Derivation of a chronic reference concentration for decalin

Regulatory Toxicology and Pharmacology 66 (2013) 38–46

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Derivation of a chronic reference concentration for decalin Leah D. Stuchal a,⇑, Roxana E. Weil a, Stephen M. Roberts a,b a b

Center for Environmental and Human Toxicology, Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32611, USA Department of Environmental and Global Health, College of Public Health and Health Professions, University of Florida, Gainesville, Florida 32611, USA

a r t i c l e

i n f o

Article history: Received 21 August 2012 Available online 28 February 2013 Keywords: Decalin Decahydronaphthalene Toxicity Reference concentration Point of departure Benchmark dose

a b s t r a c t Decalin is found naturally in crude oil and as a product of combustion. It is used commercially as a solvent due to its ability to solubilize oils and fats. Despite its widespread occurrence in consumer products and the environment that lead to inhalation exposures, an inhalation toxicity value is not currently available for decalin. To derive a reference concentration (RfC) for decalin, inhalation toxicity studies were reviewed using a weight-of-evidence approach. A 2-year mouse inhalation study was chosen as the critical study for the derivation of the chronic RfC. Benchmark dose modeling was utilized to derive a point of departure for hepatic necrosis, syncytial alteration, eosinophilic focus, and erythrophagocytosis. A BMDL10 of 44 mg/m3 was modeled for the most sensitive adverse effect, syncytial alteration. A chronic RfC for decalin of 0.08 mg/m3 was calculated by conversion of the BMDL10 to a human equivalent continuous inhalation dose of 7.9 mg/m3 and application of a total uncertainty factor of 100. Future research is needed to better characterize the toxicity associated with the chronic inhalation of decalin and refine the development of toxicity values. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction 1.1. Background Decalin (decahydronaphthalene; CAS# 91-17-8) is a dicycloalkane found naturally in crude oil and coal (Sikkema et al., 1995). It is produced and released to the environment during combustion (e.g., natural fires, gasoline engines, cigarette smoke), petroleum refining, and coal tar distillation (NTP, 1992, 2005)1 Decalin is used as a solvent for fats, oils, waxes and resins and can also be found as a thinner in paints and varnishes (O’Neil et al., 2006; Browning, 1965). Due to its ability to thin and solubilize oils, fats, and resins, it is utilized in oil paints, shoe polish, floor waxes, cleaning fluids, gasoline, and lubricating oils (NTP, 1992; Gaworski et al., 1985; Elliott et al., 1966). Decalin is also used commercially for cleaning machinery ⇑ Corresponding author. Fax: +1 352 392 4707. E-mail address: lstuchal@ufl.edu (L.D. Stuchal). BCF – bioconcentration factor; BMD – benchmark dose; BMDL – 95% lower confidence limit on the BMD; BMDL10 – BMDL at the 10% effect level; BMDLHEC – human equivalent concentration of the BMDL; DAF – dosimetric adjustment factor; HSDB – Hazardous Substances Data Bank; LC50 – concentration lethal to 50% of the population; LOAEL – lowest observed adverse effect level; NOAEL – no observable adverse effect level; NTP – National Toxicology Program; OEHHA – Office of Environmental Health Hazard Assessment; POD – point of departure; RfC – reference concentration; UF – uncertainty factor; UFA – UF for the extrapolation of animal data to humans; UFD – UF for insufficiencies in the toxicity database; UFH – UF for interindividual variability/sensitivity in humans; UFL – UF for extrapolation from a LOAEL to a NOAEL; UFS – UF for extrapolation from subchronic to chronic exposure; US EPA – United States Environmental Protection Agency. 1

0273-2300/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yrtph.2013.02.011

and as a fuel in submarine-launched cruise missile systems (NTP, 1992). Due to its wide variety of industrial applications, decalin is produced in the millions of pounds annually. Commercially available decalin is usually a mixture of two diastereomers, cis- and transdecalin (O’Neil et al., 2006). It is a colorless liquid, primarily synthesized through the complete hydrogenation of naphthalene using glacial acetic acid in the presence of a platinum catalyst at 15–25 °C and 130 atm (HSDB, 2004; NTP, 1992). This process yields a mixture of approximately 77% cis- and 23% trans-decalin. However, the percentage of each isomer varies depending on the product and manufacturer (O’Neil et al., 2006; NTP, 1992). Decalin can also be obtained through hydrogenation of tetralin under the same conditions, a process which yields almost entirely cis-decalin (NTP, 1992). The use of decalin in a number of consumer products and motor lubricants as well as fuel leads to unintentional release in the environment through point and non-point sources. Although environmental concentrations of this compound have not been reported, evidence suggests that decalin contamination may be widespread in the environment. Decalin has been detected on a number of occasions, such as in effluent released in the Gulf of Mexico from the Bucanner Gas and Oil Field, as a contaminant in drinking water in Tuscaloosa, Alabama and in the coastal water of Narragansett Bay, Rhode Island (HSDB, 2004; Middleditch, 1982). Decalin has been identified in car exhaust fumes from samples collected in the Allegheny Mountain Tunnel of the Pennsylvania Turnpike and in emissions obtained from unvented kerosene space heaters

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(Traynor et al., 1990; Hampton et al., 1982). Decalin is also continuously released into the environment in effluent from petroleum refining and coal tar distillation and in the exhaust from the combustion of gasoline and diesel fuels (HSDB, 2004; NTP, 1992). Given the prevalence of decalin in the environment, likelihood of exposure of the public to this compound by the inhalation route is high (NTP, 1992). Therefore, this paper focuses on the toxicity of decalin by inhalation.

than 2262 mg/m3). Female rats had higher blood concentrations than males at all exposure concentrations, suggesting that the male rats were more efficient at eliminating decalin from the blood. This finding is consistent with males having higher concentrations of decalin metabolites in the kidneys. Decalin is metabolized to decalone and retained in the kidneys bound to a2uglobin protein. No sex differences were noted in mice, consistent with the absence of a2u-globin protein.

