2-methylpropan-2-ol

Carcinogenicity

Description of key information

In a 2 year drinking water study, tertiary butyl alcohol produced increases in rat kidney and mouse thyroid tumors. Mechanistic information indicates tertiary butyl alcohol is unlikely to pose a significant risk for the development of tumors in humans exposed to low levels of this chemical.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

90 mg/kg bw/day

Study duration:

chronic

Species:

rat

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The carcinogenic potential of tertiary butyl alcohol has been investigated in rat and mouse. Although there were tumors observed in both species, the tumors in rats were thought to occur via a rat-specific mode of action that was not relevant to humans, whereas the tumors in mice were considered of little potential concern to humans. No classification for carcinogenicity is warranted.

Additional information

Rat

In a 2-year NTP study, groups of 60 male and 60 females F344/N rats were administered 0, 1.25 (males only), 2.5, 5 or 10 (females only) mg/ml tertiary butyl alcohol (equivalent to 0, 90, 200 and 400 mg/kg bw/day in males and 0, 180, 330 and 650 mg/kg bw/day in females) via the drinking water. The non-neoplastic effects are reported in the repeated dose section (section 5.6).

At the 15 month interim termination, a renal tubule adenoma was detected in one male of the 5 mg/L group.

At termination, the incidences of focal renal tubule hyperplasia and adenoma were increased in exposed males and a carcinoma was observed in one 5 mg/ml male. Renal tubule hyperplasia was also observed in one 10 mg/mL female. Due to the effects observed in male kidneys, further sections from all groups were examined. This identified additional male rats with hyperplasia (11, 13, 11 and 19 animals in control through to high dose groups) and renal tubule adenomas (7, 8, 15, 10 from control through to high dose groups). Renal tubule carcinomas were also identified in two 1.25 mg/L males and one 2.5 mg/L males. When the standard and extended evaluations were combined, there was a statistically significant increase in hyperplasia in males at 5 mg/L and of adenoma incidence in 2.5 mg/L males. Inclusion of the adenoma observed in the high dose males at the interim kill, lead to a statistically significant increase in adenoma incidence in the 5 mg/L males.

Table of neoplastic lesions in kidneys of males from a 2-year rat study

Dose (mg/ml)	0	1.25	2.5	5
Interim kill
Animal no	10	10	10	10
Renal tubule adenoma	0	0	0	1
Terminal kill – standard and extended investigations combined
Animal no	50	50	50	50
Renal tubule hyperplasia	14 (2.3)^a	20 (2.3)	17 (2.2)	25** (2.7)
Renal tubule adenoma	7	7	10	10
Renal tubule adenoma, multiple	1	4	9**	3
Renal tubule carcinoma	0	2	1	1
Renal tubule adenoma and carcinoma	8	13	19	13

a: Average severity of lesions in affected animals: 1=minimal; 2=mild; 3= moderate; 4 = marked.

**statistically significant (P≤ 0.01), 

Although an increase in benign tumor incidence was observed in treated groups, the increase was only marginal. Furthermore, no increase in the incidence of carcinomas was observed. The tumors were re-evaluated by a Pathology Working Group (PWG) (Hard et al, 2011). This group considered the tumors were treatment related and the results of this re-evaluation of the tumor findings are presented below. The use of contemporary diagnostic criteria by the PWG resulted in the reclassification of two control renal tumors as an amphophilic-vacuolar (A-V) adenoma and an oncocytoma; neither of which is considered to be relevant to test article administration. A lower incidence of tubule adenomas in the control group meant the combined incidence of renal tumors was now statistically significantly higher in all treated groups. One carcinoma was also downgraded to an adenoma in both the low and mid dose group.

Incidence of renal tubule tumor types in standard and step sections of kidney in male rats of the 2-year study as evaluated by the PWG.

Dose (mg/mL)	0	1.25	2.5	5
Oncocytoma	2	1	0	0
Amphophilic-vacuolar (A-V) tumor	1	0	1	1
Adenoma	3	9	9	9
Multiple	1	3	9	3
Carcinoma	0	1	0	1
Adenomas and Carcinomas combined	4	13*	18**	12*
All tumor variants	4	14	18**	13

* Statistically significant (P ≤ 0.05), **statistically significant (P≤ 0.01),

The PWG expert review of the renal tumors (Hard (2001)) suggested that the mode of action for the formation of these tumors involved two pathological processes; α2u-globulin nephropathy (α2u-N) or an enhancement of chronic progressive nephropathy (CPN). The PWG arrived at this conclusion after examining the findings from the oral 90-day and chronic studies in both rats and mice and the 90-day inhalation studies as well.

