Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Males: 50 days, Females: 38 - 57 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable without restrictions; the study was conducted according to OECD Guideline 421 and to GLPs.
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Test animals:
-Strain: Crl: CD®(SD) IGS Br strain
-Source: Charles River Laboratories, Stone Ridge (Kingston), NY, USA
-Sex: male and female
-Age at study initiation: males - 64 days, females - 67 days
-Acclimation period: 5 days
-Weight at study receipt: males 317.64 ± 11.95 g; females 232.72 ± 10.44 g
-Housing: Animals were housed singly in suspended stainless steel wire mesh cages for the duration of the study except during the matling phase during which time animals were housed in pairs. Male and female cages were housed and racked separately except during mating. Soild bottom pans containing bedding material were placed in the cages of female rats on the 19th day of gestation. Cages and racks were washed approximately once a week. Absorbant paper used to collect excreta, was changed daily throughout the study for males and daily for female rats during the pre-mating, mating and gestation phases of the study. During the lactation phase of the study bedding material in the solid bottom pans was changed only if necessary.
Due to a detectable odour from the test substance each dose group was housed in a separate animal room to reduce the potential of airborne cross-contamination. No other studies were housed in these rooms during this study.

-Diet: Certified Rodent Diet (Purina Rodent Chow #5002, meal (PMI Feed, Inc. Richmond, IN USA) available ad libitum
-Water: local ad libitum
-Method of animal identification: unique eartag
-Method of animal distribution: Stratified randomisation program

Environmental Conditions:
-Temperature (°C): 20.9 -33.7
-Humidity (%): 29.3 - 67.9
-Photoperiod: 12 hours light/dark
There were several excursions above the recommended limit of 25°C, most of which were transient (90 minutes or less). However two excursions lasting approximately 24 hours occurred in the roommshousing the low and high dose groups (2002-10-02 to 2002-10-03). During this time the animals housed in these rooms were temporarily relocated to the room housing the mid-dose group and were removed back to their respective rooms once the temperature returned to an acceptable level.

In-Life Study Dates:
-Study Initiation Date: September 10, 2002
-Experimental Start Date: September 16, 2002
-Experimental Completion Date: May 25, 2003
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Test formulations:
The test substance was mixed with the ground food to test concentrations of 15.0, 4.5 and 1.5 mg test substance per gram of feed. The mixtures were prepared five or six times and used within 14 days based on the stability of the mixture. The control animals were given the basal diet.

Stability of the test substance in test diets was determined by repeated analysis of the high (15.0 mg/g) and low dose (1.5 mg/g) mixtures on days 0, 4, 7, 10, 14, 17 and 22 days. The 15 mg/g mixture was stable after 14 days but the 1.5 mg/g mixture was not.

Where the mean analytical concentration of the mixture was <10% of the target concentration new batches were prepared and the animals provided with new feeders.

Homegeneiy of the test mixtures was evauated by measuring the concentration of the test substance in Batch 1 at three levels (top, middle and bottom of the container) for all three dose levels. It was determined that at room temperature the cis isomer is a liquid but the trans isomer is a solid. Therefore container #2 of the test material was heated slightly to ensure the isomers were mixed and then this was used to prepare all the test mixtures for the studies. Purity of the test substance was analysed at the beginning and end of the study.

Exposure:
The study consisted of four phases: pre-mating (14 days), mating (1 to 14 days), pregnancy (21-23 days) and early lactation (4-6 days). Male rats were treated throughout the study (50 days). Female rats were treated throughout the study until they were euthanised (38 to 57 days). Males were euthanised on day 51. Female rats that delivered a litter and their offspring were euthanised on days 4, 5 or 6 postpartum. Female rats that showed evidence of mating but did not deliver were euthanised on day 23 of gestation.
Details on mating procedure:
Pairing was 1 male with 1 female within the same treatment group for 1 to 14 days. Mating was confirmed either from a sperm positive vaginal smear or a copulatory plug. The day of observation of a copulatory plug in situ and/or sperm in the lavage was designated as Gestation Day 0.

Following copulation male and female rats were separated and housed individually until study termination.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The structure of the test material was confirmed using gas chromatography with mass spectrometric determination (GC/MS). Purity of the test substance was determined by gas chromatography with flame ionisation detection (GC/FID). Purity was confirmed at the start and end of the test period.

Stability of the test substance in test diets was determined by repeated analysis of the high (15.0 mg/g) and low dose (1.5 mg/g) mixtures on days 0, 4, 7, 10, 14, 17 (1.5 mg/g mixture only) and 22 days. The 15 mg/g mixture was stable after 14 days but the 1.5 mg/g mixture was not.

Where the mean analytical concentration of the mixture was <10% of the target concentration new batches were prepared and the animals provided with new feeders.

