4
2,6-Di-tert-butyl-4-nitrophenol

This chapter summarizes the relevant epidemiologic and toxicologic studies on 2,6-di-tert-butyl-4-nitrophenol (DBNP). Selected chemical and physical properties and toxicokinetic and mechanistic data are also presented. Because of the lack of available data, the committee was unable to recommend exposure guidance levels for DBNP. A discussion of the research needed to allow derivation of exposure guidance levels is provided at the end of this chapter.

PHYSICAL AND CHEMICAL PROPERTIES

DBNP is a yellow crystalline powder that is relatively soluble in organic solvents but insoluble in water (Alexander et al. 2001). Selected chemical and physical properties are listed in Table 4-1.

OCCURRENCE AND USE

A yellow substance was observed in the U.S. submarine fleet, especially in newer boats, in builders’ yards and during sea trials (MacMahon et al. 1999). Investigations during 1992-1993 by the U.S. Navy and the Electric Boat Division of General Dynamics Inc. determined that the yellow substance was DBNP, which is derived from an antioxidant, 2,6,-di-tert-butylphenol (DBP), in TEP 2190 steam-turbine lubricating oil (a synthetic turbine oil MILSPEC-L-17331). Analysis of samples of TEP 2190 lubricating oil found that it contained DBP at less than 10,000 ppm (Alexander et al. 2001). Release of small amounts of lubricating oil during operation of the steam turbine allows DBP to come into contact with electrostatic precipitators in air-handling systems that can nitrate DBP and thereby generate DBNP (Alexander et al. 2001). In 1993, the specification for TEP 2190 lubricating oil was changed by the Naval Sea Systems Command



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4 2,6-Di-tert-butyl-4-nitrophenol This chapter summarizes the relevant epidemiologic and toxicologic stud- ies on 2,6-di-tert-butyl-4-nitrophenol (DBNP). Selected chemical and physical properties and toxicokinetic and mechanistic data are also presented. Because of the lack of available data, the committee was unable to recommend exposure guidance levels for DBNP. A discussion of the research needed to allow deriva- tion of exposure guidance levels is provided at the end of this chapter. PHYSICAL AND CHEMICAL PROPERTIES DBNP is a yellow crystalline powder that is relatively soluble in organic solvents but insoluble in water (Alexander et al. 2001). Selected chemical and physical properties are listed in Table 4-1. OCCURRENCE AND USE A yellow substance was observed in the U.S. submarine fleet, especially in newer boats, in builders’ yards and during sea trials (MacMahon et al. 1999). Investigations during 1992-1993 by the U.S. Navy and the Electric Boat Divi- sion of General Dynamics Inc. determined that the yellow substance was DBNP, which is derived from an antioxidant, 2,6,-di-tert-butylphenol (DBP), in TEP 2190 steam-turbine lubricating oil (a synthetic turbine oil MILSPEC-L-17331). Analysis of samples of TEP 2190 lubricating oil found that it contained DBP at less than 10,000 ppm (Alexander et al. 2001). Release of small amounts of lu- bricating oil during operation of the steam turbine allows DBP to come into con- tact with electrostatic precipitators in air-handling systems that can nitrate DBP and thereby generate DBNP (Alexander et al. 2001). In 1993, the specification for TEP 2190 lubricating oil was changed by the Naval Sea Systems Command 88

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89 2,6-Di-tert-butyl-4-nitrophenol TABLE 4-1 Physical and Chemical Properties of 2,6-Di-tert-butyl-4- nitrophenol Synonyms 2,6-Di-t-butyl-4-nitrophenol; dibutyl-4-nitrophenol; di-tert-butyl- 4-nitrophenol; di-tertiary-butyl-4-nitrophenol; dibutyl-p- nitrophenol; dibutyl-p-nitrophenol; di-t-butyl-p-nitrophenol CAS registry number 728-40-5 Molecular formula C14H21NO3 Molecular weight 251 Boiling point NA Melting point 157.0ºC Flash point NA Explosive limits NA Specific gravity NA Vapor pressure NA Solubility NA 1 ppm = 10.27 mg/m3; 1 mg/m3 = 0.09741 ppm Conversion factors Abbreviations: NA, not available or not applicable. Source: Data on molecular weight and melting point from Alexander et al. 2001. to limit the amount of DBP in TEP 2190 oil to no more than 10 ppm (Still et al. 2005). However, the remaining stockpile of DBP-containing TEP 2190 lubricat- ing oil in inventory is being used to supply the fleet for a number of years (MacMahon et al. 1999). Still et al. (2005) indicate that exposure to DBNP may also occur in many submarines because of the mixing of “old” and “new” TEP 2190 oil in submarine turbine systems and storage tanks. Exposures to DBNP are otherwise limited. DBNP was proposed as a miticide for treatment of resis- tant mite infections in mammals (Vesselinovitch et al. 1961), but there is no indication that it was ever used for this purpose. SUMMARY OF TOXICITY Toxicity information on DBNP is limited to animal studies in as much as no case reports, experimental-exposure studies, or epidemiologic studies result- ing from human exposure to it are available. The toxicologic database on DBNP primarily includes acute-exposure studies and a few repeated-dose studies. No adequate data from inhalation-toxicity, subchronic toxicity, mutagenicity, car- cinogenicity, or reproductive-toxicity studies are available. After single or repeated oral or intraperitoneal (ip) doses, rats show clini- cal signs (prostration, rapid breathing, hyperthermia, and rapid induction of rigor mortis after death) that are consistent with inhibition of mitochondrial oxidative metabolism (Alexander et al. 2001; Carpenter et al. 1997). A single oral dose of 40 mg/kg in dimethyl sulfoxide (DMSO) and canola oil is sufficient to induce

