW 0.237-0.508 Cardiac output, L/h-kg0.75 QCC 2 Normal 11.2 Sedentary, day acclimated 11.2 Active, day-acclimated 13.2 Alveolar ventilation, L/h-kg0.75 QPC 2 Normal 9.9 Ratio = 0.9 Sedentary, day-acclimated 12.2 Ratio = 1.1 Active, day-acclimated 22.4 Ratio = 1.7 Flow (fraction QC)3 4 Lung QCC 1.0 Liver QLC 0.02 Fat QFC 0.082 Brain QBC 0.027 Gut QGC 0.16 SPTG QSC 0.257 RPTG QRC 1-Qi Volumes (fraction body weight)5 Arterial blood VABC 0.030 Venous blood VVBC 0.060 Lung VNC 0.0048 Liver VLC 0.0401 6 Fat VFC 0.08-0.16 Brain VBC 0.0054 Gut VGC 0.0249 SPTG VSC 0.60 RPTG VRC 1-Vi 1 Body weight is an experiment-specific measurement. 2 Experiment-specific based on type of acclimation and activity level of rat. “Normal” refers to rats with a normal diurnal cycle of being active and fed at night and studied during daylight hours, whether weight-maintained or free-fed. “Sedentary, day- acclimated” refers to rats acclimated to be fed and active during the light part of their diurnal cycle, but not performing a task. “Active, day-acclimated” refers to rats acclimat- ed to be fed and active during the light part of their diurnal cycle and performing a lever pressing task. Ratio is the ratio of QPC/QCC, an approximation of ventilation-perfusion ratio. 3 Values are from two main sources (Brown et al. 1997; Simmons et al. 2002). 4 Lung receives all of cardiac output. 5 Values that LE rat specific were calculated from regression equations (Simmons et al. 2002) for a 0.35 kg rat. Other values are from a standard reference (Brown et al. 1997). All calculated values were checked against reported ranges of reference values (Brown et al. 1997). 6 Experiment-specific based on body weight of rat using regression equation from Sim- mons et al. (2002). 

Toluene 397 TABLE C-3 Chemical-Specific Parameters for the Toluene Rat Model Parameter Symbol Units Value Reference Partition coefficients Blood:air PB None 21.0 Thrall et al. 2002 Lung:blood PN None 1.14 Liver:blood PL None 2.01 Brain:blood PBR None 1.72 Fat:blood PF None 86.19 Gut:blood PG None 2.62 RPTG:blood PR None 2.62 SPTG:blood PS None 1.32 Metabolism parameters Vmax VMAXC L/h/kg0.75 4.55 Tardif et al. 1993a KM KM mg/L 0.45 a Metabolism parameters are consistent with the range of published values in the literature (DeJongh and Blaauboer 1996). Physiological and Chemical-Specific Parameter Values Physiological and chemical-specific parameter values used in the present model for rats are listed in Tables C-2 and C-3, respectively. Model code is provided in Attachment 1. All details concerning model calibration and evaluation for both the rat model and the human GPAT model can be obtained from the original publica- tions (Benignus et al. 2006; Kenyon et al. 2008). Derivation of AEGLs with PBPK-Based Approach The AEGL-2 critical study was Bushnell et al. (2007a), in which the NOAEL for a doubling in choice reaction time in Long-Evans rats exposed to toluene was 1,600 ppm for 34 min. As shown in Table C-4, the internal dose metric of brain tolu- ene concentration (BrTC) in the rat was determined using a PBPK model (Kenyon et al. 2008), and the determination of the HEC used the human GPAT model (Benignus et al. 2006). The BrTC in rats at the end of exposure, calculated by the PBPK model, was 49.2 mg/L. A total uncertainty factor of 3 was applied to the rat BrTC to arrive at the target internal dose in humans of 16.4 mg/L. The uncertainty factor of 3 was applied to the internal dose metric, as recommended by the Standing Operating Pro- cedures (NRC 2001), to arrive at final human exposure values in the final row of Table C-4 which were rounded to obtain the final AEGL-2 values. The AEGL-2 values were also compared to the estimated toluene exposure concentrations (ppm) which would lead to decrements in reaction time similar to those associated with ethanol inebriation (0.08% and 0.10% blood ethanol level), with the AEGL-2 values are between those two estimates (lower portion of Table C-4). 

