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There were no chronic repeat-exposure studies conducted with tertiary butyl acetate available for review. However, results for guideline subchronic-exposure studies in which male and female rats and mice were exposed to tertiary butyl acetate for up to 13 weeks by the inhalation route suggest that the test material is unlikely to pose a significant risk for the development of a carcinogenic effect. No target organ necrosis or hyperplasia was observed in either species. The only treatment-related microscopic lesions observed were associated with hyaline droplet nephropathy, a common spontaneous lesion in male rats and frequently exacerbated by chemical exposure. While very advanced chronic progressive nephropathy may be a risk factor for the spontaneous development of renal tumors in rats, chronic progressive nephropathy has not been observed in humans. In addition, no mutagenicity/genotoxicity was observed in a bacterial reverse mutation assay, an in vitro chromosomal aberration assay conducted with human lymphocytes, or an in vivo micronucleus assay in which rats were exposed by the inhalation route to the same concentrations of tertiary butyl acetate as were used in the subchronic repeat-exposure studies cited above. All studies were conducted according to current guideline specifications.

Since absorbed tertiary butyl acetate is rapidly metabolized and the primary metabolite of tertiary butyl acetate is tertiary butyl alcohol, data from several long term studies conducted in rats and mice with tertiary butyl alcohol are relevant to the overall evaluation of carcinogenicity from exposure to tertiary butyl acetate and have therefore been included in this submission. The potential for tertiary butyl alcohol to cause cancer is well understood. In a 2-year drinking water study conducted according to NTP guidelines, male and female F344/N rats and B6C3F1 mice received the following maximum doses of tertiary butyl alcohol: male rats 288-516 mg/kg bw/day; female rats 587-886 mg/kg bw/day; male mice 1868-2896 mg/kg bw/day; female mice 1786-3488 mg/kg bw/day. There was no statistically significant increase in any tumor type in female rats. There was an increased incidence of renal tubule adenoma or carcinoma (combined) in male rats. There was no corresponding renal tumor increase in female rats at dose levels that also caused chronic progressive nephropathy and transitional epithelial hyperplasia and that were twice that administered to males. There was also no evidence of renal tumors in mice of either sex at dose levels four times that administered to male rats. There was some evidence of carcinogenic activity of tertiary butyl alcohol in female mice based on an increased incidence of follicular cell adenoma of the thyroid gland in high-dose animals only and equivocal evidence in male mice based on a marginally increased incidence of follicular cell adenoma of the thyroid gland in the mid-dose group only and a single follicular cell carcinoma in the high-dose group. There was no evidence of thyroid tumors in rats of either sex.

 

One well documented pathway leading to renal tubule tumor formation in male rats only is induction of alpha-2u-globulin (alpha-2u-g) nephropathy (Hard et al., 1993; Swenberg and Lehman-McKeeman, 1999). In this syndrome, a xenobiotic binds non-covalently to circulating alpha-2u-g synthesized in the male rat liver. Because it is a low molecular weight protein, alpha-2u-g is freely filtered through the glomerulus, and approximately half of the ultrafiltrate is absorbed into the second segment of proximal convoluted tubule and catabolized by the hydrolytic enzymes of renal lysosomes. The loose binding of xenobiotic to alpha-2u-g significantly retards the lysosomal degradation of the protein, resulting in hyaline droplet (and alpha-2u-g) accumulation in tubule cells, secondary cytotoxicity, cell loss and compensatory tubule cell regeneration, sustained during the period of exposure. Prolonged cytotoxicity and regenerative cell proliferation is considered to be the mode of action leading to renal tubule tumor formation. Additional features characteristic of this type of nephropathy are linear mineralization in the papilla.

 

Most conventional strains of laboratory rodents, including the F-344 rat used in the 2-year bioassays conducted with tertiary butyl alcohol and the Sprague-Dawley rats used in the 13-week inhalation studies conducted with tertiary butyl acetate, are subject to high morbidity from spontaneous chronic progressive nephropathy (CPN), with males being more sensitive than females. Many chemicals have been reported to exacerbate onset and severity of CPN, hastening progression to end-stage disease, which is itself a cause of mortality. It is believed that very advanced CPN is a risk factor for the spontaneous development of renal tubule tumors (Hard et al., 1997; Hard, 1998). Chemicals that induce alpha-2u-g (hyaline droplet) nephropathy generally also exacerbate CPN.