1.2. Environmental fate

3. Epidemiological data

Decalin has no or slight mobility in organic soil and sediment based on an estimated Koc of 4,600 (HSDB, 2004). Decalin volatilizes rapidly from soil and sediment with low organic content and from most environmental waters, but volatilization decreases as the organic content of the soil and sediment increase (HSDB, 2004; Baxter, 2001). Because decalin is lipophilic, it is a concern for bioaccumulation in the aquatic environment. An 6-week study using orange–red killifish (Oryzias latipes) resulted in bioconcentration factors (BCFs) of 839–2,400 for the cis-isomer and BCF’s of 1,300–2,510 for the trans-isomer, suggesting a very high potential to bioconcentrate in aquatic organisms (HSDB, 2004). Direct photolysis does not occur because of the lack of absorption of UV light above 290 nm by cyclic alkanes. Additionally, decalin lacks functional groups that hydrolyze under environmental conditions (HSDB, 2004). Because decalin does not undergo hydrolysis or photolysis in the environment, volatilization is an important fate process for terrestrial and aquatic habitats. In ambient air, decalin exists as a vapor and is degraded by reaction with photochemically produced hydroxyl radicals. The half-life of decalin in air is 19 h (HSDB, 2004; Baxter, 2001).

Systemic toxicity of decalin in humans has not been well defined and no serious industrial poisonings have been reported (Browning, 1965). Workers exposed to a mixture of decalin and tetralin have reported brownish-green urine (Sandmeyer, 1981). Decalin may cause defatting and dermatitis when in direct contact with the skin while inhalation can cause pulmonary effects such as irritation of mucous membranes, edema, pneumonitis, and hemorrhage. One case report of a man using decalin to clean paving stones described vesicular eczema and pruritus on the forearms and the sacral region, which were in closest contact with decalin. The same person also developed the presence of small amounts of albumin and urobilin as well as a few leukocytes in the urine suggesting that the kidneys may be affected (NTP, 1992; Browning, 1965). The lowest observed adverse effect level (LOAEL) for inhalation exposure in humans is 565 mg/m3 causing irritation of mucous membranes. Acute exposures to high air concentrations caused numbness, nausea, headache and vomiting (Patty, 1963). Classification concerning respiratory sensitization is not possible because data are not available for this endpoint. 4. Animal studies

2. Absorption, distribution, metabolism, and excretion No information is available on the absorption of decalin. However, like most hydrocarbons, it is believed to be well absorbed through both the oral and dermal routes. Decalin metabolism in humans has not been characterized, but a few studies have examined its metabolism in rabbits and rats. cis- and trans-Decalin orally administered to female rabbits showed that 60% of the hydrocarbons were excreted as ether-linked glucuronides in urine. Both isomers were oxidized to racemic secondary alcohols. cisDecalin was primarily metabolized to (±)-cis,cis-2-decalol and small amounts of cis,trans-2-decalol, whereas trans-decalin was metabolized to mainly (±)-trans,cis-2-decalol and trans–trans-2decalol (Elliott et al., 1966). Similar metabolites were also found in rats given an oral dose of cis- and trans-decalin (Olson et al., 1986). Interestingly however, slight differences were observed in metabolism between male and female Fischer-344 rats. The metabolites obtained from treatment with cis-decalin were cis,trans-1-decalol and cis,cis-2-decalol in both males and females, whereas the males also produced cis,cis1-decalol. Treatment with trans-decalin resulted in the production of trans,cis-2-decalol in both sexes, and trans,trans-1-decalol in males. Extracts obtained from kidneys of male rats contained cis2-decalone and trans-2-decalone, metabolites not found in the females (Olson et al., 1986). The 2-decalone metabolites of decalin were also observed in the kidneys of male F344/N rats treated with cis- and trans-decalin by the intravenous route, with no detectable levels in the females (Dill et al., 2003b). In rats, the metabolites were eliminated in urine in both the glucuronidated and sulfated forms (HSDB, 2004). A study by Dill et al. (2003b) found differences in decalin elimination between sexes and species. Mice were more efficient than rats at eliminating decalin at lower exposure concentrations (less

4.1. Non-cancer studies 4.1.1. Acute inhalation exposures The acute effects of decalin have been studied in different animal species (Table 1). In a study using six rats and an unknown number of mice, the 4-h LC50 of decalin was reported as 2827 mg/m3 for the rat and 5615 mg/m3 for the mouse (Baxter, 2001). Acute effects of decalin have been reported to include cataracts in guinea pigs exposed to vapors (Gosselin et al., 1984) and kidney and liver damage in guinea pigs exposed via the oral and inhalation routes (Browning, 1965). Three guinea pigs exposed to 1804 mg/m3 for 8 h per day died within 3 days. Necropsy revealed lung congestion, kidney injury, and liver injury (Baxter, 2001). 4.1.2. Subchronic inhalation exposures Several subchronic studies have evaluated the effects of decalin by the inhalation exposure route. These studies predominantly used male and female rats, with a few studies also using mice as model organisms (Table 2). Local effects in the lung were not reported as a result of subchronic or chronic exposures to decalin by inhalation. The NTP (2005) exposed male and female F344 rats, male NBR rats, and male and female B6C3F1 mice (five of each animal and sex per dose) to 0, 141, 283, 565, 1131, or 2262 mg/m3 decalin in exposure chambers for 6 h per day, 5 days per week, over 2 weeks. Liver weights of male and female rats (both strains) Table 1 Acute inhalation toxicity studies for decalin. Species

Number

4-h LD50 (mg/m3)

Reference

Rat Mouse

6 NA

2827 5615

Baxter (2001) Baxter (2001)

NA – not available.

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Table 2 Subchronic and chronic non-cancer inhalation toxicity studies for decalin. Species/sex

Duration

Doses

F344 rats/male and female

2 weeks

0, 141, 283, 565, 1131, or 2262 mg/ m3

NBR rats/male

2 weeks

B6C3F1 mice/male and female

2 weeks

0, 141, 283, 565, 1131, or 2262 mg/m3 0, 141, 283, 565, 1131, or 2262 mg/ m3

F344 rats/male and female

13 weeks

0, 141, 283, 565, 1131, or 2262 mg/ m3

Beagle dogs/male and female

90 days

0, 28, or 283 mg/m3

C57BL/6 mice/ female

90 days

0, 28, or 283 mg/m3

F344 rats/male and female

90 days

0, 28, or 283 mg/m3

B6C3F1 mice/male and female

14 weeks

F344 rats/male and female

2 years

B6C3F1 mice/male and female

105 weeks

0, 141, 283, 565, 1131, or 2262 mg/ m3 0, 141, 283, 565, or 2262 mg/m3 [M]; 0, 141, 565, or 2262 mg/m3 [F] 0, 141, 565, or 2262 mg/m3

Results

LOAEL

NOAEL

Reference

565 mg/m :  Significantly increased liver weight [M]: relevance uncertain 283 mg/m3:  Significantly increased kidney weight [M]  Significant increases in the incidence of granular casts of the renal medulla [M] 141 mg/m3:  Significantly increased liver weight [F]: relevance uncertain  Minimal to marked hyaline droplet accumulation [M]  Minimal to mild degeneration and regeneration of renal cortical tubules [M] 565 mg/m3:  Significantly increased liver weight: relevance uncertain

