Cyclohex-1,4-ylenedimethanol

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

GLP compliance:

yes

Limit test:

no

Justification for study design:

The study design was according to the established testing guideline, and mandated by ECHA.

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohex-1,4-ylenedimethanol

EC Number:

203-268-9

EC Name:

Cyclohex-1,4-ylenedimethanol

Cas Number:

105-08-8

Molecular formula:

C8H16O2

IUPAC Name:

cyclohexane-1,4-diyldimethanol

Specific details on test material used for the study:

Identification: 1,4-cyclohexanedimethanol (hereafter referred to as CHDM; CAS No. 105-08-08)
Batch/Lot No.: TP17030184
Receipt Date: 09 Aug 2017
Physical Description: Clear, colorless liquid
Purity: 90.4%
Water Content: 9.5%
Storage Conditions: Kept in a controlled temperature area set to maintain 18°C to 24°C
Supplier: Eastman Chemical Company

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical data for the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 443, Extended One-Generation Reproductive Toxicity Study, 28 Jul 2011, which recommends including a sufficient number of mating pairs to yield at least 20 pregnant females per dose group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test article-related moribundity and/or mortality in each generation of the study, 25 females/group was an appropriate number of animals to meet guideline recommendations.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Receipt
On 24 Aug 2017, Crl:CD(SD) rats were received from Charles River Laboratories, Inc., Raleigh, NC. The animals were 6 weeks old and weighed between 145 and 255 g at the initiation of dosing.
Justification for Test System and Number of Animals
The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical data for the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 443, Extended One-Generation Reproductive Toxicity Study, 28 Jul 2011, which recommends including a sufficient number of mating pairs to yield at least 20 pregnant females per dose group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test article-related moribundity and/or mortality in each generation of the study, 25 females/group was an appropriate number of animals to meet guideline recommendations.

Animal Identification
Upon receipt, each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Offspring were identified by tattoo markings applied to the digits after parturition and by microchip after weaning.
Pups selected for the F1 generation retained the dam number, followed by a hyphen "-" and the digit tattoo marking (i.e., 9999 01). F2 pups retained the dam number; however, the -01, -02, etc. designation in the F1 dam number was replaced with the corresponding letter of the alphabet (i.e., -01 = A, -02 = B, etc.) on some report tables.

Environmental Acclimation
After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing.

Selection, Assignment, and Disposition of Animals
F0 animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals at extremes of body weight range were not assigned to groups.
To reduce variability among the litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups.
For the F1 generation, 2 F1 pups/sex/litter from all available litters (≥20 litters/group) were randomly selected prior to weaning and were assigned to the following cohorts for subsequent reproductive/developmental toxicity testing. Assignment of same-sex littermates to a particular cohort was avoided whenever possible. In addition, if there were an insufficient number of pups to fill both cohorts, Cohort 1A was given priority.

Housing
On arrival (F0) or following weaning (F1), the animals were group housed (2 to 3 animals of the same sex). During cohabitation, animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding (heat-treated aspen bedding from Northeastern Products Corp.) equipped with an automatic watering valve. Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dosage level, and sex. Cages were arranged on the racks in group order.
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International.

Environmental Conditions
Target temperatures of 68°F to 78°F (20°C to 26°C) with a relative target humidity of 30% to 70% were maintained. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
PMI Nutrition International, LLC Certified Rodent LabDiet® 5K96 Advanced Protocol® Verified Casein Diet 10 IF was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility.It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system. Water bottles were provided, if required.Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Animal Enrichment
Animals were socially housed for psychological/environmental enrichment and were provided with environmental enrichment as appropriate to aid in maintaining the animals’ oral health. Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the Study Records and reviewed by the Study Director.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Deionized

Details on exposure:

Vehicle
Identification: Deionized water

Test Substance Characterization
The Sponsor provided to the Testing Facility documentation of the identity, strength, purity, composition, and stability for the test substance. A Certificate of Analysis was provided to the Testing Facility and is presented in Appendix 2.

eserve Samples
For each batch/lot of test substance, a reserve sample was collected and maintained under the appropriate storage conditions by the Testing Facility.

Test Substance Inventory and Disposition
Records of the receipt, distribution, and storage of test substance were maintained. With the exception of reserve sample, all unused test substance was retained for subsequent studies or discarded.

Dose Formulation and Analysis

Preparation of Vehicle
The vehicle, deionized water, was dispensed approximately weekly for administration to Group 1 control animals and adequate amounts were divided into daily aliquots, which were stored refrigerated (2°C to 8°C) until use. The vehicle was stirred continuously during dosing. Details of the dispensing of the vehicle have been retained in the Study Records.

Preparation of Test Substance
Test substance dosing formulations were prepared using appropriate methods at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared approximately weekly and an adequate amount of each formulation was dispensed into daily aliquots, which were stored refrigerated (2°C to 8°C) until use. The dosing formulations were stirred continuously during dosing. Details of the preparation and dispensing of the test substance have been retