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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
Females: 81-96 days; Males: 92 or 93 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted under GLPs and according to the OECD 421 guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley rats [Crl:CD(SD)IGS BR]
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Stone Ridge (Kingston), NY
- Age at study initiation: 52 days (male); 53 days (female)
- Housing: Males and females were housed individually in clean, suspended, stainless steel wire-mesh cages in an environmentally controlled room except during the mating phase when they were housed in pairs. No other studies were housed in the same room.
- Diet: Certified Rodent Diet [Purina Rodent Chow #5002 (meal) (PMI Feed, Inc. Richmond, IN)] available ad libitum
- Water: Water available ad libitum from water bottles with sipper tubes.
- Acclimation period: isolated for at least 5 days
-Weight at study initiation (males): 260.7 ± 12.5 g
-Weight at study initiation (females): 188.4 ± 13.7 g
-Identification method: uniquely-numbered metal ear tags
-Randomization procedure: Animals judged suitable for testing were culled from the stock population based on body weight and randomly assigned to groups using computer-generated lists. Individual body weights were within +/- 20% of the mean for each sex. Following randomization, body weights of all groups were compared by analysis of variance to ensure no statistically significant difference prior to first exposure. There were no statistically significant differences among the groups (i.e., p-value for ANOVA was not significant at p < 0.05).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.4-25.3
- Humidity (%): 30.4-67.3
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

IN-LIFE DATES:
Study Initiation Date: August 4, 1999
Experimental Start Date: August 16, 1999
Experimental Completion Date: November 18, 1999
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
Stability of test substance in dosing solutions:
Stability in drinking water was determined by repeated analysis of aqueous solutions containing 1.0 and 12.5 mg/mL concentrations of the test substance. Mixtures were analyzed using gas chromatography with flame ionization detection (GC/FID). Refrigerated samples stored either in glass or Nalgene containers were analyzed on Days 0, 7, 14, 28 and 40. Room temperature samples stored in inverted glass containers sealed with a stopper and sipper tube were analyzed on Days 0, 1, 2, 5 and 7. Mixtures were stable under all tested conditions.

Exposure:
The study consisted of four phases: pre-mating (56 days), mating (up to 14 days), gestation (21-22 days) and early lactation (4 days) for a total of 81-96 exposure days for females and 13 weeks for males until necropsy on Days 92 or 93. The rats were offered drinking water containing 0, 4.0, 8.0 or 12.5 mg/mL of the test substance resulting in concentrations of 0, 0.4, 0.8, and 1.25%, respectively, ad libitum until they were euthanized. All surviving females that delivered a litter were euthanized and necropsied on Day 4 postpartum. All surviving females that exhibited evidence of mating but did not deliver by Day 22 of gestation were euthanized and necropsied on Days 23 or 24 of gestation. All surviving females that did not exhibit evidence of mating were euthanized and necropsied on Days 94-96. Pups were exposed only in utero or during lactation. All surviving pups were euthanized on Day 4 postpartum.
Details on mating procedure:
Female rats were mated 1:1 with males within the same dose group for 1 to 14 days. Copulation was verified through identification of sperm in vaginal smears or by the appearance of a copulatory plug. The day on which copulation was verified was considered gestation day (GD) 0. Following copulation, rats were separated and housed individually until necropsy. For female animals that showed no evidence of mating, the delivery date was calculated from the last day of possible copulation with a male.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
PURITY OF TEST SUBSTANCE:
The purity of the test substance was determined by GC/FID and Karl Fischer titration. Prior to use on the study, the test substance was determined to contain 90.0% by weight of 1,4-cyclohexanedimethanol, 9.79% by weight water, and 0.21% by weight of unknown materials. At the end of the study, the purity of the test article was determined to be 89.7% by weight of 1,4-cyclohexanedimethanol, 10.0% by weight water, and 0.30% by weight of unknown materials. Based on these data, the test substance was considered to be stable during the testing period.

STRUCTURE CONFIRMATION:
Structure was confirmed using gas chromatography with mass spectrometric detection (GS/MS). The mass spectrum of the test substance was consistent with the proposed structure based on comparison with the reference library spectrum of the test substance.

HOMOGENEITY OF DOSING SOLUTIONS:
Homogeneity of the test substance in drinking water was evaluated by measuring the concentration of the test substance at three levels (top, middle, and bottom of the mixing container) of all dosing mixtures using GC/FID.