141 mg/m3 [M] None [F]

None [M] 2262 mg/m3 [F]

NTP (2005)

None

2262 mg/m3

NTP (2005)

1131 mg/m3:  Significantly increase liver weight [M]: relevance uncertain 565 mg/m3:  Significantly increase liver weight [F]: relevance uncertain 2262 mg/m3:  Significantly increased relative and absolute liver weight [F]: relevance uncertain 1131 mg/m3:  Significantly increased absolute and relative liver weight [M]  Significantly increased absolute kidney weight [M] 283 mg/m3:  Significantly increased relative kidney weight [M] No adverse effect observed

None [M&F]

2262 mg/m3 [M&F]

NTP (2005)

283 mg/m3 [M] None [F]

141 mg/m3 [M] 2262 mg/m3 [F]

Dill et al. (2003a)

None [M&F]

283 mg/m3 [M&F]

Gaworski et al. (1985)

283 mg/m3:  Post exposure significantly increased incidence of fatty liver changes: relevance uncertain  Post exposure significantly increased perivascular cuffing in the lungs: relevance uncertain  Post exposure significant increase in the incidence of mammary cysts: relevance uncertain  Post exposure increase in pituitary carcinomas: significance uncertain 28 mg/m3:  Significant increase in liver vacuolization, reversible  Post exposure significant increase in crystals and macrophages in the lungs: relevance uncertain  Post exposure significant increase in thyroid hyperplasia: relevance uncertain 283 mg/m3:  Significant increase in absolute liver weight, reversible [F]  Post exposure significant increase in CPN [F]: relevance uncertain 28 mg/m3:  Increase in the severity and incidence of nephrosis [M]  Significant increase in absolute liver weight, reversible [M]  Post exposure significant increase in CPN, mineralization, and papillary hyperplasia in the kidney [M]: relevance uncertain 1131 mg/m3:  Significant increase in liver weight [M&F]  Significant increase in centrilobular cytomegaly [M]

None

283 mg/m3

Gaworski et al. (1985)

28 mg/m3 [M] None [F]

None [M] 283 mg/m3 [F]

Gaworski et al. (1985)

1131 mg/m3 [M&F]

565 mg/m3 [M&F]

NTP (2005)

141 mg/m3:  Significant increase in renal papilla mineralization [M]  Significant increase in renal tubular hyperplasia [M]: relevance uncertain

141 mg/m3 [M] None [F]

None [M] 2262 mg/m3 [F]

Dill et al. (2003a)

2262 mg/m3:  Significant increase in centrilobular hypertrophy, necrosis, eosinophilic focus, and erythrophagocytosis [M] 565 mg/m3:  Significant increase in syncytial alteration [M]

2262 mg/m3 [M] None [F]

141 mg/m3 [M] 2262 mg/m3 [F]

NTP (2005)

3

M – male, F – female, CPN – chronic progressive nephrosis.

were significantly increased at exposures of 565 mg/m3 and greater. Liver weights of male and female B6C3F1 mice were increased at exposures of 1131 mg/m3 and greater. There were no accompany-

ing histopathologic changes in the liver of either species. Kidney weights and incidence of granular casts of the renal medulla were significantly increased for male F344 rats at exposures of

L.D. Stuchal et al. / Regulatory Toxicology and Pharmacology 66 (2013) 38–46

283 mg/m3 and greater. Dill et al. (2003a) exposed 20 male and female F344 rats per group to 0, 141, 283, 565, 1131, or 2262 mg/m3 decalin via whole-body inhalation for 6 h per day, 5 days per week, over 13 weeks. Females showed an absolute and relative increase in liver weight in the highest dose group. In male rats, decalin exposures at 1131 mg/m3 and higher increased absolute and relative liver and kidney weights. Doses of 283 mg/m3 and higher resulted in an increase in relative kidney weight, nephrotoxic lesions associated with accumulation of hyaline droplets in the cortical tubules, and granular casts in the medullary tubules. The females showed no significant nephrotoxicity. A 90-day continuous inhalation study (Bruner and Pitts, 1983; Gaworski et al., 1985) exposed three male and three female beagle dogs, 150 female C57BL/6 mice, and 75 male and 75 female F344 rats to 0, 28, or 283 mg/m3 decalin in inhalation chambers. Twothirds of the rodents were kept for study of delayed effects and were sacrificed 24 months after the exposure. The beagle dogs did not show any toxicity attributable to decalin inhalation. The female mice displayed a significant increase in reversible liver vacuolization at both decalin concentrations. The higher concentration significantly increased the incidences of fatty liver changes. Due to the reversibility of the fatty liver changes and lack of other signs of liver damage, it is unclear if the fatty liver change is a compensatory mechanism for decalin exposure. Twenty-four months post exposure, significant increases were seen in thyroid hyperplasia at both doses and in perivascular cuffing in the lungs, mammary gland cysts, thyroid cysts, and pituitary carcinoma at the highest dose. The authors attributed the significant increase in pituitary carcinoma to low occurrence rates in the control group compared with historical controls. There is no obvious explanation for latent effects from decalin exposure; therefore, the relevance of these results is questionable. Male F344 rats displayed a significant increase in absolute liver weights at all exposure concentrations, while females showed an increase in absolute liver weight only at the higher concentration. These effects were not noted 24 months post exposure suggesting the effects are reversible with cessation of exposure. At the termination of exposure, male F344 rats exhibited a significant increase in nephrosis incidence and severity at concentrations of 28 mg/m3 decalin and higher. Males also exhibited a significant increase in absolute kidney weight and toxicity characterized by hyaline droplets, necrosis, intratubular casts, tubular degeneration and medullary mineralization 24 months post exposure at both decalin concentrations in air. The histopathological analysis showed the tubules were affected at the corticomedullary junction (Bruner and Pitts, 1983). The NTP (2005) also exposed groups of 10 male and 10 female B6C3F1 mice to 0, 141, 283, 565, 1131, or 2262 mg/m3 decalin in inhalation chambers for 6 h per day, 5 days per week, over 14 weeks. Liver weights for male and female mice were significantly increased in the 1131 and 2262 mg/m3 concentration groups. The incidence of centrilobular cytomegaly of the liver was increased in males at concentrations of 283 mg/m3 and greater but only showed significance at 1131 mg/m3 and greater. Contrary to the Gaworski et al. study (1985), hepatocellular cytoplasmic vacuolization was not observed in female mice. 4.1.3. Chronic inhalation exposures Dill et al. (2003a) exposed male and female F344 rats to 0, 141, 283 (males only), 565, or 2262 mg/m3 decalin by whole-body inhalation for 6 h per day, 5 days per week, for over 2 years (Table 2). Fifty rats were used for each sex and dose group except the 2262 mg/m3 dose group for males, which only had 20 rats. Renal papilla mineralization and renal tubule hyperplasia were significantly increased in males for all exposure concentrations. Incidence of renal tubule hyperplasia did not increase in a dosedependent manner. Therefore, it is unclear if this effect is a result