Homegeneiy of the test mixtures was evauated by measuring the concentration of the test substance in Batch 1 at three levels (top, middle and bottom of the container) for all three dose levels. It was determined that at room temperature the cis isomer is a liquid but the trans isomer is a solid. Therefore container #2 of the test material was heated slightly to ensure the isomers were mixed and then this was used to prepare all the test mixtures for the studies. Purity of the test substance was analysed at the beginning and end of the study.
Duration of treatment / exposure:
Females: 38 to 57 days
Males: 50 days
Frequency of treatment:
Daily
Details on study schedule:
In-Life Study Dates:
-Study Initiation Date: September 10, 2002
-Experimental Start Date: September 16, 2002
-Experimental Completion Date: May 25, 2003
Remarks:
Doses / Concentrations:
1.5 mg/g feed
Basis:
analytical conc.
Dose level of approximately 92 mg/kg bw/day males and 111 mg/kg bw females
Remarks:
Doses / Concentrations:
4.5 mg/g feed
Basis:
analytical conc.
Dose level of approximately 276 mg/kg bw/day males and 351 mg/kg bw females
Remarks:
Doses / Concentrations:
15.0 mg/g feed
Basis:
analytical conc.
Dose level of approximately 888 mg/kg bw/day males and 1124 mg/kg bw females
No. of animals per sex per dose:
12 rats/sex/group
Control animals:
yes, plain diet
Details on study design:
Dose selection rationale:
Doses were selected in consultation with the sponsor based on the data from a previous repeated-dose oral toxicity study.

Doses received:
Males: 0, 92, 276 and 888 mg/kg bw/day
Females: 0, 111, 351 and 1124 mg/kg bw/day (time-weight average of dose levels for all phases of the study)
Parental animals: Observations and examinations:
Clinical Observations:
Clinical observations were conducted daily. Animals were also observed for moribundity/mortality each weekday afternoon. Clinical observations included but were not limited to: examination of hair, skin, eyes, mucous membranes, motor activity, feaces, urine, respiratory system, circulatory system, autonomic nervous system, central nervous system and behaviour patterns.

Body Weights:
Males: measured on days 0, 7 and weekly thereafter.
Females: measured on days 0, 7 and 14 of pre-mating phase, days 7 and 14 of mating (where applicable), days 0, 7, 14 and 20 of gestation, and days 0 and 4 postpartum.
Live pups: measured as a group on days 0 and 4 postpartum.

Food Consumption:
Feeders were weighed on days 0, 3, 7 and 14 of the premating phase for all animals. Feed consumption was not determined during mating. For male rats after mating the feeders were weighed once a week.. Additional feeder weights were collected for male rats from the mid- and high- dose groups on day 44 because a new batch of feed was prepared. For females feeders were weighed on days 0, 7, 14 and 20 of gestation, on days 0 and 4 postpartum and whenever feeders needed to be changed due to the stability of the test diet.

Water consumption:
Not monitored.
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
Motility Evaluation
On the day of necropsy, percentage motility of sperm for each animal as taken from the right epididymis was determined.

Testicular Spermatid Head Counts
Homogenisation resistant spermatid head counts were performed on the frozen left testes. Total sperm counts were determined for each animal. Data were reported as total counts (million/organ) and adjusted for organ weight (thousand/mg tissue).

Epididymal Spermatozoan Counts
Homogenisation resistant spermatiozoan counts were performed on the frozen left epididymides (caudal left epididymis). Data were reported as total counts (million/organ) and adjusted for ogan weight (thousand/mg tissue).
Litter observations:
Clinical Observations:
All pups were observed once daily for clinical observations, moribundity and mortality. The number of live pups born and the number found dead in each litter were recorded. The live pups were sexed, counted and weighed.

Body Weights:
Body weights for live pups were measured as a group by sex on Postnatal Days 0 and 4.
Postmortem examinations (parental animals):
Adult males were anesthetised by carbon dioxide and euthanised by exsanguination . Adult females and pups were euthanised with carbon dioxide.
Adult males were fasted overnight prior to necropsy on day 51. Adult females that showed evidence of mating but no litter were necropsied on day 23 of gestation. All other female rats were necropsied on days 4, 5 or 6 postpartum.
Male rat terminal body weights were collected following exsangination. Terminal body weights were not taken for adult females.

All adult animals were subjected to necropsy and a gross examination with special attention to the reproductive organs.The uteri from all adult females were examined for implantation sites, Corpora lutea were also counted.

The following tissues from adult animals were fixed in 10% buffered formalin: ovaries, uterus, vagina, Fallopian tubes, male accessory sex glands and gross lesions. After mortality analysis the right testis and right epididymis were fixed in Bouin's fixative; after approximately 24 hours, these tissues were rinsed twice with 50% ethyl alcohol and then stored in 70% ethyl alcohol.

