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Appendixes



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Page 115 Appendixes

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Page 116

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Page 117 Appendix A Application of the Recommended Evaluative Process to Specific Chemicals This appendix demonstrates the evaluative process described in Chapter 2 and Chapter 3. To do that, the Subcommittee on Reproductive and Developmental Toxicology evaluated two agents of interest to the Navy, jet fuel JP-8 and 1,1,1,2-tetrafluoroethane (HFC-134a). The Navy is concerned about the health effects, including reproductive and developmental effects, of exposure to these agents. JP-8 was selected because it is a complex mixture and because it illustrates many of the problems that attend characterization of toxic substances: There is a sparse database on the mixture and on many of its individual components, composition varies between lots, and there are few data on human exposure. Because of the wide range of environmental conditions of human exposure (e.g., extreme cold to extreme heat, variable humidity), the actual exposure to aerosolized or vaporized components of the fuel varies with the environmental circumstance. The subcommittee evaluated the toxicity of JP-8 only under standard conditions. A complete assessment by the Navy would require an evaluation of each component under the full range of environmental conditions in which human exposures occur. A number of toxicity studies, including reproductive and developmental toxicity studies, have been conducted on HFC-134a. HFC-134a was selected because more data are available for this compound than for JP8.

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Page 118 Additional examples of the application of the evaluative process to specific agents can be found in the literature (Moore et al. 1995b; 1997); An Assessment of Boric Acid and Borax Using the IEHR Evaluative process For Assessing Human Developmental and Reproductive Toxicity of Agents. Reproductive Toxicology, 11(1): 123-160; and An Assessment of Lithium Using the IEHR Evaluative process For Assessing Human Developmental and Reproductive Toxicity of Agents. Reproductive Toxicology, 9(2):175-210. JP-8 JET FUEL Jet fuel JP-8 (jet propellant-8) is a kerosene-based distillate selected by the U.S. Air Force to replace JP-4 and other predecessors, which were replaced because JP-8 has a higher flash point, is composed of higher chain hydrocarbons, and does not contain benzene. Profiles for JP-8 list the following classes of compounds exclusive of additives: alkanes (43% by weight); cycloalkanes (11%); alkylbenzenes (12%); naphthalenes (2%); and dicycloparaffins, tetralins, and olefins (% not specified) (USAF 1991). A more detailed list of hydrocarbon components is given in Table A-1. Another jet fuel, JP-5, is physically and chemically similar to JP-8, and the differences between these fuels are considered minor (ATSDR 1998). Several studies described below were conducted using JP-5. Exposure Data Human exposure to JP-8 occurs during refueling and defueling operations and during mechanical activities that deal with storage, transfer, and combustion. Military personnel can be exposed to JP-8 by the inhalation (of aerosolized or vaporized fuel), dermal, and oral routes of exposure. Occupational standards for JP-8 are primarily based on knowledge about the toxicity of kerosene and naphtha (a petroleum distillate fraction). National Institute for Occupational Safety and Health (NIOSH) guidelines include an 8-hour (hr) time-weighted-average recommended exposure limit (TWA-REL) for naphtha of 400 milligrams per cubic meter (mg/m3) (100 parts per million (ppm)) (NIOSH

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Page 119 TABLE A-1 Composition of JP-8 Jet Fuel Hydrocarbon Type Weight % JP-8 a Isooctane 3.66 Methylcyclohexane 3.51 m-Xylene 3.95 Cyclooctane 4.54 Decane 16.08 Butylbenzene 4.72 1,2,4,5-Tetramethylbenzene 4.28 Tetralin 4.14 Dodecane 22.54 1-Methylnapthalene 3.49 Tetradecane 16.87 Hexadecane 12.22 a Composition of surrogate JP-8 (USAF 1991). 1999). Naval Occupational Safety and Health recommends a permissible exposure limit (PEL) of 350 mg/m3 and a 15-minute (min) short term exposure limit (STEL) of 1,000 mg/m3 (D.T. Harris et al. 1997). Puhala et al. (1997) reported measurement of jet fuel vapors at three domestic Air Force installations. Breathing-zone samples were collected from workers involved in aircraft maintenance, fuel handling, and flightline positions. Exposures at the base that used only JP-8 are listed in Table A-2. Each exposure fell below the American Conference of Governmental Industrial Hygienists (ACGIH) TWA threshold limit values (TLVs) for the chemicals analyzed. Two recent studies measured exposure of Air Force personnel to jet fuels, including JP-8. Pleil et al. (2000) used newly developed methods to collect exhaled breath from personnel at Air Force bases and then analyzed the samples for certain volatile marker compounds for JP-8 and for aromatic hydrocarbons such as benzene. The study authors found a demonstrable JP-8 exposure for all subjects, ranging from slight elevations to greater than 100-fold when compared with a control cohort. Carlton and Smith (2000) collected breathing zone samples from workers