41

of the decalin exposure. Female rats did not show any signs of decalin-induced nephrotoxicity. No significant histopathological changes were noted in the liver. The NTP (2005) also exposed 50 male and 50 female B6C3F1 mice to 0, 141, 565, or 2262 mg/m3 decalin in inhalation chambers for 6 h per day, 5 days per week, for 105 weeks (Table 2). In the liver, the incidence of centrilobular hypertrophy, necrosis, eosinophilic focus, and erythrophagocytosis were significantly increased in males at the highest concentration. The incidence of syncytial alteration was significantly increased in males at concentrations of 565 mg/m3 and higher. 4.1.4. Reproductive and developmental exposures No reproductive or developmental exposure inhalation studies are available for decalin. This is a notable deficiency in the decalin toxicity database. An oral developmental toxicity study is available, but it utilized only one dose (Table 2). In the oral developmental toxicity study, Hardin et al. (1987) gavaged 48 pregnant CD-1 mice with 2700 mg/kg decalin in corn oil from days 6 through 13 of gestation. Litter size, birth weight, survival and growth of the offspring were normal. However, a significant increase was noted in maternal mortality and weight gain. 4.2. Cancer 4.2.1. Carcinogenicity studies The NTP (2005) conducted a 2-year inhalation study on decalin in 50 male and 50 female B6C3F1 mice exposed to 0, 141, 565 or 2262 mg/m3 decalin vapor in inhalation chambers for 6 h per day, 5 days a week for a total of 105 weeks (Table 3). The study concluded that there was no evidence of carcinogenic activity of decalin in male mice and equivocal evidence of carcinogenic activity in female mice due to a slight increased occurrence of uterine and hepatocellular neoplasms (NTP, 2005). The 141 and 2262 mg/m3 decalin concentrations resulted in increased incidences of hepatocellular neoplasms in female mice. Because the response did not correlate directly with dose, the findings were considered equivocal. Incidence of uterine stromal polyp as well as stromal polyp or stromal sarcoma (combined) also showed an upward, but non-significant trend in female mice. The combined incidence of these tumors exceeded the historical control range. The uterine stromal polyp and sarcoma data were also considered an equivocal cancer finding in female mice (NTP, 2005). The 2-year chronic inhalation study by Dill et al. (2003a) exposing male and female F344 rats to 0, 141, 283 (males only), 565, or 2262 mg/m3 decalin for 6 h per day, 5 days per week, for over 2 years found increased neoplastic lesions in the renal cortex of the male rats. Incidences of renal tubule adenoma and combined adenoma or carcinoma were significantly increased in male rats at concentrations of 283 mg/m3 decalin and higher. The incidences of renal tubule adenoma and combined adenoma or carcinoma were also significantly increased when compared with historical controls. Additionally, the incidences of benign or malignant pheochromocytoma combined were significantly increased in males at 565 and 2262 mg/m3 decalin. Pheochromocytoma occurrence was more closely correlated to the severity of nephropathy than to exposure concentration. Therefore, the authors did not consider it a result of exposure to decalin. There were no occurrences of tumors in females or in males outside of the kidney that could be attributed to decalin exposure. 4.2.2. Genotoxic and mutagenic assays Decalin was not found to be mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, or TA1535 with or without Aroclorinduced rat or hamster liver S9 enzymes (NTP, 2005). The mouse lymphoma assay was negative with and without the S9 fraction (NTP, 1992). Decalin also did not induce sister chromatid

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Table 3 Cancer inhalation toxicity studies for decalin. Species/sex

Duration

Doses

Results

Dose–response relationship

Reference

B6C3F1 mice/male and female

105 weeks

0, 141, 565, or 2262 mg/ m3

Absent

NTP (2005)

F344 rats/male and female

2 years

0, 141, 283, 565, or 2262 mg/m3 [M]; 0, 141, 565, or 2262 mg/ m3 [F]

2262 mg/m3:  Significant increase in hepatocellular neoplasms [F] (also seen at 141 mg/m3)  Increase in combined incidence of uterine stromal polyp and sarcoma [F] 565 mg/m3:  Significant increase in combined benign or malignant pheochromocytoma [M] 283 mg/m3:  Significant increase in renal tubule adenoma and combined adenoma or carcinoma [M]

Present for kidney Absent for adrenal medulla

Dill et al. (2003a)

M – male, F – female.

exchanges or micronuclei in canine peripheral lymphocytes (NTP, 1992).

female rats injected ip with male rat a2u-globin and dosed orally with 200 mg/kg decalin exhibited hyaline droplet formation and protein accumulation (Ridder et al., 1990).