For long term storage, the right testis and right epididymis were stored in 10% buffered formalin. The left testis and left epididyis were placed into individual containers, frozen with dry ice and stored at -70°C.

Organ weights:
For males, the epididymides and testes were weighed; paired organs were weighed together.

Histopathology:
The overies, right testis and right epididymis were examined microscopically for animals from the control and high-dose groups. Collected gross lesions were also examined microscopically for animals from all dose groups. Tissues were sectioned with a rotary microtome set at a thickness of either 5 or 6 µm. the resulting tissue sections were stained with hematoxylin and oesin (H&E) and examined for histopathology. In addition, duplicate sections of testicular tissue were prepared and stained with the periodic acid-Schiff procedure (PAS) and examined for histopathology.
Postmortem examinations (offspring):
The pups were euthanised with carbon dioxide.
Statistics:
Adult body weight and body weight change, litter weight, pup body weight and weight change, feed consumption, feed utilisation, organ weights, organ-to-body weight ratios, precoital interval, gestation duration, numbers of implants and corpora lutea, pre- and post-implantation loss, live-birth index, numbers of live and dead pups, numbers of male and female pups, percent of male pups, and pup survival data were evaluated using Bartlett's test (p≤0.01), one-way analysis of variance (ANOVA) (p≤0.05).

Where variances of the means were not considered equal by the Bartlett's test (p≤0.01) the data were evaluated using a Kruskal-Wallis H-test (p≤0.05) followed by Mann-Whitney U-test (p≤0.05).

The reproductive performance, litter size, and the fertility and fecundity indices were evaluated in contingency tables using a Chi-square test (p≤0.05).

Reproductive indices:
Fertility Index = (Number pregant females * 100)/ Number cohabited pairs

Fecundity Index = (Number pregnant females * 100) / Number mated pairs with copulatory plug or sperm

Offspring viability indices:
Live Birth Index = (Number of pups born alive * 100) / Number of pups in the litter


Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Minimal reductions in the amount of faeces or softened faeces, which were observed for five rats in the high-dose group for one to two days of the study. Other abnormalities observed were considered to be incidental to exposure.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean feed consumption values and body weights for males and females in the high-dose groups were lower than the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean feed consumption values and body weights for males and females in the high-dose groups were lower than the control group.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Clinical observations and mortality:
No mortality was observed during the study. There were no clinical observations that could be attributed to treatment with the test substance.

Body weights:
Lower (p≤0.05) mean body weights were observed for male rats from the high-dose group beginning on day 7 and continuing until study termination. Mean body weights also were lower (p≤0.05) for female rats from the high-dose group on day 20 of gestation when compared with the control group. Lower (p≤0.05) mean body weight gains were observed for male rats from all treated groups on day 7, and for male rats from the mid- and high-dose groups on day 42. mean body weight gains were also lower (p≤0.05) for female rats from the high-dose group on day 7 of the premating phase and on day 20 of gestation.

Although mean body weight gains were significantly higher (p≤0.05) for female rats from all treated groups on day 7 of gestation, this may be an artifact of lower control group values due to an inadvertant with-holding of feed for one day from the control group.

Overall mean bodyweight gains during gestation were comparable amongst groups.

Food consumption:
Lower (p≤0.05) mean food consumption values were observed for male rats from the high-dose group on days 7, 14, 35, 42, and 49 when compared with the control group. Mean feed consumption values were also lower (p≤0.05) for female rats from the high-dose group on days 7 of the pre-mating phase and day 14 of the gestation phase and for the mid-dose group on day 7 of gestation. Lower (p≤0.05) mean feed utilisation values were observed for male rats from the mid- and high-dose groups on days 7and 42. Mean feed utilisation values were also lower (p≤0.05) for female rats from the high-dose group on day 7 of the premating phase.
Although mean feed utilisation values were significantly higher (p≤0.05) for female rats from all treated groups on day 7 of gestation this may be an artifact of lower control group values due to an inadvertant with-holding of feed for one day from the control group.

Test substance intake:
No effects.

Reproductive performance:

Organ weights:
Mean terminal body weights were lower (p≤0.05) for male rats from the high-dose group. However, there were no differences in mean absolute and relative (to body weight) organ weights for any of the groups.

Gross pathology:
No treatment-realted gross leasions observed. All gross lesions observed considered incidental to exposure.

Histopathology:
No treatment-related changes.

