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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study was conducted under GLPs and followed the OECD 421 guideline.
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: OECD 421 (Reproduction/Developmental Toxicity Screening Test).
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
-Test substance name (as cited in study report): Tertiary butanol (t-butyl alcohol, TBA)
-Test substance category: Chemical intermediate
-Supplier: Haltermann Ltd. (Houston, TX)
-Lot number: HL30408005
-Purity: 99.6%
-Physical description: Waxy solid at room temperature. Forms a clear, colorless solution in water
-Date received: April 8, 2003
-Expiration date: No certification of stability was available. A nominal expiry date of 1 year following packaging was allocated by the Sponsor.
-Storage: Room temperature
-Analysis: Documentation of the identity, purity, composition, and other characteristics that define the test substance and the maintenance of these records was the responsibility of the Sponsor. A certificate of analysis was provided to the testing laboratory.

Test animals

other: Albino rats, Sprague-Dawley derived strain (outbred), Crl:CD(SD) IGS BR.
Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories, Inc., Kingston, New York
- Age at study initiation: approx. 8 weeks
- Acclimation period: 14 days
- Identification: Ear-Tag
- Housing: F0 animals were housed individually in clean, suspended, stainless steel wire-mesh cages in an environmentally controlled room (except during mating when one male and one female were housed together). Starting on gestation day 18 and during lactation, each dam was housed with its litter in a plastic shoebox cage with ground corncob bedding (Bed-O’-Cobs). Fresh bedding was provided every 10-11 days or as needed throughout lactation. Each cage was supplied with a glass feeder jar with a stainless steel lid. F1 weanlings retained after PND 21 were housed initially in pairs (males and females separate), then individually after PND 22 (as F0 animals).
- Diet: PMI Nutrition International, LLC Certified Rodent Lab Diet® 5002 (meal) ad libitum. Clean jars of fresh food were provided at least weekly and at each interval when feed consumption was recorded.
- Water: Tap water (Elizabethtown Water Company, Westfield NJ) was available ad libitum through an automated watering system
-Weight at study initiation: 248-300 g (males); 165-235 g (females)
-Randomization: Animals judged suitable based on body weight and other pretest evaluations were randomly assigned to control or test groups to provide similar group and individual body weight means.

- Temperature (°C): 19-24°C
- Humidity (%): 44-81%.
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

-Study initiation: May 23, 2003
-Date of animal receipt: June 3, 2003
-Dosing initiation: (F0 animals): June 17, 2003
(F1 treated animals): August 27, 2003
-Dosing termination: (F0 males): August 19, 2003
(F0 females): September 4, 2003
(F1 treated pups): September 11, 2003
-Terminal sacrifice: (F0 males): August 20, 2003
(F0 dams and excess pups): August 11-September 07, 2003
(F1 treated pups): September 3-12, 2003

Administration / exposure

Route of administration:
oral: gavage
other: deionised water.
Details on exposure:
Formulations were prepared weekly. The bulk container of tertiary butyl alcohol was heated to 30 °C to liquefy the test material. The requisite quantity of test material was weighed into a pre-warmed calibrated beaker and the necessary amount of warmed vehicle (deionized water) added with mixing to prepare the specific dose. Formulations were stored in labeled containers with nitrogen caps at room temperature. Appropriate aliquots were removed each day. Dosing formulations were re-mixed by inversion prior to opening for dosing.

Dose levels were 0, 64, 160, 400, or 1000 mg/kg bw/day of the test material. Animals were dosed by oral gavage seven days per week including holidays. The dose volume was 5 mL/kg body weight.

Dosing volumes were calculated for each animal using the most recent body weight recorded, except for F0 females during the period of gestation after gestation day (GD) 17, when the dose volume remained fixed according to the GD 17 body weight until the animal had completed parturition. If at the time of dosing an animal had started parturition, but had not completed the process by approximately 1400 hours, it was not dosed that day. For females that had completed parturition, dosage volume reverted to being determined by the most recent body weight recorded. Controls received a 5 mL/kg body weight dose of distilled water.

