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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2006 - March 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study followed OECD guidelines and was conducted under GLP assurances.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
The study director stated that "These deviations did not negatively impact the quality or integrity of the data nor the outcome of the study."
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
2,2,4,4-Tetramethyl-1,3-Cyclobutanediol
Lot no. TS060513
Exp. date: July 2009
White powder

Test animals

Species:
rat
Strain:
Crj: CD(SD)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
For the control group (Group 1), a sufficient amount of deionized water was transferred to a glass container. The vehicle was divided into aliquots for daily dispensation and stored at room temperature. The vehicle was stirred continuously throughout sampling, dispensation and dose
administration. Test article was dissolved into vehicle at the following concetrations and dosed at 10 ml/kg.

The test article in the vehicle, deionized water, was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats twice daily,
approximately 6 hours apart, from gestation days 0 through 19. Total daily dosage levels initially were 150, 300 and 600 mg/kg/day. Due to the
excessive toxicity (mortality, body weight loss, reduced food consumption and adverse clinical signs) noted in the 600 mg/kg/day group, this
dosage level was reduced to 500 mg/kg/day on 17 December 2006 (gestation days 1 through 4). Each of the 2 daily doses was administered at a
dosage volume of 10 mL/kg. A concurrent control group composed of 25 females received the vehicle (deionized water) on a comparable regimen.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
the analyzed dosing formulations were within 98.4% to 107% of the target concentration, were homogeneous and were stable when stored at room
temperature for up to 14 days. Therefore, the protocol-specified dosages of test article were administered to the animals.
Details on mating procedure:
At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the study director, each animal judged to be in good health and meeting acceptable body weight requirements (a minimum of 220 g at the initiation
of breeding) was placed in a suspended wire-mesh cage with a single resident male from the same strain and source for breeding. Resident males
were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. A breeding record containing the male and female identification numbers and the dates of cohabitation was prepared. The selected females were
approximately 12 weeks old when paired for breeding. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the
presence of sperm in a vaginal lavage. Each mating pair was examined daily. The day on which evidence of mating was identified was termed gestation day 0 and the animals were separated.
Duration of treatment / exposure:
Gestation Day 0 to Day 19
Frequency of treatment:
Twice daily by oral gavage approximately 6 hours apart
Duration of test:
20 days
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Dosage levels were based on the results of a preliminary study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual detailed clinical
observations were recorded from gestation days 0 through 20 (prior to dose administration during the treatment period). Animals were also
observed for signs of toxicity at the time of each dose administration and approximately 1 hour following each dose administration. All significant
findings were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
See cage side obseervations

BODY WEIGHT: Yes
Individual maternal body weights were recorded on gestation days 0-20 (daily).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Individual food consumption was recorded on gestation days 0-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the
corresponding body weight change intervals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
A gross necropsy was performed on females that died or were euthanized in extremis during the course of the study. Maternal tissues were retained in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. The number and location
of implantation sites, corpora lutea and viable fetuses were recorded for females dying on or following gestation day 6. Recognizable fetuses were
examined externally and preserved in 10% neutral-buffered formalin.

Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All surviving females were euthanized on gestation day 20 by carbon dioxide inhalation. The thoracic, abdominal and pelvic cavities were opened by a ventral mid-line incision, and the contents were
examined. In all instances, the post mortem findings were correlated with the ante mortem comments, and any abnormalities were recorded. The
uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions and the total number of implantation sites were recorded. The
placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All
implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded. Uteri with no
macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation
loss (Salewski, 1964).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
-Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test
article-treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.) and the number of
animals (N) used to calculate the mean.Mean maternal body weights (absolute and net), body weight changes (absolute and net) and food
consumption, gravid uterine weights, numbers of corpora lutea, implantation sites and viable fetuses, and fetal body weights (separately by sex
and combined) were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup
differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the
test article-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early
and late resorptions,total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental
variations (external, visceral, skeletal and combined) and each particular external, visceral and skeletal malformation or variation were subjected
to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test article-treated groups to the control group.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Based on the test article-related mortality, adverse clinical findings [predominantly clonic convulsions, full body twitches (sharp, involuntary and jerky movements of a majority of the body that occurred constantly but not continuously) and yellow material around the urogenital area], mean
body weight losses and reduced food consumption in the 300 and 600/500 mg/kg/day groups, a dosage level of 600 mg/kg/day was considered to exceed the maximally tolerated dose and a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level
(NOAEL) for maternal toxicity.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
not determinable due to absence of adverse toxic effects