5. Mode of action 6. Development of a reference concentration The mode of action for decalin-induced hepatotoxicity has not been reported. The mode for decalin-induced renal toxicity in male rats involves a2u-globin protein. Decalin induces the formation of a2u-globin protein complexes (called hyaline droplets) by binding to a2u-globin within the glomerular filtrate (Dill et al., 2003a,b). The formation of this complex was seen from decalin exposure by both the inhalation and oral routes (Stone et al., 1987). In order to determine whether the parent compound (decalin) or a metabolite (2-decalone) binds to a2u-globin in the kidneys, the mean concentrations of both chemicals were converted to the molar concentrations and compared individually and combined with a2uglobin concentrations. The ratio for the combined chemicals with a2u-globin was 0.99; suggesting both decalin and 2-decalone bind to a2u-globin to form the protein complexes (Dill et al., 2003a,b). When decalin is sequestered by a2u-globin, it cannot be converted to decalol and excreted in the urine. The renal tubule lesions are thought to be a result of the a2u-globin protein complex binding in the proximal convoluted tubule (Dill et al., 2003a). In the case of decalin, the presence of the protein was associated with tubular epithelia cell injury, cell degeneration, and necrosis. Subsequently, the necrotic epithelial cells were sloughed off and formed granular casts at the junction of the proximal tubule and loop of Henle, resulting in dilation of the tubule and a reduction of the epithelium where the cast was formed (NTP, 2005; Kanerva et al., 1987b; Stone et al., 1987). Continued exposure resulted in development of lesions and increased epithelium turnover in response to the cell death. The final development in decalin-induced renal toxicity was a chronic nephrosis characterized by thickening of the basement membrane, tubular dilation, and tubular epithelial cell hyperplasia (NTP, 2005; Kanerva et al., 1987a,b). This process initiated the neoplastic and non-neoplastic lesions seen in the kidney by promoting cytotoxicity and increased renal cortical epithelium cell turnover (Dill et al., 2003a). Binding to a2u-globin appears to be the principal mechanism in decalin-induced nephrotoxicity since kidney toxicity is only observed in the male rat. Young male rats administered cis-decalin displayed 2–3 times a2u-globin-bound decalone compared to trans-decalin administration (Dill et al., 2003b). This suggests either a2u-globin has a greater affinity for cis-decalone or the metabolism of cis-decalin occurs to a greater extent (or more rapidly) than trans-decalin (Dill et al., 2003b). Female rats and both sexes of mice, dogs, and guinea pigs exposed to decalin did not produce a nephrotoxic response. Additionally, NCI Black-Reiter male rats (deficient in a2u-globin) dosed with 200 mg/kg decalin by oral gavage did not form hyaline droplets, accumulate protein, or exhibit decalin induced nephrotoxicity (Ridder et al., 1990). Conversely,

6.1. Principal study and critical effects Three studies were considered relevant for the development of a chronic inhalation RfC. These studies include chronic inhalation exposures using B6C3F1 mice (NTP, 2005) and F344 rats (Dill et al., 2003a), as well as an oral developmental toxicity study in pregnant CD-1 mice (Hardin et al., 1987). No developmental studies based on inhalation of decalin could be located. The developmental study by Hardin et al. (1987) only tested one dose (2700 mg/kg) on pregnant dams by the oral route. While this study does not provide information concerning inhalation exposure or contain enough doses to formulate a dose–response curve, only the pregnant dams exhibited adverse effects at the tested dose. There were no significant effects detectible in the offspring. Based on this limited study, decalin appears to induce systemic toxicity in the pregnant dams before developmental toxicity, suggesting the fetus is not the most sensitive receptor to decalin-induced adverse effects. The lack of sufficient inhalation developmental and reproductive toxicity studies for decalin is a deficiency in the toxicity database and developmental effects remain uncertain. However, the available data suggest that developmental toxicity is unlikely to be the most sensitive adverse effect. The male and female rat study by Dill et al. (2003a) included chronic inhalation exposure, but focused primarily on the nephrotoxicity observed in males during shorter exposure durations. Even at the highest exposure concentration (2262 mg/m3), the females did not exhibit any sign of decalin-induced nephrotoxicity. The renal lesions caused by decalin are believed to be a result of a2u-globin protein accumulation in the tubules (Dill et al., 2003a,b; Stone et al., 1987). Because a2u-globin is a male rat specific protein, this method of inducing nephrotoxicity is not relevant to human health. The absence of nephrotoxicity in female rats and male and female mice suggest that the a2u-globin protein is requisite for decalin-induced nephrotoxicity (NTP, 2005; Dill et al., 2003a). Therefore, nephrotoxicity should not be considered the critical effect for extrapolation to human health. The National Toxicology Program (NTP) chronic inhalation study (2005) in B6C3F1 mice found a significant increase in centrilobular hypertrophy, necrosis, syncytial alteration, eosinophilic focus, and erythrophagocytosis in the livers of male mice at the highest concentration (2262 mg/m3). The mechanism of action for the decalin-induced hepatotoxicity has not been reported. In the absence of mechanistic data, it is assumed that the hepatotoxicity observed in male mice would be relevant to human health. Hepatotoxicity as

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a critical effect of decalin-induced toxicity is supported by some of the adverse effects seen in shorter inhalation exposure studies. In the 2-week study by the NTP (2005) both male and female F344 rats and male and female B6C3F1 mice displayed significantly increased liver weights over controls at 565 mg/m3 (and higher) for rats and 1131 mg/m3 (and higher) for mice. In a 13-week exposure of male and female F344 rats to decalin, Dill et al. (2003a) showed a significant increase in male liver weight at 1131 and 2262 mg/m3 and in female liver weight at 2262 mg/m3. The 14-week NTP (2005) exposure of male and female B6C3F1 mice also showed significantly increased liver weights over controls at decalin concentrations of 1131 and 2262 mg/m3. Histopathologic changes were seen accompanying increases in liver weight, including a significant increase in the incidence of centrilobular cytomegaly in males at 283 mg/m3 and higher. A 90-day study by Gaworski et al. (1985) using female C57BL/6 mice showed significantly increased reversible hepatocellular vacuolization at 28 mg/m3 decalin and higher. In the absence of histopathologic changes, hepatocellular vacuolization as a result of decalin exposure was considered a mild, reversible response. Also, without accompanying histopathologic changes, increases in liver weight are likely due to an increase in enzyme production induced by exposure. However, the histopathological changes seen in mice suggest an adverse effect on the liver after subchronic or chronic periods of exposure. 6.2. Benchmark dose approach Due to limitations inherent in the NOAEL/LOAEL approach, the Benchmark Dose (BMD) approach is preferred for deriving a point of departure (US EPA, 2012). Use of the BMD approach allows for the use of the entire dose–response curve to estimate a point of departure (POD) rather than using a single dose. This is especially important for decalin (NTP, 2005) because the critical study uses only three doses and 50 animals in each group, making it difficult for the NOAEL/LOAEL approach to distinguish differences in sensitivity to decalin exposure for the hepatic lesions. Gamma, Logistic, Log–logistic, Log–probit, Multistage, Probit, and Weibull models from the US EPA’s Benchmark Dose Software (Version 2.3.1) were used to fit the dichotomous data from the NTP (2005) mouse liver lesions (Table 4). For each endpoint, the best-fitting model was chosen using the methodology recommended in the Benchmark Dose Technical Guidance (US EPA, 2012). This methodology includes visual inspection of the model fit to the data and analysis of the chi-square value, p-value, Akaike’s Information Criterion, and the 95% lower confidence limit on the benchmark dose (BMDL). A point of departure of 10% was chosen for the mouse liver data because it is the default assumption for the BMD methodology and because three dose groups are insufficient to calculate a response level lower than 10% with any certainty (US EPA, 2012). The BMDL10 for increased eosinophilic foci, necrosis, syncytial alteration, and erythrophagocytosis are presented in Table 5. The centrilobular hypertrophy data were