α2u-globulin nephropathy

BACKGROUND

Alpha2u-globulin nephropathy is a male rat specific effect and, as such, tumors that occur via this mechanism are not considered relevant to human health. α-2u-Globulin is an 18.5 KDa protein that is synthesized and secreted by the liver (about 50 mg/day) of certain strains of male rats and is freely filtered by the glomeruli. About 60% of the filtered portion undergoes tubular resorption and catabolism and 40% is excreted in urine. The nephropathy evolves due to the modification of the protein in blood by a chemical ligand, so that the complex reabsorbed in the P1 segment of the proximal renal tubules is rendered more resistant to proteolysis and accumulates in the phagolysosomes of the tubule cells. This accumulation is recognized histologically as hyaline droplets, leading to lysosomal overload and the shedding of cells into the lumen. Sustained cell regeneration through chronic exposure to the eliciting chemical ensues and tumors are presumed to arise from within this proliferating cell population. Substances that induce α2u-globulin nephropathy do so by reversibly binding to the α2u protein in the renal proximal tubule. This binding prevents the α2u from being degraded by proteolytic enzymes within phagolysosomes. The α2u accumulates, resulting in proximal tubule necrosis and compensatory cell proliferation, which increases the likelihood of mutation and potentially tumorgenesis.

Criteria to determine whether a substance causes renal tumors by this mode of action have been developed by IARC (1999). The table below describes how the available information for tertiary butyl alcohol satisfies the IARC criteria. Tertiary butyl alcohol meets all the essential criteria and the majority of the supporting IARC criteria for α2u–g nephropathy. It is possible that failure to see a dose response relationship between hyaline droplet severity and renal tumor incidence may be due to the influence of the CPN (see below).

Criteria	Comparison with the criteria
Essential evidence
Renal tumors occur only in male rats	Satisfied. Renal tumors were not observed in female F344 rats or B6C3F1micefollowing chronic exposure
Acute exposure exacerbates hyaline drop formation	Satisfied. An increase in protein droplets was observed in male F344 rats following 10 days inhalation exposure to 1750 ppm T tertiary butyl alcohol. (Borghoff et al (2001))
α2u-Globulin accumulates in hyaline droplets	Satisfied. It was confirmed by Enzyme-linked immunosorbent assay (ELISA) that the accumulating protein observed in the above study was α2u protein (Borghoff et al(2001))
Sub-chronic histopathological changes including granular cast formation and linear papillary mineralization	Satisfied. The PWG (Hard,et al(2011)) reported sporadic basophilic tubules containing cellular debris were detected in 5/10 males of the 20 mg/L group, which were considered the precursor of granular casts. Linear papillary mineralization was observed in all treated males and increased in incidence and severity with dose in the chronic study. A similar observation was not noted in female rats or mice (NTP 1997 and Hard,et al (2013))
Absence of hyaline droplets and characteristic histopathological changes in female rats and in mice	Satisfied. No evidence of hyaline droplet accumulation in mice of either sex or females rats.
Negative for genotoxicity in a battery of tests	Satisfied (see section 5.7)
Supportive evidence
Reversible binding of chemical to α2u-globulin	Satisfied. Application of several biochemical techniques demonstrated that tertiary butyl alcohol reversibly binds to α2u (Williams and Borghoff (2001))
Increased and sustained proliferation in P2 segment of proximal tubules in male rat kidneys	Satisfied. BRDU immunohistochemistry stained kidney sections revealed a statistically significant increase in the labelling index in the renal cortex (where the proximal convoluted tubules are situated) in tertiary butyl alcohol -exposed male rats, but not female rats (Borghoff et al (2001))
Dose-response relationship between hyaline droplet severity and renal tumor incidence	In the 90-day study, an increased accumulation of hyaline droplet was observed with dose. In addition, via the inhalation route, a statistically significant increase in α2u concentration in male rat kidney exposed to 1750 ppm (Borghoff et al (2001)). Renal tumor incidence, however, was not as clearly dose related, with the highest incidence observed in the 2.5 mg/ml group.