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90 Exposure Guidance Levels for Selected Submarine Contaminants hyperthermia and death in male rats (Still et al. 2005). However, the toxicity of DBNP appears to be significantly affected by the solvent used as the vehicle in toxicity studies (Vesselinovitch et al. 1961; Alexander et al. 2001; Still et al. 2005). Animals that survive acute exposure to DBNP are generally reported to appear clinically normal 24 h after exposure, although decreased weight gain may persist after recovery from near-lethal exposure (Alexander et al. 2001). Histopathologic changes, typically cellular degeneration, have been reported in smooth muscle, skeletal muscle, cardiac muscle, liver, kidneys, and spleen after exposure to DBNP by gavage or ip injection (Vesselinovitch et al. 1961; MacMahon et al. 1999; Alexander et al. 2001). Repeated ip doses have been reported to cause enlargement of the liver and changes in cellular enzyme levels (Carpenter et al. 1997). Skin contact with DBNP does not result in visible signs of dermal irritation or injury even at high doses, and absorption of DBNP through the skin is minimal (Vesselinovitch et al. 1961; Alexander et al. 2001; Inman et al. 2003; Pershing et al. 2006). However, when DBNP at 2 g/kg was held in contact with the skin for 24 h, two of five rabbits showed changes in weight gain but no other clinical signs of toxicity (Alexander et al. 2001). After gavage, DBNP is poorly absorbed and slowly excreted in the urine as a glu- curonide conjugate (Holder et al. 1971; Carpenter et al. 1997). Enterohepatic recirculation may play a role in the low clearance rate of DBNP and its glu- curonide metabolite (Holder et al. 1971; Carpenter et al. 1997). The slow elimi- nation of DBNP is reported to result in evidence of cumulative toxicity after repeated oral and ip dosing (Holder et al. 1971; Vesselinovitch et al. 1961). In vitro studies with isolated mitochondria have identified deficits in stage 3 and stage 4 oxidative metabolism after exposure to DBNP (Carpenter et al. 1997). The in vitro findings are consistent with the clinical signs associated with DBNP exposure and suggest that DBNP is an inhibitor of mitochondrial respiration. The toxicity profile of DBNP is similar to that of 2,4-dinitrophenol (2,4-DNP), a well-recognized inhibitor of mitochondrial respiration (ATSDR 1995). Effects in Humans Accidental Exposures No information on accidental exposures was identified. Experimental Studies No information on experimental studies was identified. Occupational and Epidemiologic Studies In 1992-1993, as noted above, U.S. Navy submarine crews noticed a yel- lowing of bulkheads and other structures during underwater periods and sea tri-

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91 2,6-Di-tert-butyl-4-nitrophenol als. The yellowing was determined to be due to the presence of DBNP (Alexan- der et al. 2001). Crew members also reported that their skin turned yellow when they came into contact with contaminated surfaces. Deposition of the yellow material was visible on structural elements of the submarine interior and most pronounced in the engineering compartment; Alexander et al. (2001) indicated that DBNP was most likely present on clothing, bedding, eating utensils, and other surfaces in submarines because DBNP was distributed throughout the submarine by the ventilation system. Analysis of data collected by a Navy con- tractor indicated that airborne DBNP concentrations in submarines using the “old” TEP oil were less than 3.0 to 13 ppb 24-h/day for 90-day operation peri- ods (Alexander et al. 2001). Laboratory simulation of the submarine operational environment using the “old” TEP oil reported DBNP up to 122 ppb (Alexander et al. 2001). No data have been reported on whether DBNP exposure affects the health of exposed crew members. The highest DBNP surface concentration documented in submarines using the “old” TEP oil is 0.2 µg/cm2 (J. McDougal, personal communication cited in Inman et al. 2003). Although there are no reports of human toxicity associated with DBNP exposure, DBNP has toxicologic similarities in animal studies to 2,4-DNP, a dinitrophenol on which there are human data. A 1919 study and a 1946 study reported respiratory difficulty and deaths after inhalation exposure to 2,4-DNP in the occupational environment (Perkins 1919; Gisclard and Woodward 1946, both as cited in ATSDR 1995). However, no quantitative exposure data were associated with those incidents, and there was also dermal, and possibly oral, exposure to 2,4-DNP in the working environment where the cases occurred (ATSDR 1995). There are no other reports of human toxicity associated with inhalation of 2,4-DNP (ATSDR 1995). The primary database on human effects associated with 2,4-DNP stems from the oral administration of 2,4-DNP as a weight-loss medication in the 1930s. Ingestion of 2,4-DNP has been associated with agranulocytosis, cataracts, peripheral neuropathy, and serious dermatologic effects in humans (ATSDR 1995). On the basis of those human exposures, the U.S. Environmental Protection Agency (EPA) established an oral reference dose of 0.002 mg/kg per day for 2,4-DNP by the application of a safety factor of 1,000 to a lowest observed-adverse-effect level (LOAEL) of 2 mg/kg per day, a dose that caused cataracts in humans (EPA 2005). Humans appear to be more susceptible to the development of cataracts than other mammals exposed to 2,4- DNP; among other animals cataracts have been induced only in birds after 2,4- DNP administration (ATSDR 1995). No inhalation reference concentration has been calculated for inhalation exposure to 2,4-DNP because of the lack of ade- quate data (EPA 2005). Effects in Animals The oral toxicity of DBNP appears to be significantly affected by the type of vehicle used to create the solutions administered. DBNP is not soluble in wa-

