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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study was conducted according to EPA OPPTS 870.3700 and OECD 414 guidelines and the Korea National Institute of Environmental Research (KNIER) TCCA-Good Laboratory Practices Test Guidelines. This study was also conducted under the international guideline for developmental toxicity studies [International Conference on Harmonization (ICH), 1994]. The Institutional Animal Care and Use Committee of Chonnam National University approved the protocols for the animal study, and the animals were cared for in accordance with the Guidelines for Animal Experiments of Chonnam National University.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tertiary Butyl Acetate; (TBAc); tert-butyl acetate
- Source: Sigma-Aldrich Co. (Milwaukee, WI)
- Analytical purity: >99.0%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Inc. (Seoul, Republic of Korea) from a specific pathogen-free colony
- Age at receipt: 10 weeks
- Acclimation period: 7 days
- Age at study initiation: approx. 11 weeks
- Housing during acclimation period: Housed individually in clean, suspended, stainless steel wire-mesh cages for 7 days following receipt in an environmentally controlled room.
- Housing for mated females: Housed individually in clear polycarbonate cages with stainless steel wire lids.
- Diet: ad libitum access to commercial rodent chow from Samyang Feed Co, Wonju, Republic of Korea
- Water: ad libitum access to tap water that had been sterilized by UV-irradiation
-Method of animal distribution: 88 healthy females were randomly assigned to four treatment groups.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3
- Humidity (%): 50 ± 10
- Air changes (per hr): 13-18 room air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test chemical was dissolved in corn oil and was prepared fresh daily before treatment. The vehicle and test substance formulations were administered orally by gavage once daily during Gestation Days 6-19 to 22 pregnant female rats at dose levels of 0, 400, 800 and 1600 mg/kg bw/day. The test mixture was administered at a dose volume of 5 mL/kg body weight; the control rats received an equivalent volume of corn oil alone. The necessary daily volume of test chemical needed to achieve the target dose was calculated in advance based on the most recently recorded body weight of each individual animal.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
PURITY ANALYSIS: The chemical purity was determined by gas chromatography; no additional information was provided.
Details on mating procedure:
Two females were placed into a cage with a non-treated male rat overnight. Successful mating was confirmed by the presence of sperm in the vaginal smear, and the following 24 hours was designated as Day 0 of gestation (GD 0). The mated females were housed individually in clear polycarbonate cages with stainless steel wire lids.
Duration of treatment / exposure:
14 days
Frequency of treatment:
once daily
Duration of test:
21 days
No. of animals per sex per dose:
22 inseminated female animals per dose; no information on males other than 2 females were placed in a cage with a single male for mating.
Control animals:
yes, concurrent vehicle
Details on study design:
Doses of 500, 1,000, 1,500, and 2,000 mg/kg bw/day were administered to six pregnant rats per group in a dose range-finding study. At 2,000 mg/kg bw/day, all of the females died between GD 6 to 8 due to severe systemic toxicity. A dose of 1,500 mg/kg bw/day produced a tendency for both lower maternal body weight gain and fetal weight. There were no treatment-related effects on the maternal and developmental parameters at <1,000 mg/kg bw/day. For the definitive study, 1,600 mg/kg bw/day was used as the high dose, and the medium and low doses were determined using a scaling factor of 2.

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS AND SURVIVAL:
Individual detailed clinical observations were recorded from GD 0 through 20.

BODY WEIGHTS:
Individual maternal body weights were recorded on GD 0, 3, 6, 9, 12, 15, and 20.

FOOD CONSUMPTION:
Individual food consumption was recorded on GD 0, 3, 6, 9, 12, 15, and 19.