Chronic progressive nephropathy

Chronic progressive nephropathy (CPN) is a common, age-related renal disease that occurs in rats which can progress to end-stage kidney disease (Hard et al., 2013). The incidence and severity of this spontaneously occurring disease is a confounding factor in chronic toxicity and carcinogenicity bioassays, particularly if the kidney is a target organ. CPN occurs at higher incidence and with greater severity in males vs. female rats. The histological characteristics of CPN include basophilic tubules, thickened basement membranes, hyaline cast formation and glomerulosclerosis (Hard et al., 1999). Lesions of CPN are detectable in male rat kidney as early as 2 months of age, however in the most advance stages there is associated atypical tubule hyperplasia and renal tubule tumors (Hard et al., 2013). CPN is both a degenerative and a regenerative disease. The regenerative aspect is supported by studies examining the labeling of tubule cells for DNA synthesis as an index of cell proliferation. Using bromodeoxyuridine (Short et al., 1989) or proliferating cell nuclear antigen (PCNA) labeling (Hard and Seely, 2006), these investigations demonstrate that CPN-affected basophilic tubules have a high rate of cell proliferative activity. A retrospective histopathological survey of the kidneys of a substantial number of control male and female F344 rats from chronic studies has shown clear evidence of a quantitative and statistically significant association between advanced stages of CPN severity (particularly end-stage kidney) and the development of low-grade renal tubule tumors (RTT) and its precursor atypical tubule hyperplasia (ATH) (Hard et al., 2012). There is strong evidence that suggests advanced CPN is a risk factor for the spontaneous development of renal tubule tumors (Hard et al., 1997; Hard, 1998) and also with chemicals that induce α2u-N also exacerbate CPN.

The development of CPN appears to be primarily under the control of physiological factors. These factors are mainly dietary (increasing protein exacerbates CPN and decreasing caloric intake protects), and hormonal, particularly involving androgens (Baylis, 1994; Keenan et al., 2000; Hard and Khan, 2004). In 1994, The NTP program, replaced NIH-07 diet (~24% protein), with the NTP-2000 diet (~14% protein) in an effort to reduce spontaneous lesions in rats, including CPN. The tertiary butyl alcohol cancer bioassay was conducted using the NIH-07 diet where a higher incidence and severity of this lesion would be observed. Regardless of diet, CPN lesions are observed in 100% of the control male rats in a survey of 90-day studies, with increased severity of CPN in rats fed the NIH-07 diet (Travlos et al., 2011).

As described by Hard et al. (2009), CPN is a distinctive entity in rats to which there is no counterpart in human disease. A distinction in the features of nephropathy in rats compared to humans is the clinical course of the disease, the relationship to diet and immunological factors, and most distinctively, the presence of the thickened basement membrane around the basophilic, regenerative tubules is an early presentation in CPN. In humans a thickened basement membrane is seen only with atrophic tubules and not associated with degeneration or regeneration as with rats (Hard et al., 2013).

In a re-evaluation of the kidney changes in the NTP (1995) 13- week toxicity and 2-year chronic cancer assay, tertiary butyl alcohol exacerbated CPN in the high-dose males and female rats with a relationship between advanced grades of CPN and renal tumor occurrence.

In the re-elevation of the 13-week and 2-year tertiary butyl alcohol drinking water study by Hard et al. (2011), the following observations were identified:

• Cell debris in the proximal tubules at the junction of OSOM and ISOM were consistent with precursors of granular casts;
• CPN was exacerbated by tertiary butyl alcohol at 13-weeks in male rats only and at 2-years in male and female rats;
• Linear mineralization in the papillary tubules was confirmed in male rats exposed to 5 mg/ml tertiary butyl alcohol in the 2-year study, however this lesion was absent in female rats exposed to 10 mg/ml tertiary butyl alcohol;
• Hyperplasia of the epithelial cell lining of the papilla was characterized after 2-years of exposure to tertiary butyl alcohol as typical of advanced CPN in both male and female rats with no evidence of renal pelvis urothelial hyperplasia. This provided evidence that this was not a direct effect of tertiary butyl alcohol.
• Nephropathy was absent in female rats exposed to tertiary butyl alcohol for 2 years; There was evidence that the renal tubule tumors that developed were related to the advanced CPN diagnosed in male rats.

Overall the PWG concluded that both α2u-N and CPN exacerbation were the only causative factors in the development of renal tumors in male rats exposed to tertiary butyl alcohol for 2-years. Since both α2u-N and CPN do not have human counterparts, the PWG determined that the tertiary butyl alcohol-related changes in the kidney of rats could not be extrapolated for human health risk assessment since they are unlikely to pose any human risk.

The PWG went on to compare the severity of the CPN in rats with renal tumours. The mean grade of high-dose rats with renal tumors was 3.5 compared to 2.9 for rats with no tumors.