Assessment of sperm morphology and development
No treatment-related effects.
Dose descriptor:
NOEL
Remarks:
parental toxicity
Effect level:
276 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Reductions in bodyweight and feed comsumption values for anmals in the high dose group.
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
888 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Feed consumption patterns appear to be food refusal. Changes not accompanied by changes in histopathology or other signs of overt toxicity.
Dose descriptor:
NOEL
Remarks:
reproduction and developmental toxicity
Effect level:
1 124 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects related to test substance administration were noted on pup development. The dose is the highest NOEL value in females (NOEL male = 888 mg/kg bw/day (nominal))
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
The duration of the gestation phase was shorter (p≤0.05) for female rats from the mid-dose group when compared with the control group. All other parameters were comparable amongst the groups.

Clinical signs:
No effects; abnormalities occurred with similar frequency in all treated and the control groups.

Body weight:
Mean pup weight change and percent pup weight change for days 0 to 4 were significantly (p≤0.05) higher for pups from the low-dose group when compared with the control group.
Reproductive effects observed:
not specified
Conclusions:
There were no treatment-related effects on male or female fertility, pregnancy performance, or pup development in a reproductive and developmental toxicity screening study in which adult male and females rats were exposed ad libitum in the diet to dimethyl 1,4-cyclohexanedicarboxylate at dose concentrations of 0, 0.15%, 0.45% and 1.5% in feed for 38 - 57 days (females) or 50 days (males). There were no adverse effects on duration of gestation, gestation index, mean number of implant sites per pregnancy, mean number of pups born per litter, mean number of live pups per litter, live birth index, fertility and fecundity indexes, and group mean litter weights. No treatment-related abnormalities were observed in the pups. Although the duration of geatation phase was shorter for female rats in the mid-dose group there was no apparent effect on pup viability. The increased mean pup weight change and percentage pup weight change from days 0 to 4 of the low-dose group in comparison to the control group were not considered to be biologically significant. The no-observed-effect-level for reproductive and neonatal toxicity was considered to be 1124 mg/kg bw/day (females), the highest concentration level tested

In a well-conducted reproductive and developmental toxicity screening study in rats, there was no clear evidence of test substance-related effects on reproduction of parental males and females or on survival and development of F1 pups. Based on this information, dimethyl 1,4-cyclohexanedicarboxylate is not a reproductive toxicant and is not selectively toxic to the developing fetus. Dimethyl 1,4-cyclohexanedicarboxylate is not classified for Developmental or Reproductive Toxicity according to Directive 67/548/EEC, UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Executive summary:

In a reproductive/developmental toxicity screening study, 12 rats/sex/group were exposed ad libitum in the diet to dimethyl 1,4-cyclohexanedicarboxylate at dose concentrations of 0, 0.15%, 0.45% and 1.5% test substance in feed for 38 - 57days (females) or 50 days (males). There were no test substance-related effects and no effects that were considered to be of biological significance on reproductive performance of the parental females or on survival and development of the F1 pups . The highest no-observed-effect-level (NOEL) for reproductive/developmental toxicity in Sprague-Dawley rats was considered to be 1124 mg/kg bw/day for females.  

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 124 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Additional information

In a reproductive/developmental toxicity screening study, 12 rats/sex/group were exposed ad libitum in the diet to dimethyl 1,4-cyclohexanedicarboxylate at dose concentrations of 0, 0.15%, 0.45% and 1.5% test substance in feed for 38 - 57days (females) or 50 days (males). There were no test substance-related effects and no effects that were considered to be of biological significance on reproductive performance of the parental females or on survival and development of the F1 pups . The highest no-observed-effect-level (NOEL) for reproductive/developmental toxicity in Sprague-Dawley rats was considered to be 1124 mg/kg bw/day for females.


Short description of key information:
In an OECD 421 reproductive/developmental screen in rats, the NOEL (NOAEL used here as NOEL is not an available choice in the drop down) was the highest dose tested.

Effects on developmental toxicity

Description of key information
In an OECD 414 prenatal developmental toxicity study, no adverse effects were observed at the highest dose tested.
Link to relevant study records
Reference
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
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
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. 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.


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
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
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
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
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
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
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.
Key result
Abnormalities:
no effects observed
Localisation:
other: No effects observed
Key result
Developmental effects observed:
no
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.

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

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.

 

Justification for classification or non-classification

In a reproductive/developmental toxicity screening study using dimethyl 1,4-cyclohexanedicarboxylate, a no-effect-level (NOEL) of 1124 mg/kg was determined, and no test article-related effects on reproductive performance of the parental females or effects on survival and development of the F1 offspring were observed. Based upon these findings, DMCD does not satisfy the criteria for classification according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 or UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

In a pre-natal developmental toxicity study using dimethyl 1,4 -cyclohexanedicarboxylate, a NOAEL of 1000 mg/kg/day was found for developmental effects, the highest dose tested. Based on these findings,DMCD does not satisfy the criteria for classification according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 or UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).