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EC number: 200-889-7
CAS number: 75-65-0
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.
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.
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
Table of neoplastic lesions in kidneys of males from a 2-year rat
Renal tubule adenoma
Terminal kill – standard and extended investigations combined
Renal tubule hyperplasia
Renal tubule adenoma, multiple
Renal tubule carcinoma
Renal tubule adenoma and carcinoma
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.
Amphophilic-vacuolar (A-V) tumor
Adenomas and Carcinomas combined
All tumor variants
* 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
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
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).
Comparison with the criteria
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)
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
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
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.
Rats with renal tumors and 100 % grade 3 or 4 CPN
Mean CPN for rats with renal tumors
Mean CPN for rats without renal tumors
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.
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.
Follicular cell hyperplasia
Follicular cell adenoma
2/58 (3 %)
2/59 (3 %)
9/59 (15 %)
3.4 % (0-5 %)
1/60 (2 %)
0/59 (0 %)
4/59 (7 %)
1/57 (2 %)
1.7 % (1-2 %)
Follicular cell, carcinoma
Historical control range
a: Average severity of lesions in
affected animals: 1=minimal; 2=mild; 3= moderate; 4 = marked. T=
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:
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
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
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.
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
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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