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Page 120 TABLE A-2 Mean Exposure Concentrations of Jet Fuel Vapors, ppm a Analyte Overall Mean (n) Standard Deviation ACGIH TWA-TLV Naphthas 0.359 (26) .556 300 Benzene 0.003 (26) .003 10 b Heptane 0.003 (26) .006 400 m-Xylene 0.005 (26) .008 100 o-Xylene 0.003 (26) .004 100 p-Xylene 0.004 (26) .005 100 Toluene 0.006 (26) .012 50 a From Puhala et al. (1997) for base A where only JP-8 was used. b The Occupational Safety and Health Administration PEL for benzene is 1 ppm. during aircraft fuel tank entry and repair at 12 Air Force bases. They report that the highest 8-hr time-weighted average fuel exposure found was 1,304 mg/m3, and the highest 15-min short-term exposure was 10, 295 mg/m3. General Toxicological and Biological Parameters Lethality Several case studies have reported death following accidental ingestion of kerosene by children (reviewed in ATSDR 1998). The primary cause of death is respiratory effects (lipoidal pneumonia). The lowest dose of kerosene associated with death was 1,900 milligrams per kilogram (mg/kg) body weight by a 2-year-old child. Doses ranging from 120 to 870 mg/kg and as high as 1,700 mg/kg did not lead to death in children ranging from 10 months to 5 years old. No studies have reported death in humans associated with inhalation or dermal exposure to kerosene-based fuels. The acute oral lethal dose for 50% of the test animals (LD50) of JP-5 in rats (Bogo et al. 1983) and of kerosene in guinea pigs and rabbits (Deichmann et al. 1944) is greater than 10 grams (g) per kg.

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Page 121 Acute Studies Acute exposure to kerosene-based fuels, such as JP-8, has been associated with respiratory, cardiovascular, ocular, neurological, immunological, renal, and dermal effects. Those studies are briefly described below and are summarized in Table A-3. Human Studies After inhalation of JP-5 for approximately 1-hr, two individuals experienced mild hypertension, eye irritation, and neurological effects (e.g., coordination and concentration difficulties, fatigue, headache, apparent intoxication, anorexia) and one individual experienced nausea (Porter 1990). All symptoms subsided by 4 days (d) after exposure. The concentration of JP-5 was not known. Six volunteers exposed to kerosene vapor at 140 mg/m3 for 15 min did not experience any respiratory effects (Carpenter et al. 1976). Ingestion of kerosene by children and adults has been reported to cause pulmonary (e.g., pneumonia, bronchitis) and neurological (e.g., unconsciousness, semiconsciousness, drowsiness, restlessness, irritability) effects, tachycardia, cardiomegaly, vomiting, and increased leukocyte counts (reviewed in ATSDR 1998). Because in many cases ingestion of kerosene is accidental, the concentrations associated with specific effects are not reported. It has been estimated that respiratory distress will result from ingestion of 10-30 milliliters (mL) of kerosene (Zucker etal. 1986). Neurological effects (e.g., convulsions, coma) were observed in 2 of 78 children ingesting approximately 30 mL of kerosene; those effects were not observed in children ingesting from 3 to 20 mL. There are no studies assessing acute dermal exposure in humans. Experimental Animal Studies Respiratory effects, such as bronchoconstriction, were observed in rabbits and guinea pigs exposed by inhalation to kerosene (Casaco et al. 1982; Garcia et al. 1988). The rabbits were exposed to 32,500 mg/m3 for 4-9 min, and the guinea pigs were exposed to 20,400 mg/m3 for 5 min. A study exposing mice to 20 microliters (µL) of kerosene (the only dose tested) by aspiration reported that the animals showed