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92 Exposure Guidance Levels for Selected Submarine Contaminants ter but is soluble in common organic solvents (Vesselinovitch et al. 1961). As described below, the toxicity of DBNP in aqueous vehicles is less than that re- ported in studies conducted with organic solvents. The purity of the DBNP used in toxicity studies may be another confounding factor in interpreting the data from them. The studies reported by Vesselinovitch et al. (1961) did not identify the source, method of manufacture, or purity of the DBNP they used. The stud- ies undertaken by Carpenter et al. (1997), MacMahon et al. (1999), Alexander et al. (2001), Inman et al. (2003), and Still et al. (2005) have used material made with a method described by Rivera-Nevares et al. (1995), and the purity of the DBNP used in these studies is reported to be 97-99.5%, the primary impurity being DBP at concentrations of 1-3%. Acute Toxicity The oral LD50 of DBNP in male and female Sprague-Dawley rats was re- ported to be 500 and 450 mg/kg, respectively (Vesselinovitch et al. 1961) when it was given by gavage in a 0.2% aqueous carboxymethylcellulose solution. At lethal doses, rats showed general depression of activity starting 3 h after dosing; deaths occurred 4 h to 3 days after dosing. The oral LD50 value in male guinea pigs was reported to be 800 mg/kg (Vesselinovitch et al. 1961). MacMahon et al. (1998, as cited in MacMahon et al. 1999) reported that the oral LD50 of DBNP given to male F-344 rats in a corn-oil vehicle was 82 mg/kg, and the no-observed-adverse-effect level (NOAEL) was less than 50 mg/kg. Effects were hyperthermia (as determined by measurement in the ear canal) and mild histopathologic degenerative changes in multiple organs (skele- tal-, cardiac-, and smooth-muscle degeneration and minor hepatic- and renal-cell degeneration). In a study of Sprague-Dawley rats given DBNP in corn oil, the oral LD50 was 93 mg/kg (MacMahon et al. 1999). Survivors at each dose were all five at 0 mg/kg, all five at 62.5 mg/kg, four of five at 78 mg/kg, two of five at 98 mg/kg, none of five at 250 mg/kg. Clinical abnormalities at all doses included prostra- tion, hyperthermia, and labored respiration. Survivors recovered clinically (nor- mothermic) within 24 h after dosing, although rats dosed at 98 mg/kg initially lost body weight and grew at a lower rate than animals in the other groups. Histopathologic changes in animals that died after exposure at 250 mg/kg in- cluded minimal degeneration of skeletal-, cardiac- and smooth-muscle fibers with occasional contraction bands and minimal degenerative changes in individ- ual hepatocytes and renal tubule epithelial cells. The NOAEL was less than 62.5 mg/kg. The difference in LD50 values between the study of Vesselinovitch et al. (1961) and the studies of MacMahon et al. (1998, as cited in MacMahon et al. 1999) and MacMahon et al. (1999) were hypothesized to be due to differences in rat strain and the vehicle used to administer the test substances (Alexander et al. 2001).

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93 2,6-Di-tert-butyl-4-nitrophenol Alexander et al. (2001) conducted a series of exposures to address the dif- ferences observed in the Vesselinovitch et al. and MacMahon et al. studies. Those studies involved groups of five male or female F-344 rats given single oral doses of DBNP in a corn-oil vehicle (500, 275, 100, 50, or 0 mg/kg in males and 450, 275, 100, 50, or 0 mg/kg in females) and male Sprague-Dawley rats given single oral doses of DBNP in corn oil (250, 98, 78, 62.5, or 0 mg/kg) or in a 2% aqueous carboxymethylcellulose solution (DBNP at 98 mg/kg). The maximal observation period for all exposure groups was 14 days. Mortality was 100% in groups given doses of at least 250 mg/kg. Mortality was 20-80% in groups given 78-100 mg/kg. Mortality was zero in groups given 50 or 62.5 mg/kg. Maximal time to death was 4 h, 41 min for F-344 rats and 22 h, 25 min for Sprague-Dawley rats; death occurred sooner at the higher doses. Mortality was 60% in Sprague-Dawley rats given DBNP at 98 mg/kg in corn oil and 20% in rats given DBNP at 98 mg/kg in carboxymethylcellulose. Prostration, rapid breathing, increased body temperature, and muscle rigor were observed in all DBNP-exposed groups whether the animals died or survived. At 500 mg/kg, male F-344 rats convulsed before death. On histologic examination, mild, multi- focal myofiber degeneration was observed in skeletal and cardiac muscle from F-344 rats given DBNP at 100-500 mg/kg. “Less significant changes” were ob- served in “muscle and other cell types” from F-344 rats given 50 mg/kg. Among Sprague-Dawley rats given DBNP at 275 mg/kg in corn oil, hepatic and pulmo- nary congestion were common findings. Histologic examination of these Spra- gue-Dawley animals showed degenerative and necrotic changes in individual hepatocytes and renal tubule epithelial cells. Weight loss was greater in DBNP- dosed animals than in controls. Although control animals returned to normal weight gain after gavage dosing, the DBNP animals did not return to normal weight gain during the 14-day observation period. The oral LD50s calculated separately in the male F-344 rats and Sprague-Dawley rats were 80 mg/kg. The oral LD50 in the female F-344 rats was 50-100 mg/kg, or close to 80 mg/kg. Still et al. (2005) reported giving male Sprague-Dawley rats single oral doses of DBNP at 15 or 40 mg/kg, which had been prepared by first dissolving it in DMSO and then adding it to canola oil. A small amount of 14C-labeled DBNP was included in the mixture. Six of 16 of the rats dosed at 40 mg/kg died within 24 h of dosing. Necropsy examination of animals that died after DBNP dosing revealed edema or congestion of the thoracic cavity and lung hemorrhages. Animals that survived 40 mg/kg showed prostration, no auditory-startle re- sponse, reduced locomotor activity, and muscular rigidity for up to 8 days after dosing. After dosing at 15 mg/kg, rats exhibited lethargy and reduced startle response during the first 24-48 h. They were indistinguishable from controls 7-8 days after dosing. The ip LD50s in guinea pigs and mice were reported to be 580 mg/kg in males and 700 and 850 mg/kg for males and females, respectively (Vesselino- vitch et al. 1961). The ip LD50s in male and female Sprague-Dawley rats were reported to be 270 and 260 mg/kg, respectively (Vesselinovitch et al. 1961). Vesselinovitch et al (1961) also reported that rats given single lethal ip doses of