398 Acute Exposure Guideline Levels TABLE C-4 Toluene Concentrations in Air Associated with Relevant Brain Toluene Concentrations in Rats and Humans as Determined by PBPK Modeling 10 min 30 min 1h 4h 8h Duration extrapolation at the critical brain toluene concentration (BrTC) in rats, and extrapolation to humans using one-third of the critical internal dose (per guidance in NRC 2002) Rat internal dose metric (1,600 ppm 3,085 ppm 1,705 ppm 1,243 ppm 809 ppm 651 ppm for 34 min) ; BrTC = 49.2 mg/L AEGL-2 values based on GPAT 1,396 ppm 757 ppm 558 ppm 311 ppm 251 ppm estimates in humans; BrTC = (49.2/3) = 16.4 mg/L Toluene concentrations leading to effect levels comparable to blood ethanol levels (BELs) of 0.08% and 0.10% GPAT estimations of exposures 930 ppm 520 ppm 390 ppm 230 ppm 190 ppm comparable to BEL of 0.08% (standing); BrTC = 119 µM = 10.9 mg/L GPAT estimations of exposures 1,860 ppm 1,000 ppm 730 ppm 390 ppm 320 ppm comparable to BEL of 0.10% (standing); BrTC = 241 µM = 24.1 mg/L Source: Based on Bushnell et al. 2007a. For comparative purposes, alternative AEGL-2 values were also calculated us- ing the clinical study of Gamberale and Hultengren (1972) as shown in Table C-5. In this study, the threshold for narcosis was not approached as indicated by the failure to produce significant deficits in tests of perceptual speed and reaction time. In the Gamberale and Hultengren (1972) study, subjects were sequentially exposed to tolu- ene at 100, 300, 500, and 700 ppm for 20 min at each level, with a short break in the middle. The venous blood concentration calculated by the PBPK model is much greater when the full exposure regimen was simulated compared with the concentra- tion after only a 20 min exposure (about 6.5 versus 4.5 mg/L). The actual exposures were roughly equivalent to a 20-min exposure at about 1,000 ppm. Therefore, the CV determined for the actual experimental conditions was used to derive AEGL-2 values. According to the standing operating procedure for use of PBPK modeling in AEGL value development, the uncertainty factor can be applied to the HEC or to the dose metric of 6.54 mg/L, but the latter approach is generally preferred. If the total uncertainty factor is 1, the approaches become the same. The alternative AEGLs were deemed to be not as credible as the refined approach using the rat data extrapo- lated via the human GPAT model based on the comparisons with the toluene con- centrations leading to effect levels comparable to blood ethanol levels of 0.08% and 0.10%, as shown in the lower portion of Table C-4. 

Toluene 399 TABLE C-5 AEGL-2 Values for Toluene Determined with PBPK Model of Human Data 10 min 30 min 1h 4h 8h UF = 1 1,855 805 601 426 378 UF = 3, applied to human 618 268 200 142 126 equivalent concentration UF = 3, applied to dose metric 655 305 239 180 166 Source: Based on Gamberale and Hultengren 1972. The AEGL-3 values were based on a rat NOAEL for lethality and were deter- mined in the same manner as the AEGL-2 values: the determination of the internal dose metric used the rat PBPK model and the determination of the HEC used the human GPAT model. In Table C-6, the results in the first row are based on the target internal dose (BrTC) of 362 mg/L taken from the Mullin and Krivanek (1982) study in rats, then deriving the HEC with no adjustment with uncertainty factors using the GPAT model. In the second row, the HEC determined for an uncertainty factor of 1 (listed in the first row) were divided by 3. The results in the third row were obtained by dividing 362 mg/L by 3 (121 mg/L) as a new target internal dose, and then using the human GPAT model to determine the corresponding HECs. TABLE C-6 Comparison of AEGL-3 Values for Toluene Determined by PBPK Modeling 10 min 30 min 1h 4h 8h UF = 1, human BrTC = 362 mg/L 30,200 ppm 15,440 ppm 10,860 ppm 5,210 ppm 3,920 ppm UF = 3, applied to human 10,067 ppm 5,147 ppm 3,620 ppm 1,737 ppm 1,307 ppm equivalent concentration UF = 3, applied to dose metric 10,125 ppm 5,209 ppm 3,680 ppm 1,810 ppm 1,370 ppm Source: Based on Mullin and Krivanek (1982), NOAEL for lethality of 6,250 ppm (2 h) in rats. 