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Page 122 TABLE A-3 Experimental Animal Studies on the Toxicity of Kerosene and Kerosene-Based Fuels Study Type Fuel Type Species Exposure Concentration, Duration, Route Observed Effect Reference Acute Kerosene Rabbit 32,500 mg/m3, 4-9 min, inhalation (aerosol) Reduction in tidal volume and dynamic lung compliance, bronchoconstriction, increase in pulmonary resistance Casaco et al. 1982 Acute Kerosene Guinea pig 20,400 mg/m3, 5 min, inhalation (aerosol) Bronchoconstriction Garcia et al. 1988 Acute Kerosene Mouse 20 µL aspiration Pulmonary consolidation and hemorrhage, pneumonitis, decrease in pulmonary clearance of S. aureus, increase in relative lung weight, neurological effects including lack of coordination, drowsiness, behavioral changes Nouri et al. 1983 Acute Kerosene Guinea pig 3,200-8,000 mg/kg, gavage Mononuclear and polymorphonuclear cell infiltration and unspecified pathological lesion in the lungs J. Brown et al. 1974 Acute Kerosene Dog 0.5 mL/kg, aspiration Increases in arterial oxygen utilization, intrapulmonary physiological shunt fraction, respiratory rate. Decreases in arterial oxygen tension, heart rate, mean arterial blood pressure Goodwin et al. 1988

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Page 123 Acute Kerosene Rat 8,000 - 12,000 mg/kg, gavage Histopathological changes in kidneys (no change in kidney weight). Neurological effects such as unsteady gait and drowsiness observed at 12,000 mg/m3, but not 8,000 mg/m3. Muralidhara et al. 1982 Acute JP-5 Rat 18,912 mg/kg, gavage Hematological, hepatic, renal effects Parker et al. 1981 Acute JP-5 Rat 24-60 mL/kg, gavage Hepatic effects Bogo et al. 1983; Mehm and Feser 1984 Acute JP-5 Rat 19,200 mg/kg, gavage Renal effects such as hyaline droplets in cytoplasm of epithelial cells in proximal tubules; neurological effects such as reduction in food, water intake; dermal effects such as alopecia, congestion of the subcutis Bogo et al.1983 Acute JP-5 or JP-8 Rabbit 0.5 mL, dermal (undiluted fuel) No effects observed Schultz et al. 1981 Acute JP-8 Rabbit Dermal, abraded and intact skin Slight skin irritation Kinkead et al. 1984 Acute JP-5 Mouse Concentration not reported, dermal Dermatitis observed NTP/NIH 1986 Acute JP-5 Guinea pig 1% solution, dermal Mild dermal sensitization Cowan and Jenkins 1981a,b

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Page 124 Subchronic Kerosene Rat and dog 100 mg/m3, 6 hr/d, 5 d/wk for 13 wk, inhalation No respiratory, cardiovascular, gastrointestinal, hematological, musculoskeletal, hepatic, renal, body weight, neurological effects observed Carpenter et al. 1976 Repeated Kerosene Rat 1,100 mg/m3, 6 hr/d, 5 d/wk for 30 d, inhalation No hepatic effects observed Bogo et al. 1983 Repeated JP-8 Rat 497 mg/m3, 1 hr/d for 7 or 28 d, inhalation (nose only) Increased alveolar permeability USAF 1994; Chen et al. 1992 Repeated JP-8 Rat 500-1,100 mg/m3, 1 hr/d for 7, 28, 56 d, inhalation Lung epithelial permeability observed in rats exposed for 56 d USAF 1994; Hays et al. 1994 Repeated JP-8 Rat 950 mg/m3, 1 hr/d for 28 d, inhalation Disruption of epithelial and endothelial structures, convoluted airways, and alveoli filled with red blood cells and fluid USAF 1994; Pfaff et al. 1993 Repeated Kerosene Guinea pig 20,400-34,000 mg/m3, 15 min/d for 21 d, inhalation Cardiovascular effects (aortic plaques) observed Noa and Illnait 1987a,b