F0 males were dosed once daily, seven days/wk for 4 weeks prior to first pairing for mating. Dosing continued through the mating periods. F0 females were dosed once daily, seven days/wk for 4 weeks prior to first pairing for mating. Dosing continued through mating, gestation and lactation until PND 20. Females that did not produce a litter continued treatment for up to 24 days following completion of the mating period and were terminated on the 25th or 26th day after the last exposure to a male. F1 offspring were not treated directly prior to weaning. Selected F1 weanlings, 1 male and 1 female from each available litter, were dosed once daily on PND 21-27, at the same dosages used for the F0 parents, with termination on PND 28.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Formulation concentrations and stability analyses were performed by the Huntingdon Life Sciences Analytical Chemistry Laboratory. As the formulations comprised true aqueous solutions, homogeneity analysis was not necessary.
Trial formulations were prepared 8 days prior to the start of dose administration, allowed to stand in sealed containers for 4 hours at room temperature, remixed and then sampled in duplicate. One set of samples was analyzed for concentration using a method previously validated by the Huntingdon Life Sciences Analytical Chemistry Laboratory. This analysis was intended to demonstrate 4-hr stability in support of daily formulation preparation. Briefly, the analytical method involved the dilution of tertiary butyl alcohol with methanol, followed by quantification of the compound using gas chromatography-FID detection. The second set of samples was frozen at -70 °C or below pending a satisfactory outcome of the first analyses. The trial formulations were then stored at room temperature for 8 days, remixed, again sampled in duplicate and analyzed for concentration in the same manner as samples taken from the first day. These data were intended to support weekly formulation preparation. Dosing formulation samples were taken at the beginning, around week 5, and near the end of the study. These samples were analyzed for concentration in the same manner as previous samples.
Details on mating procedure:
Within each treatment group the animals were paired, 1 male to 1 female in the male’s cage, until evidence of mating was seen or for a maximum of 2 consecutive weeks. The females were observed early each morning for the presence of a vaginal plug or of sperm in a vaginal smear. The day on which positive evidence of mating was observed was defined as GD 0. Once mated, the female was removed from the mating cage and was housed individually throughout the remainder of the study. After the mating period was over, females without evidence of copulation were also returned to individual housing and monitored for visible signs of pregnancy.
Duration of treatment / exposure:
F0 males: 9 weeks
F0 females: 4 weeks prior to mating through lactation day 21
F1 directly-treated pups: PND 21-27
Frequency of treatment:
Doses / concentrations
Doses / Concentrations:
64, 160, 400, 1000mg/kgbw/day
nominal in water
No. of animals per sex per dose:
F0: 12 animals/sex/group
F1: 1 animal/sex/litter (10/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
Randomly assigned groups of 12 male and 12 female rats received tertiary butyl alcohol by oral gavage at dose levels of 0, 64, 160, 400, or 1000 mg/kg bw/day; animals were dosed 7 days a week. Males were dosed from 4 weeks prior to mating until termination (approximately 63 doses). Females were treated from the beginning of the premating period through gestation until termination on PND 21. Selected weanlings (1/sex/litter) were dosed once daily on PND 21-27 at the same dose levels received by their parents, then terminated on PND 28. Pups not selected for test substance administration were euthanized on PND day 21 or 28.

Viability and post-dose observations were checked daily. Physical observations, body weights, and feed consumption were performed on a weekly basis for males and non-gravid females throughout the study; similar examinations were made for females during the pre-mating period and at select intervals for gravid females during gestation and lactation. Feed consumption measurements were suspended during the mating trial.

From GD 18 onward, gravid dams were examined twice daily for signs of parturition. After parturition, litters were observed twice daily during lactation. Pups were given a gross physical exam and sexed on PND 0, then examined at select times throughout lactation. On PND 4, litters were culled to 10 pups/sex. Pre-weaning pups were weighed on PND 1, 4, 7, 14 and 21. Directly-dosed pups were weighed on PND 21, 24 and 28; food consumption for this group was recorded for PND 21-24 and 24-28.

At necropsy, macroscopic and microscopic examinations were performed on all parental animals. The liver, kidneys, testes and epididymides were weighed. The ovaries with oviducts, testis, epididymis, seminal vesicle with coagulating gland, prostate, thyroids with parathyroids, kidneys, and gross lesions were fixed in 10% buffered formalin. The testis, epididymis, ovaries and thyroids with parathyroids were examined microscopically from the control and high-dose animals. Sperm parameters were evaluated.

Previous studies performed under the National Toxicology Program included a 13-week and a 2-year toxicity study in rats, both by administration in the drinking water at levels of 2.5 to 40 mg/mL (13 week study) and 1.25 to 10 mg/mL (2 year study). The 40 mg/mL dosage was lethal for most animals in the 13-week study and final body weight of males at 10 and 20 mg/mL was 12% and 17% less, respectively, compared to controls. Liver and kidney weights, and the incidence of mineralization in the kidney, were increased at all dosages and there was increased incidence/severity of nephropathy. There was also increased activity of sorbitol dehydrogenase and alanine aminotransferase, with decreased urine volume and increased urine specific gravity. Hyperplasia and inflammation of the urinary bladder epithelium was observed only at dosages in excess of 10 mg/mL.