not amenable to BMD modeling. When the centrilobular hypertrophy data were analyzed in the US EPA’s Benchmark Dose Software, v2 values exceed 2 for all available models. This suggests that the available models are a poor fit to the centrilobular hypertrophy data resulting in an uncertain and non-defensible BMDL10 estimate. Therefore, a BMDL10 was not derived for this endpoint. Instead, the NOAEL/LOAEL approach was utilized to derive a POD for this effect. Based on the data reported in the NTP (2005) study, the no observable adverse effect level (NOAEL) for centrilobular hypertrophy in male mice is 565 mg/m3, which serves as the POD for this endpoint (Table 5). The most sensitive effect from decalin exposure by inhalation is syncytial alteration. The Log–logistic model provides the best fit for this data and displays a BMDL10 of 44 mg/m3 (Fig. 1). 6.3. Human equivalent concentration In the absence of a physiologically based pharmacokinetic model for the inhalation of decalin, US EPA guidance for inhalation risk assessment (US EPA, 2009) was used to derive a human equivalent concentration. The BMDL10 of 44 mg/m3 was first converted to a continuous inhalation dose of 7.9 mg/m3. The conversion is based upon determining an equivalent average concentration in air for continuous exposure (24 h a day, 7 days a week) versus exposure for 6 h a day, 5 days a week in the critical inhalation study (6 h/24 h  5d/7d  44 mg/m3). Conversion of intermittent exposures to a continuous inhalation dose is considered standard methodology for the derivation of a reference concentration (US EPA, 2002). Because the site of the adverse effects is remote from the respiratory system, decalin is considered a Category 3 gas (US EPA, 2009). The inhalation dose of a Category 3 gas is converted to a dosimetric human equivalent dose using the ratio of the mouse blood:gas partition coefficient to the human blood:gas partition coefficient. No mouse to human blood:gas partition coefficient ratio was located for decalin. Therefore, a default dosimetric adjustment factor of 1 was utilized to derive a BMDLHEC of 7.9 mg/m3; that is, the decalin blood:gas partition coefficient was assumed to be equal for mouse and human and no additional adjustment in the continuous exposure BMDL10 of 7.9 mg/m3 was made. 6.4. Uncertainty factors Uncertainty factors (UFs) were applied to the BMDLHEC of 7.9 mg/m3 to derive a chronic reference concentration for decalin. A UFH of 10 was utilized to account for biological variation in the human population that could result in sensitive subpopulations. This UF accounts for differences in susceptibility due to age, genetics, health status, and background exposures. Because the mechanism of decalin hepatotoxicity is unknown, chemical-specific variability in human susceptibility to the toxic effects cannot be estimated. Therefore, a conservative UFH of 10 (101) was chosen. Use of a dosimetric adjustment factor (DAF) to derive a human

Table 4 Liver lesion incidence in male mice from the National Toxicology Program 2-year inhalation study of decalin (NTP, 2005).

** * a

Lesion

0 mg/m3

141 mg/m3

565 mg/m3

2262 mg/m3

Number examined Eosinophilic focus Necrosis Syncytial alteration Centrilobular hypertrophy Erythrophagocytosis

50 10 0 26 (1.0) 2 (1.5) 0

50 9 1 (3.0)a 28 (1.0) 0 0

50 7 3 (1.3) 36* (1.2) 4 (1.3) 0

50 19* 19** (1.2) 44** (2.2) 36** (1.9) 9** (1.6)

Significantly different (P 6 .01) from 0 mg/m3 by the Poly-3 test. Significantly different (P 6 0.05) from 0 mg/m3 by the Poly-3 test Average severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked.

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L.D. Stuchal et al. / Regulatory Toxicology and Pharmacology 66 (2013) 38–46 Table 5 Point of departure for a 10% increase in the occurrence of liver lesions calculated from a 2-year inhalation study of decalin by the National Toxicology Program (NTP, 2005).

a b

Lesion

BMD10a (mg/m3)

BMDL10a (mg/m3)

NOAEL (mg/m3)

Eosinophilic focus Necrosis Syncytial alteration Centrilobular hypertrophy Erythrophagocytosis

1357 792 113 NAb 2057

622 447 44 NAb 1370

565 565 141 565 565

Rounded to two significant digits. Data were not amenable to BMD modeling.

BMDLHEC of 7.9 mg/m3 by the total uncertainty (100) yields a chronic decalin reference concentration of 0.08 mg/m3. 7. Discussion 7.1. Primary toxic effects

Fig. 1. Increased incidence of syncytial alteration in male mice after chronic inhalation exposure to decalin. The curve is calculated using the Log–logistic model. The BMD is the concentration that elicits a response in 10% of the tested population. The BMDL is the concentration corresponding to the lower 95% confidence interval.

The primary toxic effects from the chronic inhalation of decalin include neoplastic and non-neoplastic lesions in the kidneys of male rats and hepatotoxicity in male mice (NTP, 2005; Dill et al., 2003a). Female mice also displayed histological changes in the liver (vacuolization); however, the effects were mild and reversible with cessation of exposure (Gaworski et al., 1985). In the absence of mechanistic data, hepatotoxicity in rodents is considered applicable to human health. Therefore, the primary toxic effect of concern for decalin exposure by inhalation is hepatotoxicity. Hepatotoxicity (centrilobular cytomegaly) was also reported in male mice exposed to 1131 mg/m3 decalin for 14 weeks (NTP, 2005), suggesting that the subchronic NOAEL (565 mg/m3) is similar to the chronic NOAEL (141 mg/m3). This implies that an increase in exposure by approximately fourfold could result in similar toxicities over shorter time periods (3 months). 7.2. Uncertainties in toxic effects

equivalent dose addresses the toxicokinetic aspects of the interspecies uncertainty factor, reducing the total uncertainty by half (US EPA, 1994). Because a DAF was utilized to derive a dosimetric human equivalent dose for decalin, a UFA of 3 (100.5) was applied for the extrapolation of toxicity data from animals to humans. Although a default DAF of 1 was utilized to estimate the blood:gas partitioning of decalin, the default assumption is considered conservative and is not likely to underestimate the true DAF (US EPA, 2009). A UFS of 1 (100) was chosen for extrapolation of subchronic data to chronic effects because the critical study is a 2-year chronic mouse inhalation study. There is no extrapolation to longer exposure periods. A UFL of 1 was chosen for the extrapolation of a LOAEL to a NOAEL. To derive the BMDLHEC, a POD was calculated based on the entire dose range. Because the LOAEL is not utilized as the POD in the BMD approach, a UFL of 1 (100) is considered applicable. A UFD of 3 (100.5) was chosen for insufficiencies in the database. The toxicity data set for decalin does not contain any reproductive or multigenerational studies. The absence of reproductive and multigenerational studies implies that adverse effects for these endpoints may occur at doses near the POD, but remain uncharacterized. Additionally, the only developmental study is by the oral exposure route. Although this study showed systemic maternal toxicity before the development of fetal effects, it has not been confirmed that the same will be true for pregnant dams exposed by the inhalation route. The product of these uncertainty factors produces a total uncertainty of 100 (101  100.5  100  100  100.5 = 102) for the derivation of a reference concentration (US EPA, 2002). Dividing the