ANATOMIC PATHOLOGY AND LAPAROHYSTERECTOMY:
GESTATION DAY 20 LAPAROHYSTERECTOMY:
At the scheduled termination day (GD 20), all of the pregnant females were euthanized by ether inhalation followed by exsanguination from the aorta. A complete gross postmortem examination was then performed and the absolute and relative (organ-to-body weight ratio) weights of the lung, adrenal glands, liver, spleen, kidneys, thymus, heart, and ovaries were measured for all survivors.
Ovaries and uterine content:
The ovaries and uterus of each female were removed and examined for the number of corpora lutea and the status of all the implantation sites, (live and dead fetuses, early and late resorptions, and total implantations). Uteri with no evidence of implantation were stained with a 2% sodium hydroxide solution to identify the presence of early resorption sites; if no stained implantation sites were present, the rat was considered to be not pregnant. Resorption was classified as ‘‘early’’ when only a resorption site resembling a dark brown blood clot with no embryonic tissue was visible, and was considered ‘‘late’’ when both the placental and embryonic tissues were visible at the postmortem examination.
Fetal examinations:
All live fetuses were weighed, sexed, and examined for any morphological abnormalities, including cleft palate. Alternate fetuses were selected for either a skeletal or visceral examination. Half of the live fetuses from each litter were fixed in 5% formalin solution, eviscerated, and then processed for skeletal staining with alizarin red S using a modified Dawson’s method for subsequent skeletal examination. The other half were preserved in Bouin’s solution and examined for internal soft tissue changes using a freehand razor sectioning technique and Nishimura’s method (1974). The fetal morphological alterations observed in this study were classified as developmental malformations or variations.

Reference:
Nishimura KA, 1974. Microdissection method for detecting thoracic visceral malformations in mouse and rat fetuses. Cong. Anom., 14:23-40.
Statistics:
The unit for statistical measurement was the pregnant female or the litter. Quantitative continuous data such as the maternal body weight, food consumption, fetal body weight, and placental weight were subjected to a one-way analysis of variance (ANOVA). When the differences were significant, a Scheffe's multiple comparison test (Scheffe, 1953) was used. The number of corpora lutea, total implantations, live and dead fetuses, and fetal alterations were evaluated statistically using the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952), followed by the Mann-Whitney U-test (Hollander and Wolfe, 1973) where appropriate. The gender ratio and the proportions of litters with malformations and developmental variations were compared using a chi-square test and Fisher's exact probability test (1970). Statistical analyses were performed by comparing the treatment group with the control group using SAS software (SAS Institute, Inc., 1997). A difference with a P level of 0.05 or lower was considered to be statistically significant.

References:
Fisher RA, 1970. Statistical methods for research workers, 14th ed. Edinburgh, UK; Oliver and Boyd.

Kruskal WH and Wallis WA, 1952. Use of ranks in one criterion variance analysis. J. Am. Stat. Assoc., 47:614-617.

Hollander M and Wolfe DA, 1973. Nonparametric Statistical Methods, pp. 120-123. John Wiley and Sons, New York.

Scheffe H, 1953. A method of judging all contrasts in the analysis of variance. Biometika 40:87-104.
Indices:
No data were provided in the publication.
Historical control data:
No data were provided in the publication.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL OBSERVATIONS:
Clinical signs were observed only in the 1,600 mg/kg bw/day group and included piloerection, abnormal gait, decreased locomotor activity, a loss of fur, vocalization, reddish tears from the eyes, reddish vaginal discharge, nasal hemorrhage, and coma. Two out of 22 pregnant females treated with 1,600 mg/kg bw/day died from GD 7 to 9, at days 1–3 after the initiation of dosing with tertiary butyl acetate. No mortality or clinical signs were observed at 400 or 800 mg/kg bw/day.

BODY WEIGHTS:
Maternal body weights during pregnancy were not statistically significantly different from those of the control group but a dose-dependent decrease was noted during the treatment period. In particular, the maternal body weight on GD 9 in the 1,600 mg/kg bw/day animals was lower than the weight on GD 6 (before treatment), and the maternal body weight gain during the treatment period in the group was significantly lower than that of the control group.

FOOD CONSUMPTION:
In the 1600 mg/kg bw/day animals, the amount of food consumed was significantly lower on GD 6 and 9, than in the control group. This phenomenon appeared to occur in a dose-dependent manner.