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94 Exposure Guidance Levels for Selected Submarine Contaminants DBNP (300, 400, or 600 mg/kg) had histologic lesions in the heart (patchy, waxy degeneration of muscle fibers), liver (mild to moderate fatty change), spleen (lymphorrhexis, abortive mitosis, and other cellular changes), and kid- neys (zonal necrosis of renal tubule epithelium). In rats given 130 mg/kg ip, renal lesions were accompanied by increased blood urea nitrogen, decreased urinary concentrating capacity, decreased urinary calcium, and decreased blood pressure (Vesselinovitch et al. 1961). Intravenous (iv) administration of DBNP to rodents (species unstated) at 3.3 mg/kg in DMSO caused a marked increase in body temperature and death within 1 h, followed by the rapid onset of rigor mortis (Rivera-Nevares et al. 1995). The authors of the iv study considered the signs observed after DBNP administration to be consistent with depletion of ATP stores due to disruption of mitochondrial function. Application of 1,000 mg/kg to the shaved backs of rats resulted in no sys- temic toxicity and no evidence of dermal irritation (Vesselinovitch et al. 1961). Five New Zealand white rabbits had DBNP applied directly to their shaved backs at 2,000 mg/kg; a control group was available for comparison (Alexander et al. 2001). The DBNP was held in place with a bandage for 24 h, and the ani- mals were observed for abnormalities for 14 days. The rabbits showed no ad- verse clinical signs resulting from the exposure. On necropsy, no treatment- related lesions were reported, and histologic analysis of the DBNP-contact skin was not affected by contact with DBNP. Two of five DBNP rabbits showed sta- tistically significant changes in weight gain compared with the control group. DBNP was below the limit of detection in “the blood and the organs” of exposed rabbits. Repeated Exposure and Subchronic Toxicity Vesselinovitch et al. (1961) fed groups of six male and six female Spra- gue-Dawley rats diets containing 0, 0.05%, 0.1%, 0.2%, or 0.4% DBNP for up to 16 weeks. Deaths were zero, zero, zero, six, and 12, respectively. Feed con- sumption and body weight were reduced in the surviving animals in the 0.2% group, but not in animals at the lower doses. Vesselinovitch et al. (1961) reported studies in which groups of five fe- male Sprague-Dawley rats were dosed ip with DBNP at 0, 10, 25, 50, or 100 mg/kg in 0.2% carboxymethylcellulose daily for up to 60 days. Deaths were zero, zero, two, five, and five, respectively and occurred on days 5-30 in the 25- and 50-mg/kg groups and on days 0-5 in the 100-mg/kg group. At 10 mg/kg, animals grew normally. At 25 mg/kg, the animals maintained their starting body weight but did not gain weight. Carpenter et al. (1997) dosed groups of four male F-344 rats ip with DBNP at 0 or 10 mg/kg in DMSO for 10 days. Rats were monitored for body- weight gain, water consumption, urinary and fecal output, and behavior. Growth rate and urinary production were reported to be unaffected by DBNP. Presuma-

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95 2,6-Di-tert-butyl-4-nitrophenol bly, water consumption and behavior were unaffected as well, although they were not mentioned. Repeated ip injections of DBNP at 25 mg/kg for at least 58 days resulted in no significant inhibition of body-weight gain, but increased liver weight, in- creased the liver-to-body weight ratio, decreased the concentration of hepatic fatty acid binding protein (54% when measured as mg per gram liver or 17% when measured as mg per 100 g body weight), increased the concentration of bile acid sulfotransferase (BST) (71% when measured as mg per 100 g body weight, but no increase when measured as mg per g liver), and increased the concentration of dopamine sulfotransferase (31% when measured as mg per g liver or 28% when measured as mg per 100 g body weight) (Carpenter et al. 1997). Chromatographic analysis of hepatic BST revealed most of the BST in the DBNP rats was BST I, a typical pattern for female rats, whereas control rats showed the male profile for BST, which is three isofunctional BSTs. The change in BST suggests that DBNP exposure resulted in an altered expression of this endocrine-modulated enzyme. Chronic Toxicity No chronic-toxicity information was identified. Reproductive Toxicity in Males No reproductive-toxicity information was identified. However, the EPA Endocrine Disruptor Research Initiative includes a project to evaluate DBNP for endocrine effects, including testicular function (Still 2006). Still et al. (2005) found no 14C-DBNP or 14C-labeled metabolites in the testes of male rats after oral dosing with DBNP at 15 or 40 mg/kg. Still et al. (2005) suggested that the blood-testis barrier may prevent exposure of the testes to DBNP and its metabo- lites at the doses tested. Immunotoxicity No information on immunotoxicity was identified. Genotoxicity No genotoxicity studies of DBNP were identified. In general, activation of aromatic nitro-containing compounds requires metabolism to an aromatic hydroxylamine intermediate and eventual formation of highly reactive interme- diates, such as the nitrenium ion, that can interact with DNA (Takahashi et al. 1978). Intestinal reduction by gut microflora is involved in forming reactive intermediates from some nitro-containing compounds. Neither of the metabolic