The 10 mg/mL dosage in drinking water from the 13-week study equated to approximately 750 mg/kg bw/day received dose. Based on the results of that study, the upper dosage in the present study, 1000 mg/kg bw/day, was expected to cause some toxicity in the F0 animals, in terms of effects on body weight, liver and kidney weight and more particularly in males, possibly the exacerbation of chronic progressive nephropathy. The 400 mg/kg bw/day dosage was expected to cause some lesser/marginal toxicity, while the lower dosages were expected to be without discernible toxicity.


Maternal examinations:
Observations were made at least twice daily, prior to dosing during the treatment period, and again late in the day.

Each animal was checked for overt signs of toxicity prior to dosing and any changes from the preceding observations were recorded. Each dosed animal was observed approximately one to two hours after dosing. An additional observation was made at approximately 4 hours after dosing for the first 4 days of test substance administration.

Animals were examined prior to randomization into treatment groups, and then once weekly until euthanasia, for any abnormality or sign of toxicity. During the treatment period, this evaluation was performed prior to dosing. For mated females, this corresponded to GD 0, 7, 14, 20 and PND 1, 7, 14 and 21.

Body weights were recorded at the time of randomization into test groups, on the day treatment was initiated, and weekly thereafter until pairing for mating. Mated females were weighed on gestation day 0, 7, 14, 17, and 20 and for those animals that delivered litters on PND 1, 4, 7, 14, and 21. If parturition was complete, females were also weighed on PND 0. Females without evidence of mating were weighed weekly until termination unless they delivered a litter. A terminal body weight (non-fasted) was also recorded for each animal.

Food consumption was recorded weekly during the pre-mating period but not during the mating period. Weekly measurements continued after the mating period and for females without signs of mating until termination. Food consumption for mated females was recorded for GD 0-7, 7-14, and 14-20 and for those animals that delivered litters for PND 1-4, 4-7, and 7-14.

After GD 18, examination for signs of parturition was made twice daily (morning and afternoon). Litters were observed as soon as possible after parturition for number of live and dead pups, any pup abnormalities, and sex of each pup. After completion of parturition, litters were observed twice daily. Number of dead pups, unusual observations, signs of deficient maternal care, and pups without milk in the stomach were recorded. On PND 4, litters of more than 10 pups were randomly culled to that number with equal sex distribution where possible.

Males were terminated after at least 9 weeks of treatment. Females with litters were terminated on lactation day 21. Females that mated but failed to deliver a litter were terminated at 25 to 26 days after evidence of mating. The single female that showed total litter loss before weaning was retained on treatment until it was clear that further mating in the study was not necessary, and then terminated on lactation day 9.

A macroscopic examination was performed on all animals including examination of: external surfaces; all orifices; cranial cavity; neck and its associated tissues and organs; thoracic, abdominal and pelvic cavities and their associated tissues and organs; and external surfaces of the brain. The number of implantation sites was recorded for each female. Apparently non-pregnant status was confirmed by ammonium sulphide staining. Any gross lesions or tissues with significant findings were preserved in 10% neutral buffered formalin. The testis, epididymis, seminal vesicles with coagulating gland, prostate, ovaries with oviduct, thyroids with parathyroids, and kidneys were preserved in formalin.

The kidneys (for both sexes), liver (for both sexes), testes and epididymides were removed at necropsy, trimmed to remove fat and other contiguous tissue and were weighed as soon as possible after dissection. Paired organs were weighed separately.

The ovaries (2), testis (1), epididymis (1) and thyroids with parathyroids (for both sexes) were examined microscopically from the control and high-dose groups.
Fetal examinations:
Offspring not selected for direct treatment were sacrificed on PND 21. Offspring selected as reserves and not directly treated were sacrificed on PND 23 or 28. Offspring treated directly were sacrificed on PND 28.

A necropsy was performed on all animals. This included a macroscopic examination of: external surfaces; all orifices; cranial cavity; neck and its associated tissues and organs; thoracic, abdominal and pelvic cavities and their associated tissues and organs; and external surfaces of the brain. Any gross lesions or tissues with significant findings were preserved in 10% neutral buffered formalin.

A macroscopic external examination was performed for all pups culled on postnatal day 4. All pups were externally normal and were discarded without necropsy. Absence of milk in the stomach (visible through the skin) was noted.