ANATOMIC PATHOLOGY AND LAPAROHYSTERECTOMY:
UNSCHEDULED DEATHS:
Necropsy findings for the intercurrent deaths in the 1600 mg/kg bw/day group included expansion of the stomach, hypertrophy of the liver, and congestion/hemorrhage of the small intestine. The exact causes of these deaths were not able to be ascertained at necropsy.

GESTATION DAY 20 LAPAROHYSTERECTOMY:
At the scheduled laparohysterectomy, two cases of black/brown spotting on the lung and one case each of congestion of the thymus and hydronephrosis were observed in the control group. Hydronephrosis was present in one animal in each of the 400 and 800 mg/kg bw/day groups. Black/brown spotting on the lung and congestion of the thymus occurred in one animal in the 800 mg/kg bw/day group. Congestion/hemorrhage of the duodenum and atrophy of the spleen were observed in one animal in the 1,600 mg/kg bw/day group.

ORGAN WEIGHTS:
The absolute and relative weights of the adrenal glands and the relative weight of the liver were significantly heavier in the 1,600 mg/kg bw/day group compared to the control group, while the absolute weight of the thymus was significantly lighter than that of the controls.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
800 other: mg/kg bw/day (target)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
400 other: mg/kg bw/day (target)
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
REPRODUCTIVE TOXICITY:
There were no adverse reproductive findings for pregnant Sprague-Dawley rats treated with tertiary butyl acetate on GD 6-19 at dose levels up to 1600 mg/kg bw/day. Overall pregnancy rate, totally resorbed litters, number of corpora lutea, implantations, pre- and postimplantation loss rates, fetal deaths, litter size, and gender ratios of the live fetuses were similar in the treatment groups versus the control group. Both the male and female fetal body weights in the 1,600 mg/kg bw/day group were significantly lower than that in the control group; however, only the male fetal body weights reached statistical significance. No significant difference between treatment groups and controls was seen for the placental weight.

MALFORMATIONS (External, visceral, skeletal):
External fetal examination showed one fetus with acaudate in the 800 mg/kg bw/day group; the only visceral malformation was a single fetus with cor triloculare observed in the 800 mg/kg bw/day group. One fetus with malpositioned thoracic vertebra and scoliosis in the 800 mg/kg bw/day group and one fetus with hemicentric thoracic centrum in the 1600mg/kg bw/day group were observed, but the numbers of fetuses with skeletal malformations were not significantly increased when compared to the control group.

VISCERAL VARIATIONS:
Although the total numbers of fetuses with visceral variations and litters with affected fetuses were not significantly different among the groups, the numbers of fetuses in the 400 and 1,600 mg/kg bw/day groups with individual visceral variation, i.e., dilated ureters were significantly higher than that in the control group. Other types of visceral variations, including misshapen thymus, misshapen heart, and dilated renal pelvis, occurred at a low frequency in all of the treatment groups.