7.2.1. Cancer findings Renal tubule adenoma, renal tubule carcinoma, and pheochromocytoma were seen in male F344 rats. The renal neoplastic lesions are the final development in a2u-globin induced renal toxicity (NTP, 2005; Kanerva et al., 1987b). The significant increase in pheochromocytoma did not correlate with dose suggesting pheochromocytoma formation is not a result of inhalation exposure to decalin (Dill et al., 2003a). The formation of neoplastic lesions was not significantly increased in male B6C3F1 mice (NTP, 2005). However female B6C3F1 mice displayed an increase in the incidence of uterine and hepatocellular neoplasms. The uterine polyps and sarcomas displayed an upward trend, but not significance. The lack of significance could reflect individual variability in the female mouse tumor incidence rate or may be due to the lack of statistical power associated with the small number of mice assigned to each dose group. Despite the lack of significance, the combined occurrence of uterine stromal polyp and sarcoma in the 2262 mg/m3 concentration group exceeded the range in historical controls suggesting decalin may play a role in the occurrence of these neoplasms. The cause of the uterine neoplasms remains unclear. Further research appears necessary before the increased incidence of uterine tumors can be attributed to decalin exposure. Incidence of hepatocellular carcinoma (significantly increased at 141 mg/m3) and hepatocellular adenoma (significantly increased at 2262 mg/m3) in B6C3F1 female mice only showed a significant increase at one concentration. The significant increase in hepatocellular carcinoma at 141 mg/m3 exceeded the range of historical controls.

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Hepatocellular adenomas were only significantly increased at the highest concentration and also exceeded the range of historical controls. These data suggest decalin could play a role in the formation of hepatocellular neoplasms. 7.2.2. Low dose extrapolation One of the largest uncertainties in deriving a safe human dose is the extrapolation of the response seen at high doses utilized in toxicity testing to the much lower doses encountered in the environment. Extrapolation to low doses introduces errors because the shape of the dose–response curve in the low dose region is seldom known. Different methodologies are utilized to account for this uncertainty including extrapolation of the dose–response curve to low doses using BMD software. Using a BMD model to extrapolate the dose–response curve assumes that, at low doses, a chemical elicits a response in a manner that can be predicted based on the response at higher doses. This is not necessarily correct and the true shape of the dose–response curve at environmentally relevant concentrations remains uncertain. Additionally, in vivo toxicity tests may not contain sufficient power to accurately describe the dose–response curve, even at higher doses. Because of the limited power of most toxicity studies, the NOAEL for quantal data is, on average, representative of a 10% response (range 3–30% response) rather than a true no effect level (Barlow et al., 2009). This is exhibited in the dataset for decalin-induced hepatotoxicity where the modeled POD (based on the BMDL10) for the critical effect (syncytial alteration at 44 mg/m3) is more conservative than the NOAEL (141 mg/m3). This can occur because both the NOAEL and the BMDL10 represent similar effect levels. For the derivation of a reference dose or reference concentration, the BMD methodology is preferred because the approach utilizes the entire dose–response curve and quantifies the uncertainty in the dose–response data (US EPA, 2012), which results in a more accurate POD. 7.3. Database limitations The toxicity database for chronic inhalation exposure to decalin consists of only two studies (NTP, 2005; Dill et al., 2003a). The chronic rat inhalation study (NTP, 2005; Dill et al., 2003a) displayed a2u-globin protein-induced nephrotoxicity, which is not applicable to human health. This study did not report any histopathological changes in the liver. The chronic mouse study (NTP, 2005) reported hepatotoxicity in male, but not female, mice. A database of only two chronic studies limits the amount of toxicity information available for a weight of evidence approach to identify the critical effect. Five subchronic inhalation studies were found in the literature. A 14-week mouse inhalation study by the NTP (2005) also reported hepatotoxicity in male (but not female) mice, supporting the toxic effect seen in the chronic study. The lack of hepatotoxicity in male and female rats and female mice over a similar exposure periods and doses as the male mice suggests susceptibility of male mice to hepatotoxicity from decalin inhalation. Since the mechanism of action for decalin-induced hepatotoxicity is not known, it is unclear if hepatotoxicity in male mice can be extrapolated to humans. Histopathologic changes in the liver are usually considered relevant for extrapolation. Nevertheless, the lack of mechanistic information is a database limitation that increases uncertainty in the possible extrapolation of decalin-induced hepatotoxicity to humans. Another important limitation in the database is the lack of developmental, reproductive, and multi-generational toxicity studies for the inhalation route. The oral developmental toxicity study (Hardin et al., 1987) suggests that decalin may not cause developmental effects before inducing maternal toxicity. However, there are not enough data available to exclude these endpoints as the critical effect with certainty. Multigenerational studies may show adverse effects at lower doses