A macroscopic external examination was performed for all pups found dead or sacrificed prior to weaning. A necropsy was performed where practical, including examination of the thoracic, abdominal and cranial contents for any grossly apparent abnormalities, but no tissues were preserved. Where possible, the presence or absence of milk in the stomach was determined.
Evaluation of equality of group means was made by an appropriate statistical method, followed by a multiple comparison test if needed. Bartlett’s test was performed to determine if group variances were equivalent. For all parameters, the variances were equivalent and parametric analysis procedures were used. The parametric method used was the standard one-way analysis of variance (ANOVA) using the F ratio to assess significance. If significant differences among the means were indicated, additional tests were used to determine which means were significantly different from the control: Dunnett’s. Bartlett’s test for equality of variance was conducted at the 1% significance level; all other statistical tests were conducted at the 5% and 1% significance levels. The following parameters were analyzed statistically: body weights, body weight changes, feed consumption, organ weights, organ weight ratios, mean number of uterine implantations, pup weights [by sex and composite (each weighing interval during lactation)], and litter size.

A Fischer Exact Test with Bonferroni correction was performed to identify differences between control and treatment groups. All such tests were conducted at the 5% and 1%, two-sided risk levels. The following parameters were analyzed: mortality incidence, mating indices, pregnancy rates, and fertility indices.

Statistical analysis of pathological abnormalities was not necessary as there were no abnormalities present in the treated animals.

Female MI = No. of females mated/No. of females exposed to males
Male MI = No. of males with confirmed mating/No. of males placed with females

Female FI = No. of females pregnant/No. of females exposed to males
Male FI = No. of males with females pregnant/No. of males placed with females

PI = No. of females pregnant/No. of females mated

GI = No. of females with liveborn/No. of females with confirmed pregnancy

Offspring viability indices

VI = No. of pups alive on lactation day 4[precull]/No. of liveborn pups

LI = No. of pups alive on PND day 21/No. of pups alive on PND 4 [postcull]

LBI = Total No. of liveborn pups/total No. of pups born.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
There were no incidences of mortality during the study.

1000 mg/kg bw/day: apparent central nervous system (CNS) toxicity from the first day of dosing, characterized by unresponsiveness/lethargy and some ataxia; some animals also exhibited increased vocalization and rapid breathing. Effects were generally of mild intensity among females with the effects generally appearing 1-2 hours after dosing and resolving within the 24-hour period between doses. Intensity of symptoms was similar during the study.
400 mg/kg bw/day: similar CNS findings of mild intensity in about half of the females, occurring after about 2 weeks of dosing. Effects became indiscernible after about the fourth week of dosing.
No discernible clinical signs of toxicity at lower dosages of the test substance.

BODY WEIGHT: No significant effects in females.

Females: No effect on female body weight development at any of the dosages until pregnancy was established. At 1000 mg/kg bw/day, there was a decrease in body weight gain in the last few days of gestation, leading to an approximate 6% deficit in body weight at GD 20. The difference in weight gain but not the absolute difference was statistically significant. This slight weight difference remained until the third week of lactation when the high dose animals showed a net gain in weight, while the other groups lost some body weight.

No effect until the lactation period for females in the highest dose group when consumption over the first 2 weeks of lactation was reduced approximately 15%.

There was no statisitically significant effect on mating index, fertility index, pregnancy index, gestation index or length of gestation.

No test substance-related effects were observed.

There was no test substance-related effect on female organ weights that were collected (kidneys and liver).

There were no microscopic findings in the reproductive tissues or thyroids associated with the administration of the test substance at 1000 mg/kg bw/day. The left testis of one animal in the high-dose group contained a small number of tubules totally depleted of germ cells. This is a relatively common background finding in rats of this strain and the single occurrence was considered to be unrelated to tertiary butyl alcohol administration

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
Effect level:
160 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
There was no effect on the number of implantations per pregnancy. There was a significant reduction in the number of live born per pregnancy at 1000 mg/kg bw/day and an increase in the number of stillborn pups. The mean litter size for the high-dose group was only 10 per litter on PND 1 as compared with 14 or 15 in other groups. Subsequently, there was significantly reduced pup survival at the high dose with only 80% survival to PND 4 as compared with close to 100% in the other groups. Most of this was attributed to one early total litter loss in the group. After PND 4, pup survival was optimal or near optimal in all the groups. At the normal weaning date of PND 21, litter size in the high-dose group was substantially reduced (by 50%) for 3/10 surviving litters in the groups as a result of early pup losses. There was no meaningful inter-group differences in live pup sex ratio for the litters.