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Evidence of developmental toxicity was noted at 600/500 mg/kg/day. The mean litter proportion of postimplantation loss (primarily early resorptions) in the 9 surviving pregnant females in the 600/500 mg/kg/day group was higher than the control group value and resulted in a decrease in the mean litter proportion of viable fetuses. However, live litter size in the 600/500 mg/kg/day group was unaffected by test article
administration. Lower mean fetal body weights were also noted in this group compared to the control group, and effects on fetal skeletal
ossification were observed. Increased mean litter proportions of reduced ossification of the skull, vertebral arches and 13th rib(s) and unossified pubis, vertebral centra, entire sternum and ischium and a decreased mean litter proportion of ossified cervical centrum no. 1 were noted at
600/500 mg/kg/day and were partially due to 1 litter with severely growth-retarded fetuses. There were no test article-related effects on
intrauterine growth and survival or fetal morphology at 150 and 300 mg/kg/day.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the test article-related mortality, adverse clinical findings [predominantly clonic convulsions, full body twitches (sharp, involuntary and jerky movements of a majority of the body that occurred constantly but not continuously) and yellow material around the urogenital area], mean body weight losses and reduced food consumption in the 300 and 600/500 mg/kg/day groups, a dosage level of 600 mg/kg/day was considered to
exceed the maximally tolerated dose and a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on the increased mean litter proportion of postimplantation loss in the limited number (N = 9) of surviving pregnant females in the 600/500 mg/kg/day group and decreased mean fetal body weights with corresponding fetal skeletal developmental variations
indicative of delayed ossification in the same group, a dosage level of 300 mg/kg/day was considered to be the NOAEL for prenatal developmental toxicity when 2,2,4,4-Tetramethyl- 1,3-Cyclobutanediol was administered orally by gavage to pregnant Crl:CD(SD) rats.
Executive summary:

The test article, 2,2,4,4-Tetramethyl-1,3-Cyclobutanediol was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats twice daily, approximately 6 hours apart, from gestation days 0 through 19. Total daily dosage levels initially were 150, 300 and 600 mg/kg/day. Due to the excessive toxicity (mortality, body weight loss, reduced food consumption and adverse clinical signs) noted in the 600 mg/kg/day group, this dosage level was reduced to 500 mg/kg/day on gestation days 1 through 4. Each of the 2 daily doses was administered at a dosage volume of 10 mL/kg. A concurrent control group composed of 25 females received the vehicle (deionized water) on a comparable regimen. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each surviving female. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.

Six and 13 females in the 300 and 600/500 mg/kg/day groups, respectively, were found dead or euthanized in extremis between gestation days 1 and 19. Red matting of the skin around the nasal, buccal and/or ventral thoracic areas, which corresponded to clinical

findings of red material around the mouth and/or nose, was the only test article-related macroscopic finding for these females; no internal findings were noted. Predominant clinical findings for the found dead or euthanized in extremis females and for the females

in these groups that survived to the scheduled necropsy consisted of clonic convulsions, full body twitches (sharp, involuntary and jerky movements of a majority of the body that occurred constantly but not continuously), yellow material around the urogenital area and salivation and/or evidence of salivation (clear material around the mouth and/or nose). Additionally, popcorn seizures, tonic convulsions, labored respiration, gasping, rales, automutilation of the tail and hindlimbs, hyperactivity, hypoactivity, hypersensitivity to

handling, circling, wet dog shakes (whole body shaking of short duration) and spatial disorientation (walking or stumbling into objects), ambulating on tiptoes, excessive pawing or biting of the cage floor and/or walls, compulsive licking, repetitive movement

of the mouth and jaw, hunched posture, straub tail and piloerection were noted for a limited number of females in the 300 and 600/500 mg/kg/day groups. All other females survived to the scheduled necropsy, and there were no other test article-related clinical or macroscopic findings.Lower mean food consumption was noted in the 150, 300 and 600/500 mg/kg/day groups during gestation days 0-3 and/or 3-6. Corresponding mean maternal body weight losses were noted at 300 and 600/500 mg/kg/day during gestation days 0-3 and at 150 mg/kg/day during gestation day 0-1. Mean food consumption and body weight gains in these groups were generally similar to or slightly higher than the control group values for the remainder of the treatment period, with the exception of a slightly lower mean body weight gain in the 9 surviving pregnant females in the 600/500 mg/kg/day group during gestation days 12-20. Primarily as a result of the losses during gestation days 0-1 or 0-3, mean body weight gains during the entire treatment period and mean net body weight gains in all test article-treated groups were lower. However, only the effects observed in the 300 and 600/500 mg/kg/day groups were considered to be adverse due to the magnitude and persistency of the effects on mean body weight change and the lower mean body weights (up to 8.2% and 14.1%, respectively) and net body weights in these groups. Mean body weights and net body weights in the 150 mg/kg/day group were similar to the control group values. Evidence of developmental toxicity was noted at 600/500 mg/kg/day. The mean litter proportion of postimplantation loss (primarily early resorptions) in the 9 surviving pregnant females in the 600/500 mg/kg/day group was higher than the control group value and resulted in a decrease in the mean litter proportion of viable fetuses. However, live litter size in the 600/500 mg/kg/day group was unaffected by test article

administration. Lower mean fetal body weights were also noted in this group compared to the control group, and effects on fetal skeletal ossification were observed. Increased mean litter proportions of reduced ossification of the skull, vertebral arches and 13th

rib(s) and unossified pubis, vertebral centra, entire sternum and ischium and a decreased mean litter proportion of ossified cervical centrum no. 1 were noted at 600/500 mg/kg/day and were partially due to 1 litter with severely growth-retarded fetuses. There were no test article-related effects on intrauterine growth and survival or fetal morphology at 150 and 300 mg/kg/day.