SKELETAL VARIATIONS:
Although the total numbers of fetuses with skeletal variations and litters with affected fetuses were not significantly different among the groups, the number of fetuses in the 800 and 1600 mg/kg bw/day groups with supernumerary ribs were significantly higher than that in the control group, and occurred in a dose-dependent manner. The numbers of supernumerary ribs observed were 15 (11.8%), 23 (18.4%), 45 (33.3%), and 58 (43.9%) in the control, 400, 800, and 1600 mg/kg bw/day groups, respectively. There was some evidence of treatment-related reductions in the ossification of the fetal skeleton. The fetal ossification centers of the sacrocaudal vertebra in the 800 mg/kg bw/day group and the metacarpals and sacrocaudal vertebra in the 1,600 mg/kg bw/day group were significantly lower than those in the control, but no significant decrease in fetal ossification was observed in the 400 mg/kg bw/day group.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
400 other: mg/kg bw/day (target)
Sex:
male/female
Basis for effect level:
other: See 'remarks' field, for full detailed information.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a study conducted to investigate the maternal and developmental toxicity of tertiary butyl acetate administered orally to female Sprague-Dawley rats at dose levels of 0, 400, 800 and 1600 mg/kg bw/day from Days 6-19 of gestation, maternal mortality, adverse clinical signs, reductions in mean body weights and food consumption, and changes in mean absolute and/or relative adrenal gland, liver and thymus weights were observed in the 1600 mg/kg bw/day group. There was no dose-dependent or statistically significant increase in the incidence of other reported maternal adverse effects such as hydronephrosis, black/brown spotting of the lung and congestion of the thymus which occurred sporadically in dose groups as well as the control group. There was no adverse effect on any reported reproductive parameter. Tertiary butyl acetate was not teratogenic. Any fetal malformations occurred at a very low incidence without a dose-response relationship and were similar to those sporadically observed in normal control rat fetuses. There was a significant increase in the incidence of short supernumerary ribs and reductions in ossification of the fetal skeleton in the 800 and 1600 mg/kg bw/day groups. A dosage level of 800 mg/kg bw/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and a dosage level of 400 mg/kg bw/day was considered to be the no-observed-adverse-effect level (NOAEL) for embryo-fetal toxicity when tertiary butyl acetate was administered orally by gavage to pregnant Crl:CD(SD) rats.

When tertiary butyl acetate was administered to pregnant Sprague-Dawley rats once per day by oral gavage on Days 6-19 of gestation at doses up to 1600 mg/kg bw/day, there were no statistically significant increases in any type of external, skeletal or visceral malformation, even though that dose level caused maternal mortality, adverse clinical signs primarily related to central nervous system depression, reductions in mean body weights and food consumption, and changes in mean adrenal gland, liver and thymus weights. There was a statistically significant, exposure-related increase in the number of skeletal variations at the mid- and high-exposure levels. Variations seen were typical of embryo/fetotoxicity, particularly reduced ossification. Tertiary butyl acetate was not teratogenic under conditions of this study, even at maternally toxic doses. Tertiary butyl acetate was embryo/fetotoxic at a maternally toxic dose and minimally embryo/fetotoxic at a nonmaternally toxic dose. The developmental effects reported in this study appear to be secondary effects to maternal toxicity. Based on this information, tertiary butyl acetate is not selectively toxic to the developing fetus and is not classified for reproductive/developmental toxicity according to Directive 67/548/EEC, the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Executive summary:

In a developmental toxicity study, groups of 22 pregnant female Sprague-Dawley rats were administered tertiary butyl acetate per os on GD 6-19 at dose levels up to 1600 mg/kg bw/day. Adverse maternal effects were observed only in the high-dose group and included CNS effects, body weight reductions, statistically significant increases in the absolute and relative weights of the adrenal glands and the relative weight of the liver, and decreases in absolute weight of the thymus. The 400 and 800 mg/kg bw/day dose group dams were unaffected by test substance administration. Although the total numbers of fetuses with skeletal variations and litters with affected fetuses were not significantly different among the groups, the number of fetuses in the 800 and 1600 mg/kg bw/day groups with supernumerary ribs were significantly higher than that in the control group, and occurred in a dose-dependent manner. There was also some evidence of treatment-related reductions in the ossification of the fetal skeleton. The fetal ossification centers of the sacrocaudal vertebra in the 800 mg/kg bw/day group and the metacarpals and sacrocaudal vertebra in the 1,600 mg/kg bw/day group were significantly lower than those in the control group, but no significant decrease in fetal ossification was observed in the 400 mg/kg bw/day group. There was no evidence of any additional effects of treatment in pregnant Sprague-Dawley rats exposed to tertiary butyl acetate.   Tertiary butyl acetate was clearly embryotoxic at a maternally toxic dose (1600 mg/kg bw/day) and minimally embryotoxic at a nonmaternally toxic dose (800 mg/kg bw/day). There was no evidence of treatment-related teratogenicity in the rat, even at maternally toxic doses. Under these experimental conditions, the maternal NOAEL for this study was 800 mg/kg bw/day and the embryo-fetal NOAEL was 400 mg/kg bw/day.