400 Acute Exposure Guideline Levels Attachment 1 PBPK Model Code for Toluene AEGL Rat Model Models used were published models for the rat (Kenyon et al. 2008) and the human GPAT model (Benignus et al. 2006). All model calibration details and evalu- ation data and equations are available in these papers, except where otherwise noted (e.g., QPC and QCC values were set equal to 15 L/h-kg, so that the values for these highly influential parameters were equal to those used in the Dennison model origi- nally used for AEGL derivation). The code below is for the Kenyon el. al. (2008) model realized in acslX 3.0.1.6 (Aegis Technologies Group, Austin, TX). PROGRAM TOLRAT1.CSL !PBPK Model for Toluene (TOL)in rat !created by Elaina Kenyon 08/04/05 !incl. explicit arterial and venous compt !& non-SS lung, both inhalation & oral routes LOGICAL TOLCHX !If TOLCHX = true, then check Toluene !mass balance TOLCHX = .TRUE. LOGICAL DPSITG !Integration control variable for !precision DPSITG = TRUE. INITIAL !STATEMENTS EXECUTED ONLY AT THE BEGINNING OF THE !SIMULATION, CONSTANT COMMANDS EXECUTED ONLY at BUILD CONSTANT LIMIT = 0.1 !Limit for unbal. cmpds when checking mass balance CONSTANT DUMMY = 0.00001 !***** Physiological Parameters **** CONSTANT QPC = 16.1 !Alveolar Ventilation Rate(l/hr-kg) CONSTANT QCC = 16.1 !Cardiac Output(l/hr-kg) CONSTANT QLC = 0.02 !Fract. Blood Flow to Liver CONSTANT QFC = 0.052 !Fract. Blood Flow to Fat CONSTANT QBC = 0.114 !Fract. Blood Flow to Brain CONSTANT QGC = 0.16 !Fract. Blood Flow to Gut CONSTANT QSC = 0.249 !Fract. Blood Flow to Poorly Perfused T. QRC = 1.0 - QLC - QFC - QBC - QSC - QGC !Fract. Blood Flow to Richly Perfused T. 

Toluene 401 CONSTANT BW = 0.350 !Body Weight(kg) CONSTANT VLC = 0.026 !Volume Fraction Liver CONSTANT VFC = 0.2142 !Volume Fraction Fat CONSTANT VBC = 0.020 !Volume Fraction Brain CONSTANT VGC = 0.0249 !Volume Fraction GI Tract CONSTANT VSC = 0.4371 !Volume Fraction Poorly Perfused T. CONSTANT VNC = 0.0076 !Volume Fraction Lung CONSTANT VVBC = 0.0592 !Volume Fraction Venous Blood CONSTANT VABC = 0.0198 !Volume Fraction Arterial Blood VRC = 1.0 - VLC - VFC - VBC - VSC - VNC - VVBC - VABC - VGC !Volume Fraction Richly Perfused T. !***** Chemical Specific Parameters for TOL ***** !Partition Coefficients from Thrall et al., 2002 CONSTANT PL = 2.01 !Liver/Blood Partition Coefficient CONSTANT PF = 86.19 !Fat/Blood Partition Coefficient CONSTANT PBR = 1.72 !Brain/Blood Partition Coefficient CONSTANT PG = 2.62 !GI/Blood Partition Coefficient CONSTANT PS = 1.32 !Poorly/Blood Partition Coefficient CONSTANT PR = 3.04 !Richly/Blood Partition Coefficient CONSTANT PB = 21.0 !Blood/Air Partition Coefficient CONSTANT PN = 1.14 !Lung/Blood Partition Coefficient CONSTANT MW = 92.14 !Molecular Wt (g/mol) !***** Metabolism Parameters - VMAX/KM from Pierce et al., 1996 CONSTANT VMAXC = 4.8 !Max Rate TOL metabolism (mg/hr-kg) CONSTANT KM = 0.55 !Affinity Constant for TOL (mg/l) !***** Dosing & Exposure Variables ***** CONSTANT CONC = 250. !Inhalation Concentration (ppm) !***** Timing Commands ***** SCHEDULE DS1 .AT. TCHNG !Use discrete at discontinuities CIZONE = 1.0 !Start with inhalation on CONSTANT TSTOP = 8. !Length of experiment (hrs) CONSTANT TCHNG = 2. !Length of inhalation exposure CONSTANT POINTS = 100. !Number of points in plot CINT = TSTOP/POINTS !Communication interval !***** Inhalation Exposure Definition ***** AIO = (CONC * MW)/24450 !Initial Chamber Conc (mg/l) 

402 Acute Exposure Guideline Levels !***** Oral Exposure Definition ***** CONSTANT DV = 0.002 !Dosing Volume in L/kg CONSTANT SOCO = 100000 !Dosing Soln Conc, mg/L CONSTANT KA = 5.0 !First Order GI abs, 1/hr ODTOL = DV*BW*SOCO !Bolus gavage dose in mg ORTOL = (DV*SOCO*BW)/BW !Bolus dose in mg/kg !***** Scaled Parameters ***** QC = QCC * (BW**0.74) !Cardiac Output (l/hr) QP = QPC * (BW**0.74) !Alveolar Ventilation Rate (l/hr) QL = QLC * QC !Flow Liver Compartment (l/hr) QF = QFC * QC !Flow Fat Compartment (l/hr) QB = QBC * QC !Flow Brain Compartment (l/hr) QG = QGC * QC !Flow GI Compartment (l/hr) QS = QSC * QC !Flow Slowly Perf. Tis. Cmpt. (l/hr) QR = QRC * QC !Flow Richly Perf. Tis. Cmpt. (l/hr) VL = VLC * BW !Volume Liver Compartment, Total VF = VFC * BW !Volume Fat Compartment VB = VBC * BW !Volume Brain Compartment VG = VGC * BW !Volume GI Compartment VS = VSC * BW !Volume Slowly Perfused Tis. Cmpt. VR = VRC * BW !Volume Richly Perfused Tis. Cmpt. VMAX= VMAXC * BW**0.74 !VMAX scaled VVB = VVBC * BW !Volume Venous Blood Cmpt. VAB = VABC * BW !Volume Arterial Blood Cmpt. VN = VNC * BW !Volume Lung Cmpt. END !**** END OF INITIAL **** DYNAMIC !Code executed at end of each commun. interval ALGORITHM IALG = 2 !Gear Method for Stiff Systems (LSODE) DISCRETE DS1 !Discontinuity in Simulation executed !when inhalation exposure ends CIZONE = 0.0 !Turn inhalation off CALL LOGD(.TRUE.) !Forced logging for plots END DERIVATIVE !Gavage oral dose of Toluene in mg RDSTOM = -KA*STOMD STOMD = INTEG(RDSTOM, ODTOL) 