The analytical concentrations of the test substance in the top, middle, and bottom layers for the 12.5 mg/mL mixture varied from 12.8 to 13.67 mg/mL ± 0.272. The analytical concentrations of the test substance in the top, middle, and bottom layers for the 8.0 mg/mL mixture varied from 7.191 to 7.735 mg/mL ± 0.144. The analytical concentrations of the test substance in the top, middle, and bottom layers for the 4.0 mg/mL mixture varied from 3.766 to 4.037 mg/mL ± 0.074. Based on the results, preparations were considered to be homogeneous.

CONCENTRATION TEST SUBSTANCE IN DOSING SOLUTIONS:
The concentration of the test substance in each batch of test mixture was determined prior to use by GC/FID analysis. The mean concentrations of the test substance varied between 97-102%, 93.4 - 108%, and 95.2-106% of the target concentrations of 4.0, 8.0, and 12.5 mg/mL, respectively.
Duration of treatment / exposure:
13 weeks for males; 81-96 days for females
Frequency of treatment:
ad libitum
Remarks:
Doses / Concentrations:
0.40% (4.0 mg/mL)
Basis:
nominal in water
Remarks:
Doses / Concentrations:
0.80% (8.0 mg/mL)
Basis:
nominal in water
Remarks:
Doses / Concentrations:
1.25 % (12.5 mg/mL)
Basis:
nominal in water
No. of animals per sex per dose:
12 rats/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
Doses received:
Mean daily doses of the test substance received for the 0, 4.0, 8.0 and 12.5 mg/mL groups were approximately 0, 256, 479 and 861 mg/kg bw/day for males and 0, 385, 854 and 1360 mg/kg bw/day for females.

Dose selection rationale:
The exposure levels were based on two probe studies conducted in female rats. For both studies, the test substance was administered via drinking water for 7 consecutive days. Based on these study results, a concentration of 12.5 mg/mL (1.25%) was selected to maximize consumption of the test substance in drinking water. Concentrations of 8.0 mg/mL (0.80%) and 4.0 mg/mL (0.40%) were selected to provide evidence of possible dose-response relationships.
Positive control:
No
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS:
Clinical examinations were conducted every day for all adult animals. Clinical observations included, but were not limited to, examination of the hair, skin, eyes, mucous membranes, motor activity, feces, urine, respiratory system, circulatory system, autonomic nervous system, central nervous system, and behavior patterns. Except for holidays, all adult animals were observed each weekday afternoon for moribundity and mortality.

BODY WEIGHTS:
Body weights for males were measured on Days 0, 7 and at least weekly thereafter. The animals were fasted prior to necropsy. Terminal body weights were measured after exsanguination, but prior to necropsy.

Body weights for females were measured on Days 0, 7 and at least weekly through the pre-mating and mating phases of the study, on Days 0, 7, 14, and 20 of gestation, and on lactation Days 0 and 4. The animals were not fasted prior to necropsy and terminal body weights were not measured.

FEED CONSUMPTION:
For males, feeders were weighed on Days 0, 7 and at least weekly thereafter except for Weeks 9 and 10 during the mating phase. Food consumption for females was measured on Days 0, 7 and at least weekly during the pre-mating phase of the study; on Days 0, 7, 14, and 20 of gestation; and on lactation Days 0 and 4. Food consumption for females was not measured during the mating phase of the study.

WATER CONSUMPTION:
For males, water bottles were weighed on Days 0, 4 and at least twice weekly except for Weeks 9 and 10 during the mating phase. Water consumption for females was measured on Days 0, 4 and at least twice weekly during the pre-mating phase of the study; on Days 0, 7, 14, and 20 of gestation; and on lactation Days 0 and 4. Water consumption was not measured during the mating phase of the study.

OTHER EXAMINATIONS: A number of other examinations were performed on males and a separate group of females as part of a 13-week subchronic study. They are reported in a separate IUCLID record.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
SPERM MOTILITY:
Sperm motility was determined using the contents of the right epididymis. The epididymis was placed in a petri dish containing 1% bovine serum albumin dissolved in phosphate buffered saline. The solution was prewarmed to ~38°C and the epididymis was pierced 2-3 times and allowed at least 3 minutes of rest in the petri dish to allow the sperm to swim out. A 100 µm deep cannula was then used to obtain a sample of the mixture in the petri dish and the sample was loaded onto a pre-warmed stage of a Hamilton Thorne IVOS automated sperm analyzer. Five fields were automatically selected and each motion image was recorded and the percent motility was determined for each animal.