No effects on pups were observed during the lactation period. There were no clinical signs of toxicity or mortality among the offspring after direct tertiary butyl alcohol treatment at any of the dose levels.

Offspring born to dams treated with tertiary butyl alcohol at 1000 mg/kg bw/day exhibited lower mean body weight than the control offspring. For both sexes, there was an approximate 10% deficit in weight at PND day 1, increasing to approximately 15% by PND 7 at which point it achieved statistical significance. This deficit was still present at PND day 14 but then decreased in late lactation such that by PND 21, the mean weight for males and females was about 11% and 6% less than controls, respectively. No effects were observed at lower doses.

Pups born to dams treated with tertiary butyl alcohol at 1000 mg/kg bw/day during pregnancy and lactation, then directly treated with tertiary butyl alcohol daily at the same dose level from PND 21-27, exhibited lower mean body weight than the control group. The initial differences from control were approximately -13% and -6% for males and females, respectively, arising from the deficits acquired peri-natally. Direct tertiary butyl alcohol had no further effect on body weight gain of either male or female offspring over the one-week direct dosing period after weaning.

There appeared to have been no effect of direct tertiary butyl alcohol treatment on food consumption, although substantial contamination of the feed jars by the pups made unequivocal judgments impossible.

For all pups, both non-selected and treated, there was only a low, sporadic incidence of minor findings.

Effect levels (fetuses)

Dose descriptor:
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables


Stability: Stability was determined for the low and high dose levels. The preliminary mixes of tertiary butyl alcohol in water were stable for 8 days when stored at room temperature and the mean % nominal values of all samples were within ±10% of initial concentrations.

Dose Confirmation: All groups were assayed in duplicate on 3 occasions. The % nominal values for all dose groups were within the test laboratories SOP specified limits. The concentrations of duplicates of the samples were within ±10% of each other, and the means of concentration were within ±10% of nominal.

Applicant's summary and conclusion

There were no adverse effects on mating performance, fertility, or reproductive organs in a reproduction and developmental toxicity screening test in which male and female Sprague-Dawley rats received tertiary butyl alcohol by gavage at dose levels up to 1000 mg/kg bw/day. Developmental toxicity was evidenced by reduced body weights and increased mortality in the offspring of dams receiving 1000 mg/kg bw/day. Mild to moderate toxicity, including CNS effects, decreased body weight gain, and decreased food consumption, was observed in adults that dose level. The NOAEL for developmental/ reproductive effects was 400 mg/kg bw/day while the overall NOAEL for the study was 160 mg/kg bw/day.

In a well-conducted reproductive and developmental screening study in rats, there was no clear evidence of an adverse effect on sexual function, fertility or post-natal development in the absence of toxicity in the parental generation. Fetotoxicity was evidenced by reductions in mean litter size, a decrease in number of live born pups, an increase in the number of stillborn pups, reduced pup body weights, and increased mortality in the offspring of dams receiving approximately 1000 mg/kg bw/day by oral gavage. This dose level had been selected to cause some toxicity in the F0 animals. At this dose level, systemic toxicity, including reversible CNS effects, reduced feed consumption, and reduced weight gain was observed in both sexes of the parental generation in the present study. Based on this information, tertiary butyl alcohol is not a reproductive toxicant and is not selectively toxic to the developing fetus. Tertiary butyl alcohol is not classified for “Developmental or Reproductive Toxicity” according to the GHS guidelines.
Executive summary:

In a reproduction/developmental toxicity screening study, tertiary butyl alcohol was administered to 12 Sprague-Dawley rats/sex/group by oral gavage at dose levels of 0, 64, 160, 400, and 1000 mg/kg bw/day for up to 63 days in males and from 4 weeks prior to mating through PND 21 in females. There were no adverse effects on any reproductive parameters including mating index, fertility index, pregnancy index, or gestation index.

For dams receiving 1000 mg/kg bw/day tertiary butyl alcohol through gestation and lactation, there was a significant reduction in mean litter size, a decrease in the number of live born per pregnancy, an increase in the number of stillborn pups, increased pup mortality up to PND 4, and a decrease in mean pup body weight at birth which continued to weaning. At this dose level, mild to moderate transient systemic toxicity was observed in both sexes in the parental generation including reversible CNS effects such as lethargy and ataxia, and reduced feed consumption and weight gain.

No significant toxicity was observed at any other dose level. The NOAEL for developmental/reproductive effects was 400 mg/kg bw/day. The NOAEL for overall toxicity was 160 mg/kg bw/day