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Page 125 Subchronic JP-5 Rat and Dog 150-750 mg/m3, continuous exposure for 90 d, inhalation Female rats exposed at 150 or 750 mg/m3 and male rats exposed at 750 mg/m3 showed increased creatinine and blood urea nitrogen; dogs exposed at 750 mg/m3 had a slight (statistically significant) decrease in hemoglobin and red blood cell count, significant decreases in serum albumin, sporadic changes in blood urea nitrogen; dogs exposed at 150 or 750 mg/m3, showed hepatic effects such as lesions, mild cloudy swelling of hepatocytes USAF 1978b Repeated Kerosene Rat Average concentrations of 58 mg/m3 and 231 mg/m3, “intermediate duration exposure” inhalation At 58 mg/m3, animals had decreased blood glucose; at 231 mg/m3 had increased blood lactate, pyruvate Starek and Vojtisek 1986 Subchronic JP-5 Mouse 150 mg/m3, continuous exposure observed in the livers for 90 d, inhalation Vacuolization, hepatocellular fatty changes observed in the livers Gaworski et al. 1984 Subchronic JP-8 Rat and mouse 500 mg/m3 and 1,000 mg/m3, continuous exposure 90 d, inhalation Kidney lesion, α-2-microglobulin protein for droplet nephropathy, observed in male rats. No exposure-related increase in either sex of either species. Condition not considered relevant to humans Mattie et al. 1991

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Page 157 Peri- and postnatal Rat 1,800, 9,900, 64,400 ppm; 1 hr/d; days 17-20 pregnancy and 1-21 postpartum; inhalation (snout-only) No maternal or developmental effects observed except for a delay in F1 age at pinna detachment, eye opening and startle response at 64,400 ppm (~1/2-d delay). May be related to exposure Maternal: 64,400 Fetal: 9,900 Fetal: 64,400 Alexander et al. 1996 NOAEL, no-observed-adverse-effect level; LOAEL, lowest-observed-adverse-effect level; ppm, parts per million.

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Page 158 tive body weight gain in males exposed to 50,000 ppm over 5 wk or 10 wk was significantly reduced. There were no effects on body weight or weight gain in P females, or in F1 or F2 offspring. There appeared to be a slight increase in skeletal defects in F1 fetuses, but no significant change was reported, and some might have been cases of decreased ossification (this was unclear from the reported data). In F2 offspring, there was a slight but statistically significant increase in the age at pinna detachment (exposed at 10,000 ppm), startle response (exposed at 10,000 and 50,000 ppm), and air righting (exposed at 2,500 and 10,000 ppm); these changes were not clearly dose related, and on average they represented a ½- to 1-d delay. Because the F2 offspring were never exposed directly or indirectly, there was no change in body weight at birth or weaning, and no changes were seen in the F1 off-spring on these same measures; the changes detected in F2 animals were not considered treatment-related. As a follow-up to studies showing Leydig cell hyperplasia, Barton et al. (1994) exposed 25 male Sprague-Dawley rats per group to HFC-134a at 0, 10,000, 30,000, or 100,000 ppm for 6 hr/d for a total of 18 wk (11 wk before mating and 7 wk during and after mating). Animals were exposed snout-only for the first 9 wk to reduce the amount of material used; thereafter, whole-body exposure was used. In 10 males per group, luteinizing hormone concentrations were assessed after 16 wk, and again at 17 wk after stimulation with luteinizing hormone releasing hormone (LHRH). In another 10 males per group, at necropsy, the left testis was decapsulated, then incubated with human chorionic gonadotropin to assess androgen release; the right testis was examined histologically. High basal concentrations of luteinizing hormone were seen in all groups including controls, but there was no difference between controls and treated groups in luteinizing hormone levels before or after LHRH stimulation. At 100,000 ppm, there was no statistically significant increase in testosterone secretion and biosynthesis, but an increase in progesterone was observed. The increase in progesterone was consistent with increased Leydig cell function at this exposure level. Developmental Toxicity Studies Four developmental toxicity studies testing HFC-134a have been conducted in rats and rabbits ( Table A-5).