TESTICULAR SPERMATID HEAD COUNTS:
Testicular homogenization-resistant spermatid head counts were performed on the frozen left testis. After the tissue was thawed, the tunica was removed, the testis was weighed, homogenized, vortexed, and a 100 µL sample was transferred to a violet reaction vial containing a dye which uniquely stains the head of the sperm. A sample of stained sperm was placed onto a glass slide and loaded into the Hamilton Thorne IVOS automated sperm analyzer. Twenty fields were automatically selected by the analyzer for each animal and total sperm counts were determined.

EPIDIDYMAL SPERMATOZOAN COUNTS:
Homogenization-resistant spermatozoan counts were performed on the frozen left epididymides. The epididymal tissue was processed in a similar manner to the testicular tissue, except that the caudal left epididymis was used.
Litter observations:
CLINICAL OBSERVATIONS:
All pups were observed once daily for clinical observations, moribundity and mortality.

BODY WEIGHTS:
Body weights for live pups were measured as a group, by gender, on postnatal Days 0, 1 and 4.
Postmortem examinations (parental animals):
GROSS PATHOLOGY:
Following euthanasia, the females were necropsied and examined for gross lesions and implantation sites of the uteri were counted. The following tissues were fixed in 10% neutral buffered formalin: ovaries, Fallopian tubes, uterus, vagina, and gross lesions. Following euthanasia, the male rats were weighed and necropsied. The following reproductive tissues were fixed in 10% neutral buffered formalin: male accessory sex glands and gross lesions. The right testis and right epididymis were fixed in Bouin’s solution after sperm motility analysis. The left testis and left epididymis were frozen in dry ice. All other tissues were examined as part of the 13-week subchronic study and are reported in a separate IUCLID entry.

ORGAN WEIGHTS: For males, the epididymides and testes were weighed. No reproductive organs were weighed in the females in this study. However, in a separate group of females examined as part of a subchronic study conducted at the same dose levels, ovaries and uterus were weighed.

HISTOPATHOLOGY:
The ovaries and gross lesions from the female rats from the control and high-dose groups were examined for microscopic lesions. All reproductive tissues (except for the left testis and left epididymis) from the surviving males and males that died or were euthanized in extremis were examined for microscopic lesions. Gross lesions from all low- and mid-dose groups were examined for microscopic lesions.
Statistics:
Mean values were calculated for body weight and feed consumption. Homogeneity of body weight and feed consumption were evaluated using Bartlett’s test (p ≤ 0.01). Body weight and feed consumption were evaluated using a one-way analysis of variance (ANOVA)(p ≤ 0.05) and Duncan’s multiple range test (p ≤ 0.05).
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
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:
effects observed, treatment-related
Reproductive performance:
no effects observed
DOSES RECEIVED:
Mean daily doses of the test substance received for the 0.0, 4.0, 8.0 and 12.5 mg/mL groups were approximately 0, 256, 479 and 861 mg/kg bw/day for males and 0, 385, 854 and 1360 mg/kg bw/day for females.

MORTALITY:
One male in the low-dose group died during the study. Two rats from the high-dose group were euthanized in extremis: one female rat on Day 28 and one male on Day 39.

CLINICAL OBSERVATIONS:
Clinical abnormalities related to the test substance included hematuria and/or brown/red discoloration of the urine starting on Day 6 of the study. All discolored urine tested positive for blood. These abnormalities were observed exclusively in high-dose animals except for 2 days when these abnormalities were observed for single male rats in the other two dose groups. Minimal to severe discoloration of the urine and/or hematuria were observed for 5 males and 6 females. Because of the housing provisions, urine color could not be evaluated for the female rats after they were given nesting boxes prior to birth and during lactation. Softened or reduced amounts of feces were observed for one to two males and one to two female rats (pre-mating phase) in all groups for 1-4 days of the study. Minimal dehydration was observed in one high-dose male. Minimal to severe dehydration was observed for one to two females from the mid- and/or high-dose groups for 1 to 2 days during the pre-mating and gestation phases and for 1 to 5 days during the lactation phase.

Additional observations present in a few high-dose animals included hypothermia, pallor, partially opened eyes, piloerection, unkempt haircoat and brown discoloration of the skin of the tail and inguinal or abdominal hair. No other test substance-related clinical abnormalities were observed.