Table showing the relationship in male rats of renal tubule adenomas/carcinomas to chronic progressive nephropathy (CPN) demonstrated by NTP and PWG data.

Dose (mg/mL)	0		1.25		2.5		5
	NTP	PWG	NTP	PWG	NTP	PWG	NTP	PWG
Rats with renal tumors and 100 % grade 3 or 4 CPN	8/8 100%	6/7 86 %	13/14 93%	-	191 100 %	-	12/13 93 %	11/13 85 %
Mean CPN for rats with renal tumors	3.5	3.3	3.6	-	3.7	-	3.4	3.5
Mean CPN for rats without renal tumors	2.9	2.7	2.8	-	2.8	-	3.2	2.9

In summary, based on the tertiary butyl alcohol studies conducted by NTP, a re-evaluation of the histological endpoints in these studies with contemporary diagnosis, and associated mechanistic investigations, the data available on tertiary butyl alcohol satisfies all of the criteria outlined by IARC to identify a renal carcinogen as operating through the α2u-N mode of action. Evidence outlined above highlights the role of chronic progressive nephropathy (CPN) as a second mode of action that appears to have a dual role along with the α2u-N in the induction of the low incidence of renal tumors in male rats following chronic exposure to tertiary butyl alcohol. Therefore, the NOAEL for carcinogenicity is the top dose of 5 mg/ml.

Mice

In a 2-year NTP study, groups of 60 male and 60 females B6C3F mice were administered 0, 5, 10 or 20 mg/ml tertiary butyl alcohol (0, 540, 1040 and 2070 mg/kg bw/day in males and 0, 510, 1020 and 2110 mg/kg bw/day in females) via the drinking water. Survival was reduced in high dose males (43 natural or moribund deaths compared to 33 in the controls). As a consequence the interim kill was not conducted. Water consumption in both sexes was similar to the controls.

In this study the thyroid and liver were identified as target organs.

Thyroid gland: The incidence of follicular cell hyperplasia was significantly increased in all treated male groups and in the two top dose female groups. An increased incidence of follicular cell adenoma was observed in top dose females (15 % overall rate compared to 3 % in controls and 0-5% in historical controls). An increased adenoma incidence was also observed in mid, but not top dose males. The NTP study authors postulated that failure to see an increase in the top group males may be due to the reduced survival in this group. However, reduced survival does not appear to fully explain failure to see an increase and therefore, the increase in the mid-dose appears to be due to chance. A follicular cell carcinoma was also observed in top dose males. Overall, there is evidence of an increased incidence of adenomas in the thyroids of females.

Dose (mg/ml)	0	5	10	20
Females
Follicular cell hyperplasia	19 (1.8)^a	28 (1.9)	33* (1.7)	47**(2.2)
Follicular cell adenoma	2/58 (3 %)	3/60 (5%)	2/59 (3 %)	9/59 (15 %)
Historical control	3.4 % (0-5 %)
Males
Follicular cell hyperplasia	5 (1.2)	18** (1.6)	15*(1.4)	18** (2.1)
Follicular cell adenoma	1/60 (2 %)	0/59 (0 %)	4/59 (7 %)	1/57 (2 %)
Historical control	1.7 % (1-2 %)
Follicular cell, carcinoma	0/60	0/59	0/59	1/57 (2 %)
Historical control range	0 %

a: Average severity of lesions in affected animals: 1=minimal; 2=mild; 3= moderate; 4 = marked. T= terminal sacrifice

* Statistically significant (P ≤ 0.05), **statistically significant (P≤ 0.01),

The NTP study authors postulated that although the proliferative lesions may be due to a direct action of tertiary butyl alcohol, they may also be due to altered hepatic microsomal enzymes resulting in perturbation of hypothalamus, pituitary and thyroid (HPT) axis. In addition, an expert evaluation by McClain (2001) focused on possible causes for the thyroid gland changes observed in the mouse bioassay. 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. Administration of certain chemicals at high concentrations, cause a hormone imbalance that 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, similar to the genotoxicity profile for tertiary butyl alcohol. Several investigations have shown that excessive stimulation by TSH alone, in the absence of any chemical treatment, can cause thyroid gland neoplasia in rodents. The opinion of McClain (2001) was 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 both the rat and mouse 13-week studies is consistent with enzyme induction.

Background to mode of action

The key events for this postulated mode of action are as follows:

• Tertiary butyl alcohol induces metabolic activity in the liver that effects thyroid hormone catabolism,
• Circulating levels of thyroid hormones (T3 and T4) decrease,
• There is a compensatory increase in TSH levels leading to thyroid cell proliferation, and
• Continued thyroid follicular cell proliferation results in hyperplasia and increased formation of benign tumors.