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Page 159 Lu and Staples (1981) exposed female Sprague-Dawley rats at concentrations of 0, 30,000, 100,000, or 500,000 ppm HFC-134a for 6 hr/d, days 5-14 of gestation (11 pregnant control animals, 6 in each exposed group). All gestational ages are converted to correspond to the day of insemination as gestational day 0. Animals were killed on gestational day 20 and uterine contents were examined. Dams exposed to 100,000 or 300,000 ppm showed a reduced or absent response to sound, respectively, demonstrating the anesthetic action of HFC-134a. Animals exposed to 300,000 ppm consumed significantly less food and gained significantly less weight than did controls. There was a significant decrease in fetal weight at 300,000 ppm, and a significant increase in the incidence of skeletal variations, many of which were related to reduced ossification. Hodge et al. (1979b) exposed female rats to concentrations of 0, 1,000, 10,000, or 50,000 ppm HFC-134a for 6 hr/d on gestation days 615 and killed them on gestation day 21 for examination of uterine contents (23-29 pregnant animals per group). There were no treatmentrelated effects on maternal animals except for acute pulmonary irritation that increased in severity and incidence with exposure concentration. There was no effect on the number of implants, litter size, or litter weight. At 50,000 ppm, fetal weight was slightly but significantly decreased and there was an increased incidence of skeletal variations, primarily reduced ossification of cervical vertebrae, sternebrae, and digits. An increase in the incidence of abnormal sternebrae also was reported in the 50,000 ppm group, but these effects (bipartite or misaligned sternebrae) are often seen in controls and are likely related to reduced ossification observed in the same groups. All fetal effects reported in this study at the highest exposures can be accounted for by the fact that litter size was greater in this group than in any other, including controls. Reduced fetal weight often is associated with increased litter size, and reduced ossification of skeletal elements often accompanies reduced fetal weight. Wickramaratne et al. (1989a,b) conducted a prenatal developmental toxicity study in rabbits in two phases. In the pilot study (Wickramaratne et al. 1989a), groups of artificially inseminated rabbits were exposed to 5,000, 20,000 or 50,000 ppm (six to nine pregnant animals per group) for 6 hr/d, on gestation days 6-18, then killed on gestation day 29. Fetuses were examined only for external anomalies and cleft palate. Two animals aborted late in pregnancy, one in the

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Page 160 5,000 ppm group and one in the 50,000 ppm group. Maternal body weight loss was observed during the early dosing period in the 50,000 ppm group. The number of implantations was reduced in the 20,000 and 50,000 ppm treated groups compared with controls, with consequent reduction in litter size, gravid uterine weight, and litter weight, but increased fetus weight due to reduced litter size. In the main study (Wickramaratne et al. 1989b; same study reported in Collins et al. 1995), pregnant rabbits (18-24 per dose group) were exposed 6 hr/d to 2,500, 10,000, and 40,000 ppm gestation days 6-18. Observations were the same as in the pilot study, but fetuses were examined for external, visceral, and skeletal defects, and a single section was made through the head to examine the brain macroscopically. One control animal aborted late in pregnancy. Maternal body weight and food consumption were signficantly reduced at 10,000 and 40,000 ppm, although the effects at 2,500 ppm were within the historical control range. There was no effect on implantation number, litter size, gravid uterine weight, litter weight, or individual fetus weight. The incidence of major and minor defects either did not appear to be dose related or was within historical control ranges. The NOAEL was considered to be 10,000 ppm, based on maternal toxicity, and the NOAEL for developmental toxicity was ≥40,000 ppm. In a peri- and postnatal study design, Alexander et al. (1996) exposed rats by snout only to HFC-134a at 0, 1,800, 9,900, or 64,400 ppm (1 hr/d). Pregnant females were exposed on gestation day 17-20, then from postnatal day 1-21. F1 pups were weaned at postnatal day 21, and one male and female per litter (20 litters per group) were selected and raised to maturity. F1 animals were mated at approximately 84 d of age, killed on gestation day 20, and the uterine contents were examined. F1 offspring were examined for physical and reflex development as well as locomotor coordination, activity, learning, memory, and reversal. There were no effects on any parameter measured except for a statistically significant delay in the age at pinna detachment, eye opening, and startle response in F1 offspring at 64,400 ppm (approximately half-day delay). Although there was no effect on body weight, these animals were exposed indirectly via the milk during the period when most of these measurements were made. Exposure concentrations and blood concentrations of the dams were somewhat higher in the peri- and postnatal study than in the fertility study reported at the