BODY WEIGHTS:
For males, mean body weights were significantly lower (p ≤ 0.05) for the high-dose group during Weeks 1-7 and Week 9 when compared to control. Mean body weight gains were significantly less during Weeks 1, 2, and 5 for the high-dose animals, and higher during Week 7 for the mid-dose animals and Week 10 for the high-dose animals. There were no differences in overall study mean body weight change for any dose group compared to the control.

For females, mean body weight changes were significantly lower for the high-dose animals compared with the control during Weeks 1 and 4 of the pre-mating /mating phases, but overall mean body weight gains were comparable among groups during this phase. During gestation, mean body weights were comparable among groups, but mean body weight changes during gestation Days 14-20 and overall mean body weight gains were significantly lower for high-dose animals when compared with the control. During the lactation phase, both the mean body weights on Days 0 and 4 and overall mean body weight gains were significantly lower for the high-dose animals when compared to control.

FOOD CONSUMPTION:
Mean feed consumption was significantly lower (p ≤ 0.05) for high-dose males during Weeks 1, 2, 4, and 5 and for high dose females during Week 1 of the pre-mating phase and during gestation Days 15-20 when compared to control. Mean feed consumption values were significantly lower for mid- and high-dose females during lactation Days 0-4 when compared with controls.
.
WATER CONSUMPTION:
There were no effects in male rats. Mean water consumption values were significantly lower (p ≤ 0.05) for all female dose groups on gestation Days 9 and 12 when compared with controls but it was noted that water consumption was only measured for a few animals per group on these days and this was not considered representative of the entire group. When water consumption was measured for all animals on Days 7 and 14, no significant differences were detected.

ORGAN WEIGHTS: There were no adverse effects on testes or epididymides. In a subchronic study using non-pregnant females exposed at the same dose levels, there was no adverse effect on ovaries or uterus weights.

GROSS PATHOLOGY:
Gross lesions were observed in all groups, including controls, but none of the gross lesions were considered related to test article administration.

HISTOPATHOLOGY:
There were several microscopic lesions observed in all groups, including controls, but these lesions were considered to be incidental in nature and not related to test article administration.

SPERM PARAMETERS:
There were no significant differences in mean epididymal spermatozoa and testicular spermatid counts per tissue (total count) or mean weight adjusted epididymal spermatozoa and testicular spermatid counts among any of the groups. Percent motility was reduced in four of the eleven high-dose male rats and the mean percent motility was 69% compared with 90% in the control group. This difference did not reach statistical significance but was considered biologically significant. Mean percent motility values for the other two treated groups were comparable to the control.

LITTER OBSERVATIONS:
Viability, clinical signs and body weight changes are addressed separately in the observations for offspring. All other examined parameters, including reproductive performance, gestation length, prenatal loss, number of implantation sites, number of live and dead pups at birth, and ratio of male to female pups were comparable between the high-dose group and controls. All examined parameters, including reproductive performance, gestation length, pup survival rate, pre-natal loss, number of implantation sites, number of live and dead pups, ratio of male to female pups, pup body weights, and pup body weight changes were comparable between the control and low- and mid-dose groups.
Dose descriptor:
NOEL
Effect level:
0.8 other: %
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Histopathological findings:
not examined
VIABILITY:
Effects on viability noted in litters from high-dose dams included decreased pup survival from Day 0 to Day 1 (81.8% versus 98.4% for control), increased number of high-dose pups dying from Day 0 to Day 4, and decreased percent survival for high-dose pups from Day 0 to Day 4 (75.8% versus 97.6% for control). There was no effect on the number of live and dead pups at birth in the high-dose group. Pup survival rates and numbers of live and dead pups were comparable for the control and other test groups.

CLINICAL OBSERVATIONS:
Clinical abnormalities which may be related to test-substance exposure included pups with no or small amounts of milk in their stomachs, and pups which were missing (presumably cannibalized) or found dead. These observations appeared with greater incidences in animals treated with 1.25% 1,4-cyclohexanedimethanol compared to control. One to two pups from the high-dose group had wounds and/or cannibalized body parts. Other abnormalities considered incidental to exposure included small body size, bruises, and a blackened, dried, blunt and/or missing tip of the tail.