The liver toxicity and tumorigenicity profile of tertiary butyl alcohol provide at best only suggestive experimental evidence consistent with the general profile of thyroid carcinogens operating through an enzyme induction mode of action. Tertiary butyl alcohol treatment did not increase the incidence of liver tumors in either rats or mice (NTP, 1995). In addition, although liver weight was increased relative to body weight in the 20 and 40 mg tertiary butyl alcohol/ml male and 40 mg/ml female treatment groups in a 90-day oral drinking water study (NTP, 1995), liver weight to brain weight ratios (a more stable comparison) was not consistently affected in these treatment groups. Liver histopathology was only examined in the 40 mg/ml dose group in the oral subchronic studies and no changes were reported.

In a series of subchronic inhalation studies of tertiary butyl alcohol in rats and mice (NTP, 1997), liver weights were not altered in rats exposed to top exposures of 3500 and 2110 ppm in 18- and 90-day studies, respectively. In mice, however, absolute liver weights were increased in female mice of the 3500 ppm group as well as liver to body weight ratios in both male and females. Increased liver to body weight ratios also were identified in female mice exposed to 1080 and 2100 ppm tertiary butyl alcohol in the 13-week study. Liver to brain weight ratios were increased in the 18-day inhalation study, whereas in the 13-week inhalation study this ratio was only marginally affected in female mice. However, there was no evidence of accompanying histopathological effects in livers, and importantly in thyroids of mice as well, treated at 3500 ppm in the 18-day study or 2100 ppm in the 13-week study.

Paralleling the minimal effects of tertiary butyl alcohol on liver in short-term rodent studies, thyroid follicular cell hyperplasia (key event 3) was not observed in either rats or mice in 18-day or 13-week oral or inhalation studies (NTP, 1995; NTP, 1997). However, thyroid follicular cell hyperplasia was increased in all doses tested in the chronic tertiary butyl alcohol mouse bioassay (NTP, 1995; Cirvello et al., 1995). While the hyperplasia response was dose-related in females (19/58, 28/60, 33/59 and 49/59, respectively), in males the incidence of hyperplasia was equivalently elevated at in dose groups (5/60, 18/59, 15/59, 18/57, respectively). The follicular cell hyperplasia consisted of foci with increased numbers of closely packed follicular epithelial cells, sometimes with minimal papillary folds, that were morphologically indistinguishable from those occurring in control group mice exhibiting similar hyperplasia and tumors.

Although tertiary butyl alcohol -induced effects on liver and thyroid are not inconsistent with a possible thyroid hormone perturbation mode of action, the relatively mild effects alone are inadequate to invoke this mode of action. As noted above, however, hepatic enzyme induction is a key biochemical hallmark of the thyroid catabolism mode of action (McCLain, 1989), and the potential role of such changes has been examined in substantially more detail in a short-term study mode of action investigation in which female B6C3F1 mice were treated with 0, 2 and 20 mg/ml tertiary butyl alcohol in drinking water for 14 days (Blanck et al., 2010). This study characterized tertiary butyl alcohol treatment effects on liver and thyroid organ weights and histopathology, plasma T3, T4 and TSH, total hepatic P450 content, liver microsomal enzyme activity (7-ethoxyresorufin-O-deethylase, EROD; 7-benzoxyresorufin-O-debenzylase, BROD; 7-pentoxyresorufin-O-dealkylase, PROD), and expression levels of CYPs and conjugating enzymes measured by quantitative PCR (CYP1a1, CYP2b9, CYP2b10, CYP3a11; sulfuryltransferases ST1a1, ST2a2, STn; and glucuronyltransferases UGT1a1, UGT2b1, UGT2b5). Importantly, phenobarbital, a well characterized agent inducing thyroid tumors through the thyroid hormone catabolism mode of action (McClain, 1989), was included as a positive control in these studies (80 mg/kg/day, oral gavage).