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Page 161 same time. These minor but statistically significant effects could be the result of the exposure to HFC-134a. Integration of Toxicity and Exposure Information Interpretation of Toxicity Data General Toxicity and Pharmacokinetics Data on the toxicity of HFC-134a indicate that it is nontoxic in most circumstances. Most of the changes reported have been at high concentrations, which also induce narcosis. There is no evidence of genetic toxicity for HFC-134a. The primary effect reported is the induction of Leydig cell hyperplasia and adenomas in male rats exposed at 50,000 ppm for 6 h/d, 5 d/wk over a 2-yr period (Collins et al. 1995; Hext and Parr-Dobrzanski 1993). The NOAEL of 10,000 ppm from these data was the basis of the EPA's RfC. It should be noted that in a two-generation study (Alexander et al. 1996), the NOAEL for adult toxicity was 10,000 ppm, based on significant changes in body weight in male rats exposed snout-only at a concentration of 50,000 ppm for 1 h/d, 5 d/wk for 18 wk. The difference in results might be attributed to the snout-only exposure protocol, which might have caused stress to the rats and affected their body weight in the Alexander et al. (1996) study. Data in two human volunteers from one study (Vinegar et al. 1997) indicate severe effects on vital signs after brief exposures at concentrations of 2,000-4,000 ppm HFC-134a by inhalation. However, in a study by Emmen and Hoogendijk (1999), human volunteers exposed whole body at concentrations as high as 8000 ppm for 1 hr did not show any effects of HFC-134a. Absorption of HFC-134a was very rapid and maximal concentrations were achieved within 15-30 min. Repeated exposures in animal studies do not result in accumulation because the half-life is so short. Given the rapid absorption and excretion in rats and humans, the kinetics appear to be similar between the species. Reproductive and Developmental Toxicity Interpretation of the experimental animal data described above is

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Page 162 complicated by the fact that two exposure regimens were used: 1 hr/d snout-only exposure, to mimic MDI exposures, and 6 hr/d whole-body exposure, to mimic environmental exposures. Developmental Toxicity. No data were found from studies of developmental toxicity of HFC-134a in humans. The experimental animal data are sufficient to conclude that HFC-134a does not cause prenatal developmental toxicity when pregnant animals were exposed (whole body) for 6 hr/d during major organogenesis to HFC-134a concentrations below those associated with narcosis (<30,000 ppm). The NOAEL for rat maternal toxicity was 30,000 ppm, whereas the NOAEL for rat prenatal toxicity was 50,000 ppm (Hodge et al. 1979a; Lu and Staples 1981). The NOAEL for rabbit maternal toxicity was 10,000 ppm, whereas the NOAEL for rabbit prenatal toxicity was greater than 40,000 ppm (Wickramaratne 1989a,b; Collins et al. 1995). The data are insufficient to determine the extent to which HFC-134a causes postnatal developmental effects, as the only study addressing this issue used 1 hr/d snout-only exposures for 10 wk before mating, throughout gestation to day 20, and from postnatal days 1-21 (fertility study), or from gestation day 17-20 and postnatal days 1-21 (peri- and postnatal study); exposure to pups was not continued beyond postnatal day 21 (Alexander et al. 1996). In these studies, the adult NOAEL was 10,000 ppm (LOAEL, 50,000 ppm) in the fertility study and greater than 64,400 ppm in the peri- and postnatal study. The developmental NOAEL was greater than 50,000 ppm in the fertility study and 9,900 ppm (LOAEL = 64,400 ppm) in the peri- and postnatal study. The effects at 64,400 ppm in the peri- and postnatal study were minimal and might not be related to exposure, so 50,000 ppm is assumed to be the NOAEL for postnatal effects for snout-only exposure for 1 hr/d. The data are insufficient to determine what the extent and types of effects would be with continued exposure to pups after weaning and into the F2 generation. The experimental animal data are assumed relevant to humans. Reproductive Toxicity. No data were located from studies of the reproductive toxicity of HFC-134a in humans. The animal data were sufficient to show that HFC-134a exposures similar to those metered-dose inhalers did not affect fertility and sexual function. Although there was an effect of exposure to 50,000 ppm on male body weight gain in the fertility study mentioned above (Alexander et al. 1996), fertility and reproductive function were not affected.