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96 Exposure Guidance Levels for Selected Submarine Contaminants studies reported to date identified formation of DNA-reactive intermediates from DBNP (Holder et al. 1971; Carpenter et al. 1997). Carcinogenicity No documentation of any U.S. federal or federal advisory group review of DBNP for carcinogenicity was identified. The above discussion on genotoxicity is pertinent to consideration of DBNP for carcinogenicity. TOXICOKINETIC AND MECHANISTIC CONSIDERATIONS Holder et al. (1971) found that 28.1% of a 20-mg oral dose of 14C-DBNP (randomly labeled in tert-butyl groups and given in carboxymethylcellulose) was excreted unchanged in a methanol extract of feces collected from Wistar rats (body weight 250-390 g) over a 5-day period. Maximal excretion was ob- served 48 h after dosing and ceased by 72 h. A dose of 1.0 mg 14C-DBNP orally resulted in excretion of about 33% and 20% of the radiolabel in the urine and feces, respectively, after 5 days. When rats were pretreated with neomycin to reduce microflora in the gut, excretion rates were altered to 23% and 34% in the urine and feces, respectively. Bile contained only small amounts of radiolabel (1.4%) whether or not rats received neomycin. After ip administration, about 60% of a dose (0.192 mg or 1.0 mg) of 14C-DBNP was eliminated in the urine; and biliary excretion amounted to about 30% of the dose after ip (0.192 mg) or iv (0.096 mg) administration in an aqueous ethanol solution. Overall, the data indicate that DBNP is slowly absorbed from the gastrointestinal tract with a large amount of it unchanged in the feces after oral dosing. Absorption through the gut after oral administration was about 50% of the dose. Metabolism of DBNP by the gut microflora appears to aid absorption: excretion of unabsorbed 14 C-DBNP in the feces increased after neomycin treatment. Failure to detect unchanged DBNP in the urine or bile indicates that once DBNP is absorbed, it is excreted only after metabolism. Still et al. (2005) found that a single dose of DBNP at 15 mg/kg contain- ing 1.5 µCi of 14C-DBNP in DMSO and canola oil resulted in a significant in- crease in radioactivity counts after 24 h in the fat > liver > kidneys > heart > lungs > brain > striated muscle > spleen. Six days after dosing, radioactivity remained increased in liver > brain > fat > kidneys > heart; at 10 days after dos- ing only the liver continued to show a significant increase in radioactivity. 14C in the blood peaked 7.5 h after dosing and was cleared from the blood by 24 h after dosing. 14C in the urine peaked at 96 h and decreased incrementally thereafter. Some 23% of the oral dose was excreted in the feces within the first 24 h, and 54% was excreted in 96 h. An additional 3% of the radiolabel was excreted over the next 72 h. The authors suggested that DBNP or its metabolites may be able to accumulate in the body after continuous or repeated exposures.

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97 2,6-Di-tert-butyl-4-nitrophenol Carpenter et al. (1997) studied several biochemical measures associated with exposure of F-344 rats to DBNP. Ring-labeled 14C-DBNP reached peak blood concentrations in 5-10 min when given to male F-344 rats by ip injection (91.2 µg). Oral administration of a similar dose resulted in peak blood 14C- DBNP in about 1 h. The rapid phase of clearance that occurs in the first hour after ip dosing is followed by a pseudosteady state that continues for a week and results in the removal of 4-6% of the radiolabel from the blood. The rapid phase is due to distribution of DBNP to body tissues and elimination in the urine and feces. Within 24 h after ip dosing with DBNP at 0.4 mg/kg, 20% and 12% of the dose is excreted in the urine and feces, respectively, and tissue distribution in- cludes the liver (14-16%), spleen (3-5%), kidneys (8-10%), heart (2-5%), brain (0.8-1.2%), muscle (0.5-1%), fat (11-13%), and blood (6-8%). DBNP is slowly excreted from the body: 82-90% is excreted in the urine and feces within 10 days after a single ip dose of 0.4 mg/kg. Urinary and fecal excretion is 18-20% and 12-15%, respectively, during the first 2 days after dosing and decreases con- siderably thereafter. A single metabolite isolated from the urine, bile, and feces was a glucuronide conjugate of DBNP; no parent DBNP was isolated from these media, and no metabolite with a reduced nitro group was found. Those results are in agreement with those of Holder et al. (1971) and indicate that, once ab- sorbed, DBNP is excreted after phase II metabolism as a glucuronide conjugate. Enterohepatic circulation of DBNP glucuronide is likely to contribute to the low rate of elimination of DBNP. As described above, five New Zealand white rabbits had DBNP at 2,000 mg/kg applied directly to their shaved backs for 24 h. DBNP was below the limit of detection in “the blood and the organs” of exposed rabbits (Alexander et al. 2001). In vitro dermal absorption studies using rat skin also found very little ab- sorption of DBNP (J. McDougal, personal communication cited in Inman et al. 2003). Using isolated perfused porcine skin flaps exposed to 14C-DBNP, Inman et al. (2003) found that among several exposure scenarios (40.0 µg/cm2 in 100% ethanol, 40.0 µg/cm2 in 85% ethanol and 15% water, 4.0 µg/cm2 in 100% etha- nol, and 4.0 µg/cm2 in 85% ethanol and 15% water) the highest absorption measured was 1.08%. The highest mass of 14C-DBNP absorbed was 0.5 µg. That most of the 14C-DBNP applied was found on the surface of the skin where it was applied indicates very low skin absorption of DBNP. Disposition and uptake-elimination profiles of a single dose of DBNP were quantified by using human skin grafted onto athymic nude mice (Pershing et al. 2006). DBNP was measured in samples of stratum corneum, epidermis, and dermis after exposure for 0.5, 1, 2, 4, 8, and 24 h. The Cmax (maximal con- centration) of DBNP in the stratum corneum was 1,663 " 602 µg/cm3. The Cmax in the epidermis and dermis was about 0.03 and 0.003 times that, respectively. Tmax (time to maximal concentration) occurred at 0.5-1.0 h in the three com- partments. Over a 24-h interval, the greatest amount of DBNP remained in the stratum corneum, and elimination half-lives were 9-11 h in the three layers.