The profile of tertiary butyl alcohol-induced effects in this study was strongly suggestive of a liver enzyme induction mode of action, albeit at a lesser potency than phenobarbital; the top dose of tertiary butyl alcohol in female mice in the NTP chronic drinking water bioassay was 2110 mg/kg/day (28 mmol/kg) compared to 80 mg/kg/day (0.34 mmol/kg) phenobarbital. After 14 days of treatment, tertiary butyl alcohol did not increase liver weights, while phenobarbital increased absolute and relative weights by 13 and 20 percent, respectively. However, diffuse centrilobular hepatocellular hypertrophy was observed after both tertiary butyl alcohol and phenobarbital treatment. This response was graded as minimal in 1/5 and slight in 1/5 tertiary butyl alcohol treated mice and was restricted to the 20 mg/ml high-dose group; for phenobarbital, minimal and slight grade responses were noted in 4/5 and 1/5 mice, respectively. Plasma thyroid hormones were significantly reduced at both tertiary butyl alcohol doses after 14 days of treatment (T3: 12 and 13%; T4: 15 and 22%, respectively, at 2 and 20 mg/ml); phenobarbital decreased T3 and T4 by 21 and 48 percent, respectively, at 14 days. Compensatory increases in plasma TSH were not observed for either dose of tertiary butyl alcohol and for phenobarbital and there were no changes in thyroid histopathology with either agent.

Tertiary butyl alcohol induced a pattern of changes in metabolic enzyme activity and expression similar to that of phenobarbital. After 14 days of treatment in the 20 mg/ml dose only, tertiary butyl alcohol significantly increased liver P450 content 56 percent, PROD 109 percent, and BROD 1129 percent; a slight but statistically significant increase in BROD was found in the 2 mg/ml dose. EROD activity was not altered at any tertiary butyl alcohol dose. In comparison, phenobarbital respectively elevated changes in P450 content and PROD and BROD activities of 130, 710 and 7075 percent and also produced a modest 1.5-fold in EROD activity. Quantitative PCR transcript analysis revealed that after 14 days of treatment TBA at 20 mg/ml significantly increased expression of CYP2b10 (2085%), CYP2b9 (21%) and Sult1a1 (28%), while only CYP2b10 (68%) and Sult1a1 (20%) were increased at 2 mg/ml. Phenobarbital increased CYP2b10 and Sult1a1 3201% and 58%, respectively, while CYP2b9 was unchanged. Phenobarbital treatment also significantly elevated transcript levels of CYP3a11 (113%), Sult2a2 (825%), Sultn (96%), UGT1a1 (83%) and UGT2b1 (29%). No changes in transcript expression were found at either tertiary butyl alcohol dose following 3 days of treatment; however, phenobarbital-induced expression levels at 3 days were approximately equivalent to those seen at 14 days.

Dose-dependent non-linear toxicokinetic behavior of tertiary butyl alcohol: Implications for human hazard and risk of high-dose specific mouse thyroid follicular cell adenomas.

Since the thyroid adenoma response was restricted only to female mice and the top dose tested, it is reasonable to consider whether the species, sex and dose-dependent thyroid adenoma response might have been associated with onset of high-dose specific non-linear toxicokinetic behavior. In recent years several comprehensive reviews have emphasized that rodent carcinogenicity responses restricted to high test doses may have questionable human health hazard and risk relevance. Thus, high-dose specific saturation of metabolic processes, including toxicokinetics, may result in transition to novel modes of action unique to those high dose levels and which are not related to alternative modes of action operating at lower animal doses and substantially lower real-world human exposures (Foran et al., 1997; Slikker et al., 2004a,b; Barton et al., 2006; Carmichael et al., 2006; Doe et al., 2006). Recently, the likely lack of human relevance of high-dose toxicity findings observed only under conditions of doses that exhibit saturation of metabolic processes has been recognized in OECD guidance for dose selection in animal bioassays (OECD, 2011). In dose selection guidance offered for the Extended One-generation Reproduction Test, OECD recommended the following regarding appropriate dose selection:

“If TK data are available which indicate dose-dependent saturation of TK processes, care should be taken to avoid high dose levels which clearly exhibit saturation, provided of course, that human exposures are expected to be well below the point of saturation. In such cases, the highest dose level should be at, or just slightly above the inflection point for transition to nonlinear TK behaviour.”

Although the above guidance was offered for a reproduction study, this recommendation also is appropriate for dose selection in other animal bioassays including carcinogenicity studies, and particularly so for substances that are not regarded as genotoxic as is the case for tertiary butyl alcohol (McGregor, 2010).

