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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
White solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Sexually mature, virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. This species and strain of animal is recognized as appropriate for developmental toxicity studies. WIL Research has historical control data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Crl:CD(SD) rats (125 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 16-Jan-2015. The animals were approximately 80 days old upon receipt. Each female was examined by a qualified biologist on the day of receipt, and 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 a minimum of 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 Following positive evidence of mating, the females were individually housed in clean, solid-bottom cages with bedding material.
The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Municipal water supplying the facility was sampled for contaminants according to WIL Research’s SOPs. Reverse osmosis purified (on site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study.
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.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
The vehicle was dispensed approximately weekly for administration to the control group and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light. The vehicle was mixed throughout the sampling and dose administration procedures.
Dosing formulations were prepared at the test item concentrations indicated as follows: 0, 250, 500 and 1000 mg/kg/day.
The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
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 (National Research Council, 2011).
Duration of treatment / exposure:
The test item, 1,4 cyclohexanedicarboxylate (CHDA), 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
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
250 mg/kg
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
analytical conc.
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.
Ovaries and uterine content:
Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All surviving 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.
Maternal tissues with gross lesions were preserved in 10% neutral buffered formalin for possible future histopathologic examination. For gross lesions, 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. 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. Crown rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.
Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood 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).
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison.
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. Where applicable, the litter was used as the experimental unit. Maternal body weights (absolute and net), body weight changes (absolute and net), organ weights, 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 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

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The presence of rales as a clinical sign was considered adverse at 1000 mg/kg/day, along with decreased body weights in that same group.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on adverse clinical findings and body weight effects, a dosage level of 500 mg/kg/day was considered to be the no observed adverse effect level (NOAEL) for maternal toxicity. Due to the lack of developmental effects at any dosage level, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the NOAEL for embryo/fetal development when CHDA was administered orally by gavage to bred Crl:CD(SD) rats.
Executive summary:

The objective of the study was to determine the potential of 1,4‑cyclohexanedicarboxylate (CHDA), 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.

1,4‑Cyclohexanedicarboxylate (CHDA), 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 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. Selected organs were weighed. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

No test item-related mortality or moribundity was noted. No mortality or moribundity occurred at the 1000 mg/kg/day dose level. Test item-related clinical findings of rales were noted for females in the 500 and 1000 mg/kg/day groups at the daily examination and approximately 1 hour following dose administration. These findings were considered adverse in the 1000 mg/kg/day group; however, due to the limited frequency of these findings in the 500 mg/kg/day group, this finding was not considered adverse in this group. In addition, findings of red and clear material around the mouth were noted for females in the 1000 mg/kg/day group at approximately 1 hour following dose administration generally throughout the treatment period.

Test item-related lower mean body weight gains, without corresponding effects on food consumption, were noted in the 1000 mg/kg/day group during gestation days 2-6, 15-20, and when the entire treatment period (gestation days 2‑20) was evaluated. As a result, mean body weights in this group were lower (5.2% to 6.8%) than the control group on gestation days 19 and 20. Lower net body weight and net body weight gain were also noted in the 1000 mg/kg/day group. Mean gravid uterine weight in this group was similar to the control group. Mean body weights, body weight gains, net body weights, net body weights, gravid uterine weights, and food consumption in the 250 and 500 mg/kg/day groups were unaffected by test item administration. 

No test item-related macroscopic findings or organ weight effects were noted at any dosage level.

Intrauterine growth and survival and fetal morphology in the test item groups were unaffected by test item administration.

Based on adverse clinical findings and body weight effects, a dosage level of 500 mg/kg/day was considered to be the no‑observed‑adverse‑effect level (NOAEL) for maternal toxicity. Due to the lack of developmental effects at any dosage level, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the NOAEL for embryo/fetal development when CHDAwas administered orally by gavage to bred Crl:CD(SD) rats.