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than are exhibited in other studies. An analysis by Martin et al. (2009), revealed 94 out of 316 chemicals in the Toxicity Reference Database displayed specific reproductive or offspring toxicities in multigenerational studies when compared to systemic effect under chronic exposure periods. In general, however, the multigenerational toxicities were less sensitive than adverse effects seen in the 2-year chronic studies. Due to the lack of inhalation studies for developmental, reproductive, and multi-generational toxicity, the importance of these endpoints in deriving a safe exposure concentration for decalin remains unclear. 8. Conclusions Adequate toxicity data are available from a 2-year inhalation study in mice to derive a chronic RfC for decalin. The principal effect is hepatotoxicity, and a chronic RfC of 0.08 mg/m3 was calculated for decalin by conversion of the BMDL10 to a human equivalent continuous inhalation concentration of 7.9 mg/m3 and application of a total uncertainty of 100. There are several toxicity database limitations and uncertainties, however, and these limitations should be acknowledged and qualified when using this chronic RfC. Future research into decalin is needed to better characterize the toxicity associated with chronic inhalation exposure (especially developmental and reproductive effects) and will add to the refinement of this value. Conflict of interest statement The authors declare that there are no conflicts of interest. References Barlow, S., Chesson, A., Collins, J.D., et al., 2009. Use of the benchmark dose approach in risk assessment. EFSA J. 1150, 1–72. Baxter, C.S., 2001. Alicyclic hydrocarbons. In: Bingham, E., Cohrssen, B., Powell, C.H. (Eds.), Patty’s Toxicology. John Wiley & Sons, Inc., New York, pp. 151–231. Browning, E., 1965. Toxicity and Metabolism of Industrial Solvents. American Elsevier, New York, pp. 138–140. Bruner, R.H., Pitts, L.L., 1983. Nephrotoxicity of Hydrocarbon Propellants to Male, Fishcer-344 Rats. Air Force Aerospace Medical Research Lab, Wright-Patterson AFB, OH, pp. 337–349. Dill, J.A., Lee, K.M., Renne, R.A., et al., 2003a. A2u-Globulin nephropathy and carcinogenicity following exposure to decalin (decahydronaphthalene) in F344/ N rats. Toxicol. Sci. 72, 223–234. Dill, J.A., Fuciarelli, A.F., Lee, K.M., et al., 2003b. Single administration toxicokinetic studies of decalin (decahydronaphthalene) in rats and mice. Toxicol. Sci. 72, 210–222. Elliott, T.H., Robertson, J.S., Williams, R.T., 1966. The metabolism of cis- and transdecalin. J. Biochem. 100, 403–406. Gaworski, C.L., Haun, C.C., MacEwen, J.D., et al., 1985. A 90-day vapor inhalation toxicity study of decalin. Fundam. Appl. Toxicol. 5, 785–793. Gosselin, R.E., Smith, R.P., Hodge, H.C., et al., 1984. Clinical Toxicology of Commercial Products, 5th ed. Williams and Wilkins, Baltimore, pp. II-153. Hampton, C.V., Pierson, W.R., Harvey, T.M., et al., 1982. Hydrocarbon gases emitted from vehicles on the road. 1. A qualitative gas chromatography/mass spectrometry survey. Environ. Sci. Technol. 16, 287–298. Hardin, B.D., Schuler, R.L., Burg, J.R., et al., 1987. Evaluation of 60 chemicals in a preliminary developmental toxicity test. Teratogen. Carcinogen. Mutagen. 7, 29–48. Hazardous Substances Data Bank (HSDB) [Internet], 2004 – [updated 2004 Mar 2]. Decahydronaphthalene; HSDB number: 287. National Library of Medicine, Bethesda, [about 25 pp.]. Available from: . Kanerva, R.L., McCracken, M.S., Alden, C.L., et al., 1987a. Morphogenesis of decalininduced renal alteration in the male rat. Food Chem. Toxicol. 25, 53–61. Kanerva, R.L., Ridder, G.M., Lefever, F.R., et al., 1987b. Comparison of short-term renal effects due to oral administration of decalin or d-limonene in young adult male Fischer-344 rats. Food Chem. Toxicol. 25, 345–353. Martin, M.T., Mendez, E., Corum, D.G., et al., 2009. Profiling the reproductive toxicity of chemicals from multigeneration studies in the toxicity reference database. Toxicol. Sci. 110, 181–190. Middleditch, B.S., 1982. Volatile constituents of the produced water effluent from the Buccaneer gas and oil-field. J. Chromatogr. 239, 159–171. National Toxicology Program (NTP), 1992. CAS# 119-64-2/91-17-8, Tetralin/ Decalin, Summary of Data for Chemical Selection. US Department of Health and Human Services, National Institutes of Health, Research Triangle Park, NC.

46

L.D. Stuchal et al. / Regulatory Toxicology and Pharmacology 66 (2013) 38–46

NTP, 2005. Toxicology and Carcinogenisis Studies of Decalin (CAS No. 91–17-8) in F344/N Rats and B6C3F1 Mice and Toxicology Study of Decalin in Male NBR Rats (Inhalation Studies). US Department of Health and Human Services, National Institutes of Health, Research Triangle Park, NC. O’Neil, M.J., Heckelman, P.E., Koch, C.B., et al. (Eds.), 2006. The Merck Index, 14th ed. Merck and Co., Inc., Whitehouse Station, NJ, p. 2849. Olson, C.T., Yu, K.O., Serve, M.P., 1986. Metabolism of nephrotoxic cis- and transdecalin in Fischer-344 rats. J. Toxicol. Environ. Health 18, 285–292. Patty, F. (Ed.), 1963. Industrial Hygene and Toxicology Volume II: Toxicology, 2nd ed. John Wiley & Sons, New York, pp. 1214–1215. Ridder, G.M., Von Bargen, E.C., Alden, C.L., et al., 1990. Increased hyaline droplet formation in male rats exposed to decalin is dependent of the presence of a2uglobulin. Fundam. Appl. Toxicol. 15, 732–743. Sandmeyer, E.E., 1981. Alicyclic hydrocabons. In: Clayton, G.D., Clayton, F.E. (Eds.), . 3rd ed., Patty’s Industrial Hygiene and Toxicology 3rd ed., vol. 2B John Wiley & Sons, Inc., New York, pp. 3221–3241. Sikkema, J., de Bont, J.A., Poolman, B., 1995. Mechanisms of membrane toxicity of hydrocarbons. Microbiol. Rev. 59, 201–222.

Stone, L.C., Kanerva, R.L., Burns, J.L., et al., 1987. Decalin-induced nephrotoxicity: light and electron microscopic examination of the effects of oral dosing on the development of kidney lesions in the rat. Food Chem. Toxicol. 25, 43–52. Traynor, G.W., Apte, M.G., Sokol, H.A., 1990. Selected organic pollutant emissions from unvented kerosene space heaters. Environ. Sci. Technol. 24, 1265–1270. US EPA, 1994. Methods for Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry. Office of Health and Enviornmental Assessment, Research Triangle Park, NC. US EPA, 2002. A Review of the Reference Dose and Reference Concentration Processes. Risk Assessment Forum, Washington, D.C.. US EPA, 2009. Risk Assessment Guidance for Superfund Volume I: Human Health Evaluation Manual, (Part F, Supplemental Guidance for Inhalation Risk Assessment). Office of Superfund Remediation and Technology Innovation, Washington, D.C.. US EPA, 2012. Benchmark Dose Technical Guidance. Risk Assessment Forum, Washington, D.C..