Dimethyl cyclohexane-1,4-dicarboxylate

Developmental toxicity / teratogenicity

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: The study was conducted at a GLP facility to OECD guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl cyclohexane-1,4-dicarboxylate

EC Number:

202-347-5

EC Name:

Dimethyl cyclohexane-1,4-dicarboxylate

Cas Number:

94-60-0

Molecular formula:

C10H16O4

IUPAC Name:

1,4-dimethyl cyclohexane-1,4-dicarboxylate

Constituent 2

Reference substance name:

1,4-Cyclohexanedicarboxylic acid, 1,4-dimethyl ester

IUPAC Name:

1,4-Cyclohexanedicarboxylic acid, 1,4-dimethyl ester

Constituent 3

Reference substance name:

DMCD

IUPAC Name:

DMCD

Details on test material:

Clear, colorless liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Sexually mature, virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. Crl:CD(SD) rats (125 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC. The animals were approximately 80 days old upon receipt. Each female was examined by a qualified biologist on the day of receipt; clinical observations and body weights were recorded. Each animal was uniquely identified using a programmable microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region during the acclimation period. The animals were housed for 12 days for acclimation purposes. During the acclimation period, the rats were observed twice daily for mortality and changes in general appearance and behavior.
Upon arrival, all rats were housed 2-3 per cage in clean, solid-bottom cages with bedding material (Bed O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were individually housed in clean, solid-bottom cages with bedding material. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.
All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Actual mean daily temperature ranged from 70.0°F to 70.8°F (21.1°C to 21.6°C) and mean daily relative humidity ranged from 39.3% to 50.5% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test item, dimethyl-1,4-cyclohexanedicarboxylate (DMCD), in the vehicle (corn oil) was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 2 through 19. Dosage levels were 250, 500, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen.

Analytical verification of doses or concentrations:

yes

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 was placed in a solid-bottom cage with bedding material with a 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 maintained. The selected females were approximately 13 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 and verified by a second biologist. 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:

The test item, dimethyl-1,4-cyclohexanedicarboxylate (DMCD), in the vehicle (corn oil) was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 2 through 19.

Frequency of treatment:

Daily

Duration of test:

17 days

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from gestation days 0 through 20 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals.
Individual maternal body weights were recorded on gestation days 0 and 2-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 2-6, 6-9, 9-12, 12-15, 15-20, and 2-20.
Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0 20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.
Individual food consumption was recorded on gestation days 0 and 2-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” on the individual report tables.

Ovaries and uterine content:

Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All females were euthanized on gestation day 20 by carbon dioxide inhalation. The cranial, thoracic, abdominal, and pelvic cavities were opened by a ventral mid line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, 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.
Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.
Following organ weight collection the brain, liver, spleen, and thymus gland were preserved in 10% neutral-buffered formalin for possible future histopathologic examination; other maternal tissues were preserved 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.

Fetal examinations:

Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the WIL Research study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded.
Each viable fetus was subjected to a visceral examination including the heart and major blood vessels. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development). Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.
Following fixation in alcohol, each fetus was stained with Alizarin Red S and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-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. Data obtained from nongravid animals were excluded from statistical analyses. Where applicable, the litter was used as the experimental unit. Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, organ weights (absolute and relative to brain weights), and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test item-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 to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test item-treated groups to the control group

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Details on maternal toxic effects:
None noted

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
None noted

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects found at the limit dose.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Localisation:

other: No effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the lack of adverse maternal or developmental toxicity observed at any dosage level, a dosage level of 1000 mg/kg/day, the highest dosage evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when dimethyl-1,4-cyclohexanedicarboxylate (DMCD) was administered orally by gavage to bred Crl:CD(SD) rats.

Executive summary:

The objectives of the study were to determine the potential of the test item, dimethyl‑1,4‑cyclohexanedicarboxylate (DMCD), to induce developmental toxicity after maternal exposure prior to implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no‑observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity. The test item, dimethyl-1,4-cyclohexanedicarboxylate (DMCD), in the vehicle (corn oil) was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 2 through 19. Dosage levels were 250, 500, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 13 weeks of age at the initiation of dose administration. 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 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. Selected organs were weighed. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

All females in the control, 250, 500, and 1000 mg/kg/day groups survived to the scheduled necropsy on gestation day 20. Test item‑related clinical findings were limited to dose-dependent clear and red material around the mouth in all test item-treated groups approximately 1 hour following dose administration generally throughout the treatment period. These findings did not persist to the daily examinations (prior to dose administration) on the following day and were not considered to be adverse. Test item-related slightly lower mean body weight gains were noted in the 250, 500, and 1000 mg/kg/day groups during gestation days 2-6. Mean body weight gains in the test item-treated groups were similar to the control group throughout the remainder of the treatment period (gestation days 6-19) and there was no corresponding effects on mean food consumption or mean absolute body weights. Therefore, the initial body weight decrements in the 250, 500, and 1000 mg/kg/day groups were not considered to be adverse. Mean maternal body weights, net body weights, net body weight gains, gravid uterine weights, and food consumption in the 250, 500, and 1000 mg/kg/day groups were unaffected by test item administration.

No test item-related macroscopic findings or effects on brain, liver, thymus, and spleen weights were noted at any dosage level. 

Intrauterine growth and survival and fetal morphology in the 250, 500, and 1000 mg/kg/day groups were unaffected by maternal test item administration. 

Based on the lack of adverse maternal or developmental toxicity observed at any dosage level, a dosage level of 1000 mg/kg/day, the highest dosage evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when dimethyl-1,4-cyclohexanedicarboxylate (DMCD) was administered orally by gavage to bred Crl:CD(SD) rats.