 

In an expert evaluation by Hard (2001), the author evaluated the renal effects observed in the NTP 13-week and 2-year carcinogenicity studies of tertiary butyl alcohol. The author focused on two possible modes of action to explain the occurrence of renal tubule tumors in male rats only and concluded that the available data strongly suggest that alpha-2u-g nephropathy plays a role in the renal tubule carcinogenesis observed in the 2-year study. These data include: occurrence of renal tubule tumors in male rats only; induction of hyaline droplet accumulation upon acute exposure; identification of the presence of alpha-2u-g; chronic microscopic changes which included linear papillary mineralization; no similar renal toxicity observed in female rats or either sex of mice; and negative genotoxicity studies. Further evidence of an alpha-2u-g-mediated mechanism of action was a binding study conducted by William and Borghoff (2001) which demonstrated reversible binding of tertiary butyl alcohol to alpha-2u-g in the male rat kidney. These results would explain the accumulation of alpha-2u-g in the male rat kidney following exposure to tertiary butyl alcohol. Hard postulated a second possible mode of action involving end stage chronic progressive nephropathy, itself a risk factor for renal tubule neoplasia. In the 2-year bioassay, there was a dose-related enhancement of CPN by tertiary butyl alcohol in male rats and the severity was greater in male rats than in female rats. No enhancement of CPN was observed in either sex of B6C3F1 mice. Based on the Hard (2001) evaluation, one or both of these modes of action may have played a role in the observed renal carcinogenesis in male rats.  Hard et al. (2009) also did an extensive comparison of rat CPN with human renal disease and observed that chronic progressive nephropathy and alpha-2u-g nephropathy have not been observed in humans.

 

An expert evaluation by McClain (2001) focused on possible causes for the thyroid gland changes observed in the mouse bioassay conducted with tertiary butyl alcohol. It discussed the possible role of microsomal enzyme induction, various intra- and extrathyroidal mechanisms whereby chemicals alter thyroid function, and important species differences between rodents and humans in thyroid gland physiology and susceptibility to thyroid follicular cell neoplasia secondary to hormone imbalance. A mode of action involving indirect interference with thyroid hormone production has been proposed to explain a slight increase in the number of thyroid tumors observed in mice at high oral exposures. Rats and mice are highly sensitive to chemicals that disrupt the normal synthesis and secretion of thyroid hormones. A number of these chemicals, when tested at high concentrations, cause a hormone imbalance which results in increased secretion of pituitary thyroid stimulating hormone (TSH) to stimulate thyroid function. This in turn can cause follicular cell hyperplasia, increased thyroid weights, and in long-term studies produce an increased incidence of thyroid tumors by an indirect mechanism associated with hormone imbalance and chronic hyper-secretion of TSH. Compounds acting by this mechanism usually show little or no evidence of genotoxicity. It should be noted that tertiary butyl alcohol was negative in in vitro and in vivo mutagenicity tests as was tertiary butyl acetate. Several investigations have shown that excessive stimulation by TSH alone, in the absence of any chemical treatment, can cause thyroid gland neoplasia in rodents. It is the author’s opinion that “the most likely hypothesis for the thyroid gland proliferative lesions is hormone imbalance secondary to microsomal enzyme induction of thyroid hormone metabolism.” An increase in liver weights in rat and mouse 13-week studies conducted with both tertiary butyl alcohol and tertiary butyl acetate is consistent with enzyme induction.

 

Microsomal enzyme induction was demonstrated in a recently completed study which investigated hepatotoxicity and thyroid hormone production in B6C3F1 mice administered tertiary butyl alcohol in the drinking water for 3 or 14 days as a possible mechanism to explain thyroid tumors observed in mice in the 2-year NTP drinking water studies (Blanck et al., 2009). Phenobarbital, known to cause enzyme induction, was used as a positive control. This study demonstrated that at high oral doses, tertiary butyl alcohol is a weak liver enzyme inducer (weaker than phenobarbital) in mice. Induction was specific for the CYP2b10 and CYP2b9 enzymes as well as the Phase II enzyme sulfotransferase 1a1, which is involved in thyroid hormone metabolism.