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98 Exposure Guidance Levels for Selected Submarine Contaminants DBNP was quickly absorbed into the stratum corneum, but overall absorption across the skin graft was minimal. DBNP-dosed animals have a clinical appearance (hyperthermia, rigor of skeletal and cardiac muscle, convulsions, and weight loss) that is shared by ani- mals dosed with dinitrophenols, such as 2,4-DNP, and other agents that act as uncouplers of oxidative metabolism (Alexander et al. 2001). On the basis of quantum-mechanics calculations, Rivera-Nevares et al. (1995) suggested that DBNP might be second only to 3,5-di-tert-butyl-4-hydroxybenzylidene maloni- trile (the most potent known uncoupler of oxidative phosphorylation) as an un- coupler of ATP. However, in vitro data reported by Carpenter et al. (1997) found DBNP to be one-third less potent than 2,4-DNP as an inhibitor of mito- chondrial respiration. Other in vitro studies have explored relative effects of exposure to DBNP on protein synthesis, mitochondrial respiration, and cell toxicity and species differences in these end points. When liver slices were used, human tissue was less sensitive than rat tissue to DBNP (Carpenter et al. 1997). End-point suscep- tibility in both species was ATP content > protein synthesis > LDH release > K+ leakage. At 50 µM DBNP, the ATP content of rat liver slices was reduced by 70% compared with a 30% reduction in human liver slices. At the same concen- tration, protein synthesis was reduced by 60% in rat liver slices and 30% in hu- man tissue. When hepatocytes (WP-344 cells) were cultured in the presence of DBNP, loss of cellular viability was 8, 12, or 100 % after a 24-h exposure at 0.1, 0.2, or 2 µg/mL. Isolated rat liver mitochondria showed reduction in both stage 3 (at 200 or 300 nmol) and stage 4 (at 1, 2, or 5 nmol) respiration. INHALATION EXPOSURE LEVELS FROM THE NATIONAL RESEARCH COUNCIL AND OTHER ORGANIZATIONS The occurrence of DBNP in the submarine environment is due to the unin- tended release of DBP from submarine steam-turbine systems that use TEP 2190 as a lubricating oil. The nitration of DBP by the electrostatic precipitator in the submarine air-handling system results in the formation of DBNP. DBNP has no known industrial or commercial use and is not reported to have natural sources. There are no published recommendations for inhalation exposure to DBNP from other national or international bodies. COMMITTEE RECOMMENDATIONS Contact with DBNP in the submarine environment potentially involves oral, dermal, and inhalation exposure. Significant dermal absorption of DBNP appears to be unlikely: studies reported by Vesselinovitch et al. (1961), Alexan- der et al. (2001), Inman et al. (2003), and Pershing et al. (2006) indicate that DBNP is absorbed to only a minor extent, if at all, through the intact skin. Al- though Alexander et al. (2001) reported reduced weight gain in a proportion of