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Page 163 The data were sufficient to show that whole-body exposure to HFC-134a for 6 hr/d for 2 yr could affect the testis. Although a dominant lethal study showed no effects at concentrations as high as 50,000 ppm (Hodge et al. 1979a), chronic exposure of rats at 50,000 ppm HFC-134a resulted in a statistically significant increase in the incidence of Leydig cell hyperplasia and adenoma (Hext and Parr-Dobrzanski 1993; Collins et al. 1995). The NOAEL was 10,000 ppm for a 6 hr/d exposure. In a follow-up study to Hext and Parr-Dobrzanski (1993), Barton et al. (1994) showed that HFC-134a (6 hr/d, 18 wk) increased the synthesis and release of testosterone and progesterone from testes incubated with human chorionic gonadotropin but did not alter the qualitative aspects of androgen biosynthesis. The changes in testosterone and progesterone secretion were consistent with the increased Leydig cell activity in the chronic study. Given the significant increase in Leydig cell hyperplasia and adenoma in rats, EPA considered these effects adverse and based the RfC for HFC-134a on this effect. The experimental animal data are assumed relevant to humans, because the data are inadequate to show that the effects are irrelevant. Default Assumptions The data on Leydig cell hyperplasia and adenoma are assumed to be relevant to humans. Likewise, the lack of developmental toxicity is also assumed to be relevant to humans. The pharmacokinetics in humans and animals are very similar, as is the lack of toxicity from acute exposures, notwithstanding the data from the two subjects reported by Vinegar et al. (1997). Because of the similarities in human and animal pharmacokinetics, this portion of the interspecies uncertainty factor can be reduced from 10 to 3. The intraspecies uncertainty factor of 10 should be retained because of the unusual findings in the Vinegar et al. (1997) study. A database deficiency factor of 10 should be applied due to the lack of a 6 hr/d exposure in a multigeneration study with exposure continuing throughout the two generations, as well as lack of a developmental neurotoxicity study with a 6 hr/d exposure. It appears that there is little or no effect of HFC-134a in any of the studies reviewed except at doses that induce narcosis, but additional data with longer exposure durations and additional endpoints are needed to confirm this observation.

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Page 164 Quantitative Evaluation In the two-generation study by Alexander et al. (1996), no adverse reproductive or developmental effects were observed in rats exposed at a concentration of 50,000 ppm for 1 hr/d during gametogenesis and mating (males), or gametogenesis, mating, pregnancy, and lactation (females). Since the effects at 64,400 ppm (exposed for 1 hr/d) in the peri- and postnatal study (Alexander et al. 1996) were minimal, and the next lower dose was 9,900 ppm, the subcommittee has identified 50,000 ppm as the NOAEL for reproductive and developmental effects for less than a chronic exposure. The NOAEL of 50,000 ppm for a 1 hr/d exposure converted to a 6 hr/d exposure is 8,300 ppm. The UEL for reproductive and developmental effects is then calculated using the adjusted NOAEL of 8,300 ppm divided by an aggregate uncertainty factor of 300 (3 for interspecies extrapolation, 10 for intraspecies differences, and 10 for deficiencies in the database). Thus, the UEL for reproductive and developmental toxicity is ~ enlarge ~ For chronic exposure, the subcommittee chose the NOAEL of 10,000 ppm for a 6-hr/d, 2-yr exposure based on a significant increase in the incidence of Leydig cell hyperplasia in treated rats (Collins et al. 1995; Hext and Parr-Dobrzanski 1993). Applying an aggregate uncertainty factor of 300 (3 for interspecies extrapolation, 10 for intraspecies differences, and 10 for deficiencies in the database), the UEL for reproductive and developmental toxicity is ~ enlarge ~ The chronic exposure UEL is higher than the EPA's chronic RfC of 80 mg/m3, which is based on the same study and endpoints (see above). There are several differences in the way the UEL was derived compared with the RfC. To calculate the chronic RfC, EPA derived a BMC10 of 11,030 ppm and adjusted it from a 6-hr/d, 5-d/wk exposure to a continuous exposure by multiplying by 6/24 hr and 5/7 d. A total