BODY WEIGHTS:
Biologically significant effects noted in litters from high-dose dams included lower mean body weights on Day 0 postpartum, decreased high-dose male and female pup weights on Day 1, decreased high-dose male pup body weight change and % body weight change on Day 1, decreased high-dose male and female pup body weight change and % body weight change on Day 4, and decreased high-dose male and female pup body weight change from Day 0 to Day 4. Statistically significant differences were limited to lower mean body weights for the high-dose group female pups on Day 0. Pup body weights and pup body weight changes were comparable among the control and other test groups.
Reproductive effects observed:
not specified

Stability of test substance:

For refrigerated samples stored in glass containers, concentrations of test substance in the 12.5 and 1.0 mg/mL solutions were 12.5 ± 0.25 and 0.949 ± 0.0194 prior to storage and 11.1 ± 0.26 and 0.873 ± 0.0196 after 40 days of storage. For refrigerated samples stored in Nalgene containers, concentrations of test substance were 11.6 ± 0.13 and 0.960 ± 0.0146 prior to storage and 11.2 ± 0.08 and 0.910 ± 0.0264 after 40 days of storage. For samples stored in glass containers at room temperature, concentrations of test substance were 12.2 ± 0.24 and 0.980 ± 0.0183 prior to storage and 12.5 ± 0.14 and 0.987 ± 0.0133 after 7 days of storage. Based on these results, the refrigerated mixtures were stored in either Nalgene or glass containers during the study, and the room temperature mixtures were stored in glass containers during the study.

Probe Studies:

In the first study, 12 female rats were offered drinking water containing 0 mg/mL (0%), 5.0 mg/mL (0.50%), 10.0 mg/mL (1.00%), or 15.0 mg/mL (1.50%) of 1,4-cyclohexanedimethanol for seven days. No mortality was observed in the study; animals appeared clinically normal; slight decreases in body weight were reported for the first 3 days of the study but all animals had gained weight by Day 7; and there were no effects on feed consumption. Water consumption in the high-dose group was 50% of the control, possibly because the animals found the water unpalatable. Therefore, a second probe study was conducted to determine the maximum dose possible by the drinking water route.

For the second study, six females were offered drinking water containing either 0.0 or 12.5 mg/mL (0 and 1.25%, respectively) of the test substance for seven days. No mortality was observed; clinical observations were normal; slight decreases in body weight were reported for the first 4 days of the study but all animals had gained weight by Day 7. Significant decreases in food consumption were noted on Day 2 for the treated group; and water consumption was comparable among the treated and control groups. The animals treated with concentrations of 12.5 mg/mL received the largest dose in the probe study so this concentration was chosen to achieve a target dose level of 1000 mg/kg bw/day for the definitive study.

Conclusions:
In the current study, clinical abnormalities including mortality, hematuria and/or brown/red discoloration of the urine, dehydration, softened or reduced amounts of feces, reduced body weights, and decreased food consumption were observed in male and female high-dose animals when 1,4-cyclohexanedimethanol was administered at dose concentrations of 0, 0.40% (4.0 mg/mL), 0.80% (8.0 mg/mL), or 1.25 % (12.5 mg/mL) in the drinking water for 91 days in males and 81-96 days in females. In addition, high-dose group males had decreased sperm motility although this did not affect reproductive performance. Reduced postnatal pup survival and lower mean pup body weights were observed in litters from high-dose group dams although these abnormalities were generally observed in dams that also exhibited systemic effects. The NOEL for reproductive/developmental toxicity in Sprague-Dawley rats was considered to be 479 mg/kg bw/day for males and 854 mg/kg bw/day for females. The NOEL for the entire study was 479 mg/kg bw/day.

In a well-conducted combined repeated dose and reproductive/developmental toxicity screening study in which groups of 12/rats/sex were administered up to 1.25% of the test material ad libitum in drinking water for 91 (males) or 81-96 (females) consecutive days during premating, mating, pregnancy and early lactation, there were no gross or microscopic effects on any reproductive organ in either sex. Mean daily doses were approximately 0, 256, 479 and 861 mg/kg bw/day for males and 0, 385, 854 and 1360 mg/kg bw/day for females. There were no adverse effects on any reproductive parameters and all pregnant animals delivered normally. Although high-dose group males had decreased sperm motility, this did not affect reproductive performance. Reduced postnatal pup survival and lower mean pup body weights were observed in litters from high-dose group dams although these effects were generally observed in dams that also exhibited systemic effects (i.e., reduced body weights and feed consumption, dehydration, hematuria). All other examined parameters, including reproductive performance, gestation length, prenatal loss, number of implantation sites, number of live and dead pups at birth, and ratio of male to female pups were comparable between the high-dose group and controls. There were no adverse effects on litters from dams in the low and mid-dose groups. Based on an absence of significant reproductive effects in the parental generation at dose levels up to 861 mg/kg bw/day in males and 1360 mg/kg bw/day for females and the limitation of adverse effects on the offspring to dose levels that caused systemic effects in the dams, i.e., 1360 mg/kg bw/day, 1,4-cyclohexanedimethanol is not classified for “Reproductive or Developmental Toxicity” according to GHS.
Executive summary:

In a reproductive/developmental toxicity screening study, 1,4-cyclohexanedimethanol was administered to 12 Sprague-Dawley rats/sex/group at dose concentrations of 0, 0.40% (4.0 mg/mL), 0.80% (8.0 mg/mL), or 1.25 % (12.5 mg/mL) in the drinking water for 91 days in males and 81-96 days in females. Dose levels of the test substance were approximately 0, 256, 479 and 861 mg/kg bw/day for males and 0, 385, 854 and 1360 mg/kg bw/day for females. Clinical abnormalities were observed primarily in the high-dose groups and included mortality, bloody or brown/red discolored urine, softened and/or reduced feces, dehydration, reductions in body weight and body weight gains, and decreased feed consumption. High-dose group males had decreased sperm motility, although this did not affect reproductive performance. Several high-dose group dams had litters that showed decreased postnatal pup survival and clinical abnormalities; these abnormalities were mostly observed in litters from mothers that also exhibited clinical abnormalities (i.e., reduced body weights and feed consumption, dehydration, discolored/bloody urine). Pups from high-dose dams had decreased pup body weights and pup body weight gains. Administration of the test material did not affect the reproductive performance in this study in terms of gestation length, prenatal loss, number of implantation sites, number of live and dead pups at birth, and ratio of male to female pups. The No-Observed-Effect Levels for reproductive/developmental effects in this study were considered to be 479 mg/kg bw/day for male rats based on decreased sperm motility in high-dose males and 854 mg/kg bw/day for female rats based on effects in pups from dams in the high-dose group. The NOEL for the entire study was 479 mg/kg bw/day.

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

1,4-cyclohexanedimethanol (CHDM, CAS 105 -08 -8) was administered to 12 Sprague-Dawley rats/sex/group at dose concentrations of 0, 0.40% (4.0 mg/mL), 0.80% (8.0 mg/mL), or 1.25 % (12.5 mg/mL) in the drinking water for 91 days in males and 81-96 days in females. Dose levels of the test substance were approximately 0, 256, 479 and 861 mg/kg bw/day for males and 0, 385, 854 and 1360 mg/kg bw/day for females. Clinical abnormalities were observed primarily in the high-dose groups and included mortality, bloody or brown/red discolored urine, softened and/or reduced feces, dehydration, reductions in body weight and body weight gains, and decreased feed consumption. High-dose group males had decreased sperm motility, although this did not affect reproductive performance. Several high-dose group dams had litters that showed decreased postnatal pup survival and clinical abnormalities; these abnormalities were mostly observed in litters from mothers that also exhibited clinical abnormalities (i.e., reduced body weights and feed consumption, dehydration, discolored/bloody urine). Pups from high-dose dams had decreased pup body weights and pup body weight gains. Administration of the test material did not affect the reproductive performance in this study in terms of gestation length, prenatal loss, number of implantation sites, number of live and dead pups at birth, and ratio of male to female pups. The No-Observed-Effect Levels for reproductive/developmental effects in this study were considered to be 479 mg/kg bw/day for male rats based on decreased sperm motility in high-dose males and 854 mg/kg bw/day for female rats based on effects in pups from dams in the high-dose group. The NOEL for the entire study was 479 mg/kg bw/day.


Short description of key information:
The NOEL for cyclohexanedimethanol (CHDM, CAS 105-08-8; CHDA is a putative metabolite of CHDA, and that is supported by a CHDM PK study) is 854 mg/kg/day for developmental effects

Justification for selection of Effect on fertility via oral route:
The study was chosen from 2 potential read-across studies due to its higher level of concern

Effects on developmental toxicity

Description of key information
The developmental NOAEL in a pre-natal developmental study was 1000 mg/kg/day, 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 following 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. 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.
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
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
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.
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.
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
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified
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.

 

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

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

1,4Cyclohexanedicarboxylate (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 220) 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 noobservedadverseeffect 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.

Justification for classification or non-classification

As there were no significant findings for reproduction or development, CHDA is not classified under GHS.