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99 2,6-Di-tert-butyl-4-nitrophenol the animals exposed at 2,000 mg/kg under occlusion for 24 h, it is unlikely that crew members would encounter such high exposure, because the maximal sur- face concentration determined in a submarine environment is reported to be 0.2 µg/cm2 (J. McDougal, personal communication cited in Inman et al. 2003). Oral exposure to DBNP is considered possible, but the degree of exposure by this route will depend to some extent on personal and environmental hygiene in the submarine. With the small amount of information available, it is not pos- sible to estimate DBNP exposure by the oral route reliably. Because the DBP in TEP 2190 oil has been reduced from no more than 10,000 ppm to no more than 10 ppm (Alexander et al. 2001; Still et al. 2005), the surface contamination lev- els in submarines that have converted to using TEP 2190 oil manufactured with reduced DBP levels are likely to be significantly lower than the highest deter- mined before the current (post-1993) TEP oil MILSPEC went into effect, 0.2 µg/cm2. There are no reports of the study of inhalation exposure to DBNP. Air- borne DBNP in submarines using “old” TEP oil has been measured at less than 3.0 to 13 ppb 24 h/day for 90-day operation periods (Alexander et al. 2001). Laboratory simulation of the submarine operational environment reported DBNP concentrations from “old” TEP oil as high as 122 ppb (Alexander et al. 2001). The routes of exposure used in animal studies that could be a basis for cal- culation of EEGL and CEGL values are oral, ip, and iv administration. The ad- verse clinical signs in rats associated with ip, iv, and oral administration of DBNP are similar. None of the iv studies include sufficient detail for deriving EEGL and CEGL values. Available data (and their limitations) from oral and ip studies considered in deriving EEGL and CEGL values are discussed in the fol- lowing paragraphs. Exposure to DBNP at acutely toxic doses results in prostration, rapid breathing, hyperthermia, and rapid induction of rigor mortis after death—signs that are consistent with inhibition of mitochondrial oxidative metabolism (Alex- ander et al. 2001; Carpenter et al. 1997). The lowest lethal single dose of DBNP reported is 40 mg/kg given by oral gavage. The oral LD50s in rats given DBNP vary widely. Much of the variability may be attributable to the use of aqueous vs organic solvent vehicles, inasmuch as some studies with aqueous vehicles report larger LD50s. However, rat strain and DBNP purity may also be sources of vari- ability in the determination of LD50s. Histopathologic changes, primarily cell degeneration, have been described in skeletal muscle, cardiac muscle, smooth muscle, liver, kidneys, spleen, and lungs of rats that died after lethal exposures to DBNP. Residual signs in animals that survive the acute phase of toxicity at near lethal doses are typically reported to include reduction in body weight gain. In vitro studies of DBNP have demonstrated that the compound has the ability to uncouple oxidative phosphorylation; this is a property shared with 2,4- DNP (ATSDR 1995). Clinical signs and histopathologic lesions associated with DBNP and 2,4-DNP are very similar; the male rat oral LD50s of DBNP (50 mg/kg) and 2,4-DNP (38 mg/kg) are also similar (ATSDR 1995). Significant

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100 Exposure Guidance Levels for Selected Submarine Contaminants differences between the two chemicals are the induction of cataracts and periph- eral neuropathy by 2,4-DNP but not by DBNP. However, those difference may be due to inadequacies in the testing of DBNP or the use of test species (rats, mice, guinea pigs, and rabbits) insensitive to the development of chemically induced cataracts. None of the studies conducted with DBNP have been of suffi- cient duration to rule out the possibility that DBNP may induce peripheral neu- ropathy similar to that induced by 2,4-DNP. No studies have demonstrated a convincing NOAEL or LOAEL of acute oral exposure to DBNP. The lowest LOAEL of a single dose of DBNP is 15 mg/kg given to male rats in a study reported by Still et al. (2005). After dosing with DBNP, rats exhibited lethargy and reduced startle response during the first 24-48 h; recovery appeared complete within 7-8 days, when test animals were regarded as indistinguishable from controls. The presence of central nervous system (CNS) effects in rats given DBNP at 15 mg/kg correlates with the pres- ence of 14C label derived from DBNP in the brain for the first 24 h after dosing (Still et al. 2005). The reduction of acoustic-startle response in the rats given 15 mg/kg is an indicator that CNS performance may be seriously impaired at this exposure. Most of the reports identify ip DBNP at 25 mg/kg as toxic in rats (Vesselinovitch et al. 1961; Carpenter et al. 1997). Repeated ip doses of 10 mg/kg are reported to be without effect on mortality and body weight for 60 days of exposure. Rats have also been fed DBNP at doses at about 25 and 50 mg/kg per day for 16 weeks without effect on mortality or body weight (Vesselinovitch et al. 1961). However, none of the data were considered to be adequate for use in recommending EEGL or CEGL values. The Navy’s Bureau of Medicine and Surgery (BUMED) has attempted to set allowable exposure limits for DBNP in the submarine environment (Alexan- der et al. 2001). However, inadequacies of the DBNP database were so great as to preclude the development of an inhalation reference value. Recognizing the deficiencies, Alexander et al. (2001) calculated a range of maximal allowable DBNP exposure levels for submarine crews by applying safety factors to the rat oral LD50 of 80 mg/kg and LOAEL of 50 mg/kg. On the basis of the application of safety factors of 1,000 and 100,000 to the rat LD50, they calculated a maximal allowable atmospheric concentration of 27.3-0.273 ppb. Application of the same safety factors to the LOAEL resulted in the calculation of a maximal allowable atmospheric concentrations of 17.53-0.1753 ppb. Alexander et al. (2001) com- pared the data with the measured and simulated concentrations of DBNP in the submarine atmosphere, which were less than 3.0 ppb to 122 ppb, and concluded that more research was necessary to clarify the risk associated with DBNP. If the maximal allowable atmospheric concentrations by Alexander et al. (2001) were recalculated with the more recent data reported by Still et al. (2005) in which effects were seen at 15 mg/kg, the recalculated maximal allowable concentra- tions would be lower than those identified in Alexander et al. (2001).