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Page 165 uncertainty factor of 100 was applied to the BMC10: 3 for interspecies extrapolation, 10 to protect sensitive individuals, and 3 for a database deficiency of a two-generation study. The NOAEL of 10,000 ppm was used as the basis for the chronic UEL and the UEL was not adjusted because it was developed for an occupational exposure scenario. A total uncertainty factor of 300 was applied to the NOAEL: 3 for interspecies extrapolation, 10 to protect sensitive individuals, and 10 for database deficiencies due to the lack of both a two-generation study and a developmental neurotoxicity study. The latter concern was raised by the Alexander et al. (1996) peri- and postnatal study that suggested the possibility of developmental neurotoxicity in offspring of animals exposed for 1 hr/d, 5 d/wk during late gestation and lactation. Had the BMC10 derived by EPA been used in the calculation, the final value would have been slightly higher (i.e., 37 ppm or 155 mg/m3). Critical Data Needs A two-generation reproduction study is needed with at least 6 hr/d exposure continuing to pups after weaning and into the F2 generation to determine the effects of long-term exposures. Developmental neurotoxicity endpoints should be included in this study based on the types of effects seen in the peri- and postnatal study with the snout-only 1 hr/d exposure. Summary HFCs, including HFC-134a, have been developed as an alternative to CFCs, which are known to contribute to the breakdown of ozone to oxygen in the stratosphere. HFCs do not contribute to the destruction of stratospheric ozone, but some HFCs have global warming potential. They primarily serve as replacements for CFCs in refrigeration equipment and mobile air conditioning; they also have pharmaceutical applications (e.g., as propellants for metered-dose inhalers used to treat asthma).

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Page 166 Human Exposure Human exposure to HFC-134a occurs via inhalation from accidental leaks of air conditioning units and refrigerators, from spills or industrial use, and from use of metered-dose inhalers such as those that deliver medication for the treatment of asthma. Toxicology Developmental Toxicity There are no human data on the effects of HFC-134a on development. The animal data are sufficient to support a conclusion that exposures to HFC-134a does not cause prenatal developmental toxicity when pregnant animals are exposed for 6 hr/d during major organogenesis to concentrations of HFC-134a below those associated with narcosis (less than 30,000 ppm). HFC-134a also did not cause peri- and postnatal effects in rats with snout-only exposure for 1hr/d during gestation days 17-20 and postnatal days 1-21 at 50,000 ppm. However, the data are insufficient to determine the extent to which HFC-134a caused postnatal developmental effects including endpoints of developmental neurotoxicity, as there are no studies with at least a 6 hr/d exposure throughout two generations. These toxicity findings are assumed to be relevant for the prediction of risk to humans. Reproductive Toxicity There are no human data on the effects of HFC-134a on male or female reproduction. The animal data are sufficient to conclude that HFC-134a exposures similar to those used in MDIs will not cause affect sexual function and fertility. There was no effect on dominant lethality after treatment of males and mating with unexposed females. However, data are insufficient to determine the effects of whole-body exposure for 6 hr/d, as a multigeneration study with exposure continuing through two generations is not available. Chronic exposure of rats at concentrations as high as 50,000 ppm resulted in a significant in-

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Page 167 crease in the incidence of Leydig cell hyperplasia. These toxicity findings are assumed to be relevant for the prediction of human risk. Quantitative Evaluation Reproductiveand Developmental Toxicity There are no human data from which to develop a quantitative evaluation. Laboratory studies in rats identified a NOAEL of 50,000 ppm for reproductive and developmental effects for a less than chronic exposure. The NOAEL of 50,000 ppm was adjusted to convert from a 1hr/d exposure to a 6hr/d exposure; the adjusted NOAEL is 8,300 ppm. Dividing the adjusted NOAEL by an aggregate uncertainty factor of 300 (3 for interspecies extrapolation, 10 for intraindividual differences, and 10 for an incomplete database), the UEL for reproductive and developmental toxicity for less than a chronic exposure is 28 ppm. A similar UEL is calculated for chronic exposures. Laboratory studies in rats identified a NOAEL of 10,000 ppm based on a significant increase in the incidence of Leydig cell hyperplasia. Applying an aggregate uncertainty factor of 300 (3 for interspecies extrapolation, 10 for intraindividual differences, and 10 for an incomplete database), the UEL for reproductive and developmental toxicity for a chronic exposure is 33.3 ppm. Certainty of Judgment and Data Needs Data on the toxicity and disposition of HFC-134a suggest that it is a compound with little or no reproductive or developmental toxicity except at very high exposure concentrations that induce narcosis. However, data are needed from postnatal evaluations and from a multigeneration study with 6 hr/d exposure. The similarities in pharmacokinetics between humans and laboratory animals provide confidence that the data are relevant for predicting human risk.