The metabolism of tertiary butyl alcohol suggests that it is reasonable to predict potential high-dose specific metabolic saturation. As reviewed in McGregor (2010), the primary metabolism of tertiary butyl alcohol in both rodents and humans is mediated through cytochrome P450 oxidation of the tertiary butyl alcohol methyl group, resulting in formation of 2-methyl-1,2-propanediol and 2-hydroxyisobutyrate. Cytochrome P450 metabolic oxidation is commonly associated with vulnerability to high-dose metabolic saturation. Tertiary butyl alcohol is not a substrate for alcohol dehydrogenase, and lesser amounts of tertiary butyl alcohol metabolism proceed through direct glucuronidation of the parent molecule.

There is ample evidence that tertiary butyl alcohol exhibits dose-dependent non-linear toxicokinetics in rats following intravenous or inhalation dosing. Following intravenous dosing of tertiary butyl alcohol at 37.5, 75, 150, or 300 mg/kg bw, the AUC0-∞ increased disproportionately at the 300 mg/kg bw dose relative to the next lowest dose of 150 mg/kg bw (Poet et al., 1997). The top intravenous dose exhibiting metabolic saturation in this study was lower than the top oral drinking water doses of 420 and 650 mg/kg bw/day for male and female rats, respectively, in the tertiary butyl alcohol carcinogenicity bioassays (McGregor, 2010), suggesting that the high oral doses in the rat bioassay were likely above metabolic saturation. Because tertiary butyl alcohol is rapidly absorbed following oral administration (maximum blood concentrations are reached within 1 hour after dosing), and its primary metabolism is through oxidative hepatic metabolism, the systemic toxicokinetics of tertiary butyl alcohol are likely equivalent following dosing via either the intravenous or oral routes of administration. This possibility is substantiated by the observation that the terminal blood elimination half-life (t1/2β) of tertiary butyl alcohol following oral administration of tertiary butyl alcohol in rats is in the order of days versus hours when single oral doses are in the range of 1500 to 2000 mg/kg bw (reviewed in McGregor, 2010).

Leavens and Borghoff (2009) also demonstrated high-dose specific non-linear toxicokinetic behavior of tertiary butyl alcohol in rats following single 6 hour inhalation exposures to 250, 450 and 1750 ppm tertiary butyl alcohol. An examination of the graphically-presented toxicokinetic data in this study indicate that a 7-fold increase in exposure (250 ppm to 1750 ppm) resulted in an approximate 14-fold disproportionate increase in tertiary butyl alcohol Cmax blood concentrations in male rats. In addition, repeated inhalation exposures for 8 days reduced the blood Cmax in male rats relative to single dose values (2250 µM vs 4250 µM), suggesting a metabolic compensation (induction) response associated with saturated metabolism. McGregor (2010) estimated that a 2100 ppm 6 hour inhalation exposure to male rats (greater than the 1750 ppm exposures used in Leavens and Borghoff) resulted in an approximate systemic tertiary butyl alcohol dose of 312 mg/kg bw, further supporting the conclusion that the top oral dose of 420 mg/kg bw/day in male rats in the NTP rat bioassay likely exceeded metabolic saturation.

Evidence of non-linear toxicokinetic behavior in mice also has been clearly demonstrated in mice in which single dose intraperitoneal administration of tertiary butyl alcohol at doses of 5, 10 and 20 mmol/kg bw (370, 741 and 1482 mg/kg bw) resulted in respective AUC values of 28, 96 and 324 mmol.hrs/L (Faulkner and Hussain, 1989). Thus, a 4-fold increase in dose (5 to 20 mmol/kg bw) resulted in an 11.6-fold increase in systemic AUC; metabolic saturation may have been present even at the next lowest dose of 10 mmol/kg bw in which a 2-fold increase in dose (5 to 10 mmol/kg bw) resulted in a 3.4-fold increase in AUC. Importantly, the top intraperitoneal dose of 1482 mg/kg bw used in this study was substantially lower than the top bioassay of 2110 mg/kg bw/day in female mice exhibiting thyroid adenomas in the NTP bioassay. These data indicate that the high-dose specific thyroid adenomas occurred under conditions of non-linear toxicokinetic behavior due to saturated oxidative metabolism. Again, due to rapid oral absorption of tertiary butyl alcohol, the systemic toxicokinetics following intraperitoneal administration are likely to parallel that of oral absorption.