 

In addition, McClain points out species differences in thyroid gland physiology and concludes that, as a result, “the rodent thyroid gland is markedly more active and operates at a considerably higher functional level with respect to thyroid hormone turnover as compared to the primate.” Thyroid tumors in rodents occur in response to increased hepatic hormone clearance to a much greater degree than in humans. Therefore, a thyroid hyperplasia response in rodents may not be relevant for assessment of human carcinogenicity, especially since neoplastic effects in mice in the NTP study were observed only at extremely high oral doses, i.e., mean compound consumption in mg tertiary butyl alcohol/kg bw/day was 3488 (wk 1-13), 2043 (wk 14-52) and 1786 (wk 53-101) for females and 1386 (wk 1-13), 910 (wk 14-52) and 1042 (wk 53-101) for males. Although transitional epithelia hyperplasia of the urinary bladder was also observed in mice in this study, there was no progression to neoplasia.

 

Based on the rapid metabolism of tertiary butyl acetate to tertiary butyl alcohol, the alpha-2u-globulin nephropathy present at 13 weeks in male rats exposed to both tertiary butyl acetate and tertiary butyl alcohol, and increased liver weights in both rats and mice exposed to tertiary butyl acetate and tertiary butyl alcohol, it is reasonable to assume that chronic exposure to tertiary butyl acetate would cause the same tumorigenic effects observed in the chronic NTP studies conducted with tertiary butyl alcohol.

An overarching review was conducted by Bus et al (2015)* which examined its primary use as a solvent in industrial and consumer products. Derivations of acute and chronic reference (RfC) values were made to develop hazard quotients (HQ) for various exposure scenarios in the US EPA risk assessment of tertiary butyl acetate; most HQ values less than 1, confirming low risks to human health. The publication also discusses the use of metabolic surrogate read-across in reducing animal testing.

 

The assessment found that tertiary butyl acetate has a low order of toxicity following subchronic inhalation exposure, and neurobehavioral changes (hyperactivity) in mice observed immediately after termination of exposure were the effects used as conservative endpoints for derivation of the acute and chronic RfC values. Tertiary butyl acetate was concluded to not be genotoxic and, although this substance has not been tested for carcinogenicity, it is unlikely to be a human carcinogen in that its non-genotoxic metabolic surrogates tertiary butyl alcohol and methyl tertiary butyl ether (MTBE) produced only male rat α -2u-globulin-mediated kidney cancer and high-dose specific mouse thyroid tumors, both of which have little qualitative or quantitative relevance to humans.

* Bus JS, Banton MI, Faber WD, Kirman CR, McGregor DB & Pourreau DB (2015) Human health screening level risk assessments of tertiary-butyl acetate (TBAC): Calculated acute and chronic reference concentration (RfC) and Hazard Quotient (HQ) values based on toxicity and exposure scenario evaluations, Critical Reviews in Toxicology, 45:2, 142-171

Justification for classification or non-classification

There was no evidence of necrosis or hyperplasia observed in groups of male and female rats and mice exposed to tertiary butyl acetate by the inhalation route for up to 13 weeks and all target organ effects were similar to those observed following oral administration of the metabolite tertiary butyl alcohol. Genotoxicity studies conducted with tertiary butyl acetate were negative both in the presence and absence of metabolic activation. 

 

The tumorigenic responses of male rats and male and female mice to tertiary butyl alcohol is unlikely to be by a genotoxic mode of action since results were negative for induction of genetic damage in a series of in vitro and in vivo studies. The observed tumor types were not uncommon for the species/sex used in the study, increased tumor incidence occurred in only one species for each type of tumor, statistically significant increases in hyperplasia occurred in organs subsequently developing tumors, and tumor incidence in male rats reached statistical significance only following an extended evaluation of the kidney and then only in the mid-dose group. All available data suggest that the kidney tumors observed in male rats exposed to tertiary butyl alcohol in the drinking water for two years most likely occurred through a mechanism related to alpha-2u-g nephropathy (a condition unique to male rats) and/or a dose-related enhancement of chronic progressive nephropathy (end-stage CPN is itself a risk factor for renal tubule neoplasia). The CPN observed in rats also has no counterpart in humans and therefore is not relevant for species extrapolation to humans. Likewise, the thyroid follicular hyperplasia progressing to neoplasia observed in mice in the 2-year drinking water study most likely also occurred by a mode of action not relevant for human risk assessment. Because of marked species differences between rodent and human thyroid gland physiology, microsomal enzyme induction of thyroid hormone metabolism is much more likely to result in neoplasia in rodents.

 

Based on a weight-of-the-evidence evaluation, tertiary butyl acetate is unlikely to pose a significant risk for the development of any tumor type in humans exposed to this chemical and is not considered to be classified for “Carcinogenicity” according to EU CLP (Regulation (EC) No. 1272/2008) and UN GHS.