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101 2,6-Di-tert-butyl-4-nitrophenol Because of the inadequacy of the DBNP database, the committee agrees with Alexander et al. (2001) regarding the need for further information and is unable to recommend EEGL and CEGL values for DBNP exposure. DATA ADEQUACY AND RESEARCH NEEDS The U.S. Navy is reported to have reduced the DBP in TEP 2190 lubrica- tion oil to less than 10 ppm. Presumably, the reduction has led to a substantial reduction in the potential exposure of DBNP in the submarine environment. However, no document substantiating that presumption was made available to the committee. The animal-toxicity database available for assessment of hazard and risk includes only single or repeat-dose studies, primarily via the oral route, with a small number of end points assessed. The committee considered deriving exposure guidance levels on the basis of noninhalation exposure routes, but there were insufficient data to support the route-to-route extrapolation. Further- more, the overall database available for determining EEGL and CEGL values is small, and many of the data points are conflicting. Thus, much uncertainty is associated with any attempt to estimate exposure guidance levels for this com- pound. The committee recommends that, at a minimum, a short-term inhalation study be conducted that looks at a comprehensive set of end points before an EEGL or CEGL value is established. REFERENCES Alexander, W.K., G.B. Briggs, K.R. Still, W.W. Jederberg, K. MacMahon, W.H. Baker, and C. Mackerer. 2001. Toxicity of 2,6-di-tert-butyl-4-nitrophenol (DBNP). Appl. Occup. Environ. Hyg. 16(4):487-495. ATSDR (Agency for Toxic Substances and Disease Registry). 1995. Toxicological Pro- file for Dinitrophenols. Agency for Toxic Substances and Disease Registry, Public Health Service, U.S. Department of Health and Human Services, Atlanta, GA. Au- gust 1995 [online]. Available: http://www.atsdr.cdc.gov/toxprofiles/tp64.html [ac- cessed June 8, 2007]. Carpenter, R.L., T.K. Narayanan, A.E. Jung, S. Prues, and K.R. Still. 1997. Characteriza- tion of the Metabolism, Distribution and Toxicity of 2,6-di-t-butyl-4-nitrophenol for Purposes of Health Hazard Assessment. Technical Report NMRL-97-39. Naval Medical Research Institute, Wright-Patterson Air Force Base, OH. EPA (U.S. Environmental Protection Agency). 2005. 2,4-Dinitrophenol (CASRN 51-28- 5). Integrated Risk Information System, U.S. Environmental Protection Agency [online]. Available: http://www.epa.gov/iris/subst/0152.htm [accessed June 8, 2007]. Gisclard, J.B., and M.M. Woodward. 1946. 2,4-Dinitrophenol poisoning: A case report. J. Ind. Hyg. Toxicol 28(2):47-51. Holder, G.M., A.J. Ryan, T.R. Watson, and L.I. Wiebe. 1971. A note on the excretion of 2,6-di-tert-butyl-4-nitrophenol in the rat. Food Cosmet. Toxicol. 9(4):531-535. Inman, A.O., K.R. Still, W.W. Jederberg, R.L. Carpenter, J.E. Riviere, J.D. Brooks, and N.A. Monteiro-Riviere. 2003. Percutaneous absorption of 2,6-di-tert-butyl-4-

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102 Exposure Guidance Levels for Selected Submarine Contaminants nitrophenol (DBNP) in isolated perfused porcine skin. Toxicol. In Vitro 17(3):289- 292. MacMahon, K.L., W.H. Baker, W.K. Alexander, W.W. Jederberg, K.R. Still, G.B. Briggs, and R,J, Godfrey. 1998. Acute Oral and Dermal Toxicity Evaluation of 2,6-dibutyl-4-nitrophenol. Technical report AFRL/HEST- TR-1998. U.S. Air Force Research Laboratory, Wright-Patterson Air Force Base, OH. MacMahon, K.L., W.H. Baker, W.K. Alexander, K.R. Still, and G.B. Briggs. 1999. Acute Oral Toxicity Evaluation of 2,6-dibutyl-4-nitrophenol in Male Sprague- Dawley Rats. Technical Report AFRL-HE-WP-TR-1999-0173. U.S. Air Force Re- search Laboratory, Wright-Patterson Air Force Base, OH. Perkins, R.G. 1919. A study of the munitions intoxications in France. Public Health Rep 34:2335-2374. Pershing, L.K., J.L. Nelson, J.L. Corlett, G.B. Briggs, K.R. Still, and W.W. Jederberg. 2006. Disposition and pharmacokinetics of a lubricant contaminant, 2,6-di-tert-4- nitrophenol, in grafted human skin. J. Appl. Toxicol. 26(5):402-409. Rivera-Nevares, J.A., J.F. Wyman, D.L. von Minden, N. Lacy, M.L. Chabinyc, A.V. Fratini, and D.A. Macys. 1995. Facile synthesis and physical and spectral charac- teristics of 2,6-di-tert-butyl-4-nitrophenol (DBNP): A potentially powerful uncou- pler of oxidative phosphorylation. Environ. Toxicol. Chem. 14(2):251-256. Still, K.R. 2006. The Evaluation of DBNP for Endocrine Effects. EDRI Federal Project Inventory [online]. Available: http://www.epa.gov/endocrine/inventory/DOD- 018.html [accessed June 11, 2007]. Still, K.R., A.E. Jung, G.D. Ritchie, W.W. Jederberg, E.R. Wilfong, G.B. Briggs, and D.P. Arfsten. 2005. Disposition of 2,6-di-tert-butyl-4-nitrophenol (DBNP), a sub- marine atmosphere contaminant, in male Sprague-Dawley rats. Environ. Res. 98(3):363-367. Takahashi, K., Y. Kawazoe, M. Tada, N. Ito, and M. Okada. 1978. Carcinogenicity of 4- nitrosoquinoline 1-oxide and its possible role in carcinogenesis by 4-nitroquinoline 1-oxide. Gann 69(4):499-505. Vesselinovitch, D., K.P. Dubois, F.W. Fitch, and J. Doull. 1961. Mammalian toxicity and histopathologic effects of 2,6-dibutyl-4-nitrophenol. Toxicol. Appl. Pharmacol. 3:713-725.