Direct human exposures to tertiary butyl alcohol are primarily expected in industrial applications due to its extensive and primary uses as a closed-system intermediate in the manufacture of other products and in various solvent applications (McGregor, 2010). Occupational German MAK and ACGIH TLV values of 20 ppm (62 mg/m3) have been established for tertiary butyl alcohol. Exposure to a 20 ppm occupational exposure limit would result in an estimated systemic dose of 8.9 mg/kg bw (62 mg/m3 x 10 m3 inhaled air per 8 hr workshift = 620 mg tertiary butyl alcohol inhaled ; 620 mg/70 kg body weight worker = 8.9 mg/kg), which is 237-fold lower than the top oral drinking water dose resulting in thyroid adenomas in the NTP mouse bioassay. The estimated inhaled occupational dose of 8.9 mg/kg bw likely overestimates actual systemic doses of tertiary butyl alcohol in that it assumes 100 percent retention of inhaled tertiary butyl alcohol. Consideration of the DNEL for workers applied to this assessment of 2.7 mg/m3 for long term, inhalation, systemic effects, reduces the estimated exposure by a further factor of 20.

As noted above, both reviews addressing appropriate dose selection strategies for animal toxicity testing and an OECD toxicity test dose selection guideline suggest caution in selection of high-dose toxicity test doses that exhibit evidence of metabolic saturation and associated high-dose specific non-linear toxicokinetics in that toxicity findings doses observed under such conditions have limited human health hazard and risk implications if the inflection point for onset of non-linear toxicokinetics is well separated from real-world human exposures. These conditions are fulfilled with tertiary butyl alcohol with respect to the health implications of high-dose and female mouse specific thyroid adenomas. The NTP oral bioassay dose producing female mouse thyroid adenomas (2110 mg/kg bw/day) is likely well above the onset of metabolic saturation for oxidative metabolism of tertiary butyl alcohol, the primary route of tertiary butyl alcohol metabolism and systemic clearance in animals and humans. Importantly, this dose also is well above a human occupational dose of 8.9 mg/kg bw/day conservatively estimated to result with exposures to the MAK and ACGIH TLV values of 20 ppm tertiary butyl alcohol.

Use of toxicokinetic data to provide a data-informed selection of the appropriate top dose in animal toxicity tests has recently been described as a Kinetically Derived Maximum (KMD) dose selection strategy (Saghir et al., 2012). The KMD dose selection strategy specifically emphasizes that toxicokinetic data, when available, can and should be used as an alternative to conventional top dose selection strategies based on Maximum Tolerated Dose (MTD). KMD considerations are specifically designed to proactively avoid generation of animal toxicity findings that ultimately have limited if any value to identification of true human health hazards and risks. The KMD approach also has great value for reducing significant laboratory and animal resources (including reduction in unnecessary animal suffering) that otherwise might be applied to further explore the potential (lack of) health relevance of toxicity findings observed at MTD doses where a KMD approach could have been used as a dosing alternative.

The toxicokinetic data described above indicate tertiary butyl alcohol would have been a strong candidate for a KMD-based dose selection strategy for the chronic bioassay. Application of the OECD guidance to select the top test dose based “at, or just slightly above the inflection point for transition to nonlinear TK behaviour” would likely have justified a top drinking water dose in the NTP bioassay for mice in the range of 10 mg/L versus the 20 mg/L that was actually used. Had such a KMD strategy been employed, the end result would have been that tertiary butyl alcohol may not have been identified as a high-dose specific mouse thyroid tumorigen.

In conclusion, consideration of the high-dose specific non-linear toxicokinetic behavior of tertiary butyl alcohol relative to real-world human exposures provides valuable additional weight-of-evidence information addressing the likely lack of human health relevance of high-dose specific female mouse thyroid adenomas observed in the NTP oral drinking water tertiary butyl alcohol carcinogenicity bioassay.

A NOAEL for carcinogenicity of 10 mg/ml is derived based on the increase in thyroid tumors observed in top dose females.

Summary

The carcinogenicity of tertiary butyl alcohol has been investigated in rat and mouse. In the rat study, a very slight increase in renal tumors was observed in the kidneys of male rats. It has been demonstrated that tertiary butyl alcohol causes α2u–g nephropathy and also exacerbates the progression of CPN. Failure to see tumors in female rats suggests the α2u–g nephropathy is an important mechanism in the formation of these tumors in males. On this basis, the tumors are not considered relevant to humans.

In mice, tumors were observed in the thyroid of female mice. It was postulated that these tumors occur as a result of increased metabolism of thyroid hormone; to which humans are considerably less sensitive. Given that the tumors were benign and were observed in a single species and sex at the maximum tolerated dose the extent of the concern is low. Additionally, high-dose specific non-linear toxicokinetic behavior of tertiary butyl alcohol further supports the likely lack of human relevance of the high-dose female mouse specific thyroid tumors.

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Carcinogenicity: via oral route (target organ): urogenital: kidneys