Toluene 403 !CI - Concentration TOL in inhaled air (mg/l) RAI = QP * C !Rate TOL to lung (mg/hr) AI = INTEG (RAI, 0.) !Amount TOL entering lung, mg CI = AIO * CIZONE !CA - Concentration TOL in arterial blood (mg/l) ! CA1 - Concentration part to lung !AAB - Amount TOL in arterial blood (mg) RAAB = QC * CA1 - QC * CA AAB = INTEG(RAAB, 0.) CA = AAB/VAB !AX - Amount TOL exhaled (mg) RAX = QP * CX !Rate TOL exhaled (mg/hr) AX = INTEG (RAX, 0.) CX = CA1/PB !Conc. TOL in exhaled air(mg/l) CXPPM = ((0.7 * CX) + (0.3 * CI)) * (24450/MW) !ppm AXKG = AX/BW !mg exhaled/kg body weight !AN - Amount TOL in lung (mg) RAN = QC * CV + QP * CI - QC * CA1 - QP * CX AN = INTEG (RAN, 0.) CA1 = AN/(VN * PN) CN = AN/VN !AS - Amount TOL in slowly perfused tissues (mg) RAS = QS * (CA - CVS) !Rate of change in conc. (mg/hr) AS = INTEG (RAS, 0.) CVS = AS / (VS * PS) !Conc partition to slow per. tis.(mgl) CS = AS / VS !Conc in volume slow per. tis.(mg/l) !AR - Amount TOL in rapidly perfused tissues (mg) RAR = QR * (CA - CVR) !Rate of change in conc. (mg/hr) AR = INTEG (RAR, 0.) CVR = AR / (VR * PR) !Conc partition to rap per. tis.(mg/l) CR = AR / VR !Conc in volume rap per. tis.(mg/l) !ABR - Amount TOL in brain (mg) RABR = QB * (CA - CVBR) !Rate of change in conc (mg/hr) ABR = INTEG (RABR, 0.) CVBR = ABR / (VB * PBR) !Conc partition to brain(mg/l) CBR = ABR / VB !Conc in volume brain(mg/l) AUCBR = INTEG(CBR, 0.) !AUC for TOL in brain !AF - Amount TOL in fat tissue(mg) RAF = QF * (CA - CVF) !Rate of change in conc(mg/hr) AF = INTEG (RAF, 0.) CVF = AF / (VF * PF) !Conc partition to fat(mg/l) CF = AF / VF !Conc in fat volume(mg/l) 

404 Acute Exposure Guideline Levels !AG - Amount TOL in gut tissue(mg) !RAG - rate of change TOL in gut conc, mg/hr RAG = QG * (CA - CVG) - RDSTOM AG = INTEG (RAG, 0.) CVG = AG / (VG * PG) !Conc partition to gut (mg/l) CG = AG / VG !Conc in gut volume(mg/l) !AL - Amount TOL in liver(mg) !RAL - rate of change in liver conc, mg/hr RAL = (QL * CA) + (QG * CVG) - ((QL+QG) * CVL) - RAM AL = INTEG (RAL, 0.) CVL = AL / (VL * PL) !Conc partition to liver(mg/l) CL = AL / VL !Conc in liver volume(mg/l) !AUCPO - Amount TOL oxidatively metabolized (mg) !RAM - Rate of oxidative metabolism of TOL (mg/hr) RAM = (VMAX * CVL) / (KM + CVL) AUCPO = INTEG (RAM, 0.) AUCKG = AUCPO / BW !Amount TOL metabolized/kg body weight !CV - TOL mixed venous blood concentration (mg/l) !RAVB - Rate of change in concentration (mg/hr) RAVB = (QL+QG)*CVL) + QF*CVF + QS*CVS + QR*CVR + QB*CVBR - QC * CV AVB = INTEG(RAVB, 0.) CV = AVB/VVB !Conc in venous blood volume (mg/l) AUCV = INTEG(CV,0.) !AUC for toluene in venous blood !PMASS - TOL Mass balance(mg) PMASS = AN + AF + AG + AL + AS + AR + ABR + AUCPO + AX + AVB + AAB + STOMD TERMT (T .GE. TSTOP) !terminate solution END !END OF DERIVATIVE !**Code to calculate variables used in checking mass balance** BALPC = (AI + ODTOL) - PMASS TBALPC = ABS(BALPC) END !END OF DYNAMIC END !END OF PROGRAM 

Toluene 405 APPENDIX D TOLUENE SIMULATIONS Vernon Benignus, Elaina M. Kenyon, William Boyes, and Philip Bushnell 2/22/2013 Acute exposure to toluene vapor impairs neurologic function. The degrees of neurologic impairment produced by exposures to toluene, and dose response- relationships, have been determined using a variety of behavioral procedures. Some of these behavioral procedures, such as choice reaction time tasks, have also been used to evaluate the acute consequences of exposure to another common intoxicant, ethanol. This circumstance allows the potency of toluene and ethanol to be compared relative to a common degree of behavioral impairment. Because the behavioral impairments caused by ethanol intoxication have also been associated with fatal automobile acci- dents in a dose-effect manner, it is possible to estimate the degree of toluene exposure associated with a level of impairment that might increase the probability of causing a fatal automobile accident if an exposed person were also driving at the same time. In addition to the probability of an automobile accident, this level of impairment would also be expected to put a person who was not driving at risk for other accidents or dan- gers given their unique circumstances and surroundings. With this approach, it is pos- sible to estimate the potential severity of acute behavioral impairments caused by tolu- ene in terms that are relevant for AEGL value determinations. Behavioral Consequences of Toluene Exposure Simulations comparing the behavioral effects of toluene and ethanol were made to provide context for the danger of toluene exposure in terms of the danger associated with comparable levels of intoxication with ethanol. These simulations used a behavioral effect (speed of choice reaction time) as a dependent variable. In these tests, the subject must make a decision about which response is correct as quickly as possible. Table D-1 and Figure D-1 show the severity of a decrement in reaction time (percent of maximum possible effect) as a function of brain toluene concentration, and also the blood ethanol concentrations equivalent to these brain toluene concentrations. Brain toluene concentrations are related quantitatively to blood ethanol concentrations in terms of the magnitude of change in reaction time associated with each chemical (Benignus et al. 2005). In addition, Table D-1 and Figure D-1 show corresponding increases in fatal automobile crashes as a function of degree of intoxication with either toluene or ethanol. These functions were derived from data relating the increased risk of a fatal car crash to blood ethanol concentration and the quantitative relationship between the effects of toluene and ethanol on reaction time. These relationships allow one to 

406 Acute Exposure Guideline Levels estimate the number of fatal automobile accidents that would be produced by toluene exposure as a function of the degree of intoxication (Benignus et al. 2011). It should be noted that the increase in fatal automobile accidents is given as the number of fatalities per 1,000 drivers who had the toxicant in their bodies while driving. From Table D-1 it may be concluded that a concentration of toluene in brain of 119 µM produces a degree of intoxication associated with about 13% reduction in speed of choice reaction time. This degree of impairment is also associated with 0.08 g/dl of venous ethanol, a concentration that defines legal intoxication and is associ- ated with an increase over baseline of 305 fatal automobile accidents per 1,000 driv- ers. Almost half of drivers with 0.10 g/dl venous ethanol or a brain toluene concen- tration of 241 µM would be expected to have fatal automobile accidents. Inhaled Concentrations of Toluene Required to Produce Specific Brain Concentrations In addition to the duration of exposure, alveolar ventilation, which depends upon physical activity, affects the concentration of toluene in the inhaled air that is required to produce a given brain toluene concentration. Table D-2 and Figure D-2 give approximate concentrations of toluene in inhaled air for a person exercising at four levels of exertion for two brain concentrations and for five exposure durations of interest. This illustrates the large impact of exertion on toluene uptake. TABLE D-1 The Importance of the Behavioral Effects of Toluene Exposure Estimated via the Alcohol Effects on Fatal Automobile Accidents and Employing the Equivalent Dose of Brain Toluene Proportion of Fatal Effect on Choice Venous Blood Automobile Accidents Equivalent Brain Reaction Time Test Alcohol (g/dL) Greater Than Baselinea Toluene (µM)b (% of Maximum)c 0.10 0.478 241.0 26.2 0.09 0.386 172.0 19.1 0.08 0.305 119.0 13.1 0.07 0.216 83.1 8.9 0.06 0.132 48.8 4.9 a Fatal automobile accident estimates were obtained from Zador (1991) and Zador et al. (2000) as expressed in Figure 2 of Benignus et al. (2011). The proportion of persons who were killed in a single-car crash with a measured post-mortem blood-ethanol concentra- tion, relative to persons driving with that same blood-ethanol concentration who did not crash, is expressed as a proportional increment above baseline (0.00 g/dL). b Toluene/alcohol equivalence was computed using Equation 2 in Benignus et al. (2011). c Behavioral effect magnitudes were computed using Equation 1, with human parameters for the low motivation case, from Table 4 of Benignus et al. (2009). 

Toluene 4407 FIGUR RE D-1 The effecct of alcohol intoxication (top hhorizontal axis) oon fatal automobbile accidents (per 1,000 driivers per year) compared with thhe equivalent am mount of toluenee in the brain (µM). This grraph is a modifieed form of Figurre 3 in Benignuss et al. (2011). T The alcohol axis was construucted from Figru ure 1c from the ssame source. Thhe plotted functioons reflect the t mean estimaate (solid line) and the upper aand lower 95% % confidence lim mits (dashedd lines). TABLEE D-2 Approxim mate Exposuress Required to Prroduce Brain Toluene Concen ntrations Given in Table D-1 ass Determined byy the GPAT Moodel Concentration Required R for 241 µM Conccentration Requireed for 119 µM Brain Toluene (ppm) Brainn Toluene (ppm) Time Lying Stan nding 1 mph 2 mph Lyingg Standing 1 mph 2 mph 10 min 2,290 1,860 1,350 1,000 1,1500 930 670 500 30 min 1,320 1,000 680 480 700 520 350 240 1h 1,010 730 460 320 540 390 240 165 4h 555 390 250 200 325 230 140 110 8h 445 320 220 190 275 190 130 100 The valu ues in the table were w approximaated by iterativelly applying GPA AT (Benignus et al. 2006). The T modeled sub bject was lying down, d standing, or walking at 1 oor 2 mph. 

408 Accute Exposure Guideline Levels FIGUR RE D-2 Concentrration of inhaled d toluene (ppm) required to prooduce brain tolueene concenttrations of 241 orr 119 µM at four exercise levelss. The two brain toluene concenttra- tions are equivalent to venous concentrrations of alcohhol of 0.1 or 0.08 g/dl. Those coon- centratio ons are roughly approximate vaalues of ppm geenerated by the GPAT model (B Be- nignus et e al. 2006). Inhaleed Air Concen ntrations Requiired to Producce Toluene Concentrationns of 119 or 2441 µM in the Brrain All A calculations so far were madem using the GGPAT model, because the Deen- nison model m does nott have a brain compartment. IIn order to maake exposure essti- w the accepted Dennison model, mates with m PAT model wass used to estimate (a) a GP the vennous concentratiions of toluene required to pro duce brain conccentrations of 1110 or 241 µM and (b) the Dennison mod del was used to determine the pppm values neces- sary to produce these venous concen ntrations. Thesee venous and innhaled-air conceen- trationss are presented in i Table D-3. 

Toluene 409 TABLE D-3 Inhaled Air and Venous Concentrations of Toluene Required to Produce Either 119 or 241 µM Brain Toluene Concentrations Computed from the Dennison PBPK Model Time For 119 µM For 241 µM Air (ppm) Venous (mg/L) 10 min 625 2.07 1,498 5.24 30 min 372 2.75 729 5.87 1h 312 3.05 597 6.49 4h 258 3.55 481 7.52 8h 239 3.66 436 7.73 Behavioral Contingencies For both rats and humans, sensitivity to a given concentration of a solvent in the brain depends upon the situation under which the exposed individual was tested (Benignus et al. 2007, 2009). When a behavioral decrement has little consequence to the subject, the behavioral disruption by the toxicant will be maximal. If the subject loses a reward for poor performance, the effect of the tested toxicant will be reduced, or more toxicant will be necessary to disrupt the behavior. If painful punishment follows poor performance, the effect of the tested toxicant is greatly reduced. For example, rats working to avoid painful shocks are less susceptible to toluene expo- sure than are rats working for a food reward. Rats working for a food reward are less susceptible than are some nonmotivated neurophysiologic procedures that are not subject to either reward or punishment. It is important to consider behavioral meas- urements that are reasonably comparable if sensitivity is to be compared across fac- tors such as different behavioral tasks or across different species. Quantitative Rat-to-Human Extrapolation Using ED10 values for various situations in rats and humans from Figure 3 in Benignus et al. (2009), a table of extrapolation ratios was created (see Table D-4). Table D-4 shows that if experimental procedures are the same in rats and humans, the same brain solvent concentrations in the two species should produce the same ED10. In contrast, if the contingencies on performance differ, then ED10s will differ accordingly. Thus, modeling studies with rats suggest that the ED10 of a subject whose behavior is minimally constrained by contingencies will be lower by a factor of 86 compared to a subject avoiding punishment (Benignus et al. 2009). Thus quan- titative rat-human extrapolation requires information about the experimental contin- gencies applied during the tests; data from animal studies suggest that if the contin- gencies differ, then the extrapolation must take into account their relative impacts of the behavioral contingencies applied to each species. 

410 Acute Exposure Guideline Levels TABLE D-4 Extrapolating Factorsa from Rats to Humans Depending on Experimental Conditions None Minimum Withhold reward Painful punishment None 1.00 7.30 19.40 86.30 Minimum – 1.00 2.70 11.80 Withhold Reward – – 1.00 4.40 Painful punishment – – – 1.00 a Divide rat ED10 for the appropriate rat experimental condition by the extrapolation factor to calculate the human ED10. Factors calculated from the data to produce Figure 3 in Be- nignus et al. (2009). REFERENCES Benignus, V.A., P.J. Bushnell, and W.K. Boyes. 2005. Toward cost-benefit analysis of acute behavioral effects of toluene in humans. Risk Anal 25(2):447-456. Benignus, V.A., T. Coleman, C.R. Eklund, and E.M. Kenyon. 2006. A general physiolog- ical and toxicokinetic (GPAT) model for simulating complex toluene exposure scenarios in humans. Toxicol. Mech. Methods 16(1):27-36. Benignus, V.A., W.K. Boyes, E.M. Kenyon, and P.J. Bushnell. 2007. Quantitative com- parison of the acute neurotoxicity of toluene in rats and humans. Toxicol. Sci. 100(1):146-155. Benignus, V.A., P.J. Bushnell, W.K. Boyes, C. Eklund, and E.M. Kenyon. 2009. Neuro- behavioral effects of acute exposure to four solvents: Meta-analyses. Toxicol. Sci. 109(2):296-305. Benignus, V.A., P.J. Bushnell, and W.K. Boyes. 2011. Estimated rate of fatal automobile accidents attributable to acute solvent exposure at low inhaled concentrations. Risk Anal. 31(12):1935-1948. Zador, P.L. 1991. Alcohol-related relative risk of fatal driver injuries in relation to driver age and sex. J. Stud. Alcohol 52(4):302-310. Zador, P.L., S.A. Krawchuck, and R.B. Voas. 2000. Alcohol-related relative risk of driv- er fatalities and driver involvement in fatal crashes in relation to driver age and gender: An update using 1996 data. J. Stud. Alcohol 61(3):387-395. 

Toluene 411 APPENDIX E ACUTE EXPOSURE GUIDELINE LEVELS FOR TOLUENE AEGL-1 VALUES 10 min 30 min 1h 4h 8h 67 ppm 67 ppm 67 ppm 67 ppm 67 ppm (250 mg/m3) (250 mg/m3) (250 mg/m3) (250 mg/m3) (250 mg/m3) Key references: Multiple clinical studies, including: (1) Astrand, I., H. Ehrner-Samuel, A. Kilbom, and P. Ovrum. 1972. Toluene exposure. I. Concentration in alveolar air and blood at rest and during exercise. Scand. Work Environ. Health 9:119-130. (2) Gamberale, F., and M. Hultengren. 1972. Toluene exposure. II. Psychophysiological functions. Work Environ. Health. 9(3):131-139. (3) Baelum, J., G.R. Lundqvist, L. Molhave, and N.T. Andersen. 1990. Human response to varying concentrations of toluene. Int. Arch. Occup. Environ. Health 62(1):65-72. Test species/Strain/Number: Humans, (1) 15, both sexes; (2) 12 males; (3) 71 subjects Exposure route/Concentrations/Durations: Inhalation; (1) 100 or 200 ppm for 60 min, exercise incorporated into protocol; (2) 100, 300, 500, or 700 ppm, successive 20-min exposures for a total of 85 min (one 5-min break), exposure via a mouthpiece; (3) 100 ppm for 7.5 h, varying exposures of 50-300 ppm (TWA of 100 ppm) for 7.5 h. Effects: (1) 100 or 200 ppm with exercise: no effect on heart rate, pulmonary ventilation, oxygen consumption, or blood lactate; subjective symptoms not assessed. (2) One of 12 subjects able to distinguish between control and toluene exposure 100 ppm: no effect on reaction time 300 ppm: increase in simple reaction time 500 ppm: increase in complex reaction time 700 ppm: decrease in perceptual speed at end of exposure (3) 100 ppm: no ocular irritation, complaints of “poor air quality,” irritation of nose and lower airways. 50-300 ppm (TWA of 100 ppm): same symptoms as above. End point/Concentration/Rationale: Weight of evidence from multiple clinical studies indicated that toluene at 200 ppm for up to 8 h would be without effects that exceed the definition of AEGL-1. Slight irritation reported in some studies, but not others. Uncertainty factors/Rationale: Intraspecies: 3, the minimum alveolar concentration for volatile anesthetics differs by no more than 2- to 3-fold in the human population. Modifying factor: None Animal-to-human dosimetric adjustment: None Time scaling: Not applied; steady-state at 67 ppm is approached fairly rapidly. Data adequacy: Twenty clinical studies, many of them recent and well-conducted, addressed sensory irritation and the threshold for CNS effects. Metabolism and monitoring studies also indicate a lack of substantial effects at 200 ppm. 

412 Acute Exposure Guideline Levels AEGL-2 VALUES 10 min 30 min 1h 4h 8h 1,400 ppma 760 ppm 560 ppm 310 ppm 250 ppm (5,300 mg/m3) (2,900 mg/m3) (2,100 mg/m3) (1,200 mg/m3) (940 mg/m3) Reference: Bushnell, P.J., W.M. Ohiro, T.E. Samsam, V.A. Benignus, Q.T. Krantz, and E.M. Kenyon. 2007a. A Dosimetric analysis of the acute behavioral effects of inhaled toluene in rats. Toxicol. Sci. 99(1):181-189. Test species/Strain/Number: Rat; Long-Evans; 16 male rats exposed in groups of 4. Exposure route/Concentrations/Durations: Inhalation; 0, 1,200, 1,600, 2,000, or 2,400 ppm for up to 70 min. Effects: Reaction time doubled compared with air-exposed controls. Concentration- related increase in reaction time. NOAEL for a doubling of reaction time was 1,600 ppm for a 34-min exposure. End point/Concentration/Rationale: Doubling of reaction time, threshold of 1,600 ppm for 34-min exposure Uncertainty factors/Rationale: Interspecies: 1, PBPK modeling eliminated the toxicokinetic component of the uncertainty factor; the pharmacodynamic component was assigned a factor of 1 because similar central-nervous-system effects were observed in rodents and humans. Intraspecies: 3, the minimum alveolar concentration for volatile anesthetics differs by no more than 2- to 3-fold in the human population. Modifying factor: None Animal-to-human dosimetric adjustment: PBPK modeling performed Time scaling: PBPK modeling was used to determine the equivalent exposure concentrations that yield the dose metric at each of the AEGL exposure durations. Data adequacy: The values are supported by a comparison of the AEGL values and corresponding effect levels from ethanol consumption in humans (Benignus et al. 2011). a Concentration is one-tenth or more of the lower explosive limit of 14,000 ppm for tolu- ene in air. Therefore, safety considerations against the hazard of explosion must be taken into account. AEGL-3 VALUES 10 min 30 min 1h 4h 8h b b b 5,200 ppm 3,700 ppm 1,800 ppm 1,400 ppmb –a (20,000 mg/m3) (14,000 mg/m3) (6,800 mg/m3) (5,300 mg/m3) Reference: Mullin, L.S., and N.D. Krivanek. 1982. Comparison of unconditioned reflex and conditioned avoidance tests in rats exposure by inhalation to carbon monoxide, 1,1,1- trichloroethane, toluene or ethanol. Neurotoxicity 3(1):126-137. Test species/Strain/Number: Rats, CD, 6 males per group Exposure route/Concentrations/Durations: Inhalation, 810, 1,660, or 3,100 ppm for 4 h; 6,250 ppm for 2 h Effects: No deaths after a 2-h exposure at 6,250 ppm End point/Concentration/Rationale: NOAEL for lethality, 6,250 ppm for 2 h 

Toluene 413 Uncertainty factors/Rationale: Interspecies: 1, PBPK modeling eliminated the toxicokinetic component of the uncertainty factor; the pharmacodynamic component was assigned a factor of 1 because similar central-nervous-system effects were observed in rodents and humans. Intraspecies: 3, the minimum alveolar concentration for volatile anesthetics differs by no more than 2- to 3-fold in the human population. Modifying factor: None Animal-to-human dosimetric adjustment: PBPK modeling performed Time scaling: PBPK modeling was used to determine the equivalent exposure concentrations that yield the dose metric at each of the AEGL exposure durations. Data adequacy: There are multiple lethality studies in rats and mice. The AEGL-3 values are supported by the 20-min NOAEL of 12,000 ppm for lethality in the mouse (Bruckner and Peterson 1981a), the 2-h NOAEL of 5,000 ppm for lethality in rats (Kojima and Kobayashi 1973), the 4-h NOAEL of 6,000 ppm for lethality in rats (Wada et al. 1989), the NOAEL of 6,000 ppm in mice repeatedly exposed for 30 min/day (Moser and Balster 1981), and the chronic NOAEL of 1,200 ppm for mice and rats (NTP 1990). a The 10-min AEGL-3 value of 10,000 ppm (38,000 mg/m3) is higher than 50% of the lower explosive limit of 14,000 ppm for toluene in air. Therefore, extreme safety consid- erations against the hazard of explosion must be taken into account. b Concentration is one-tenth or more of the lower explosive limit of 14,000 ppm for tolu- ene in air. Therefore, safety considerations against the hazard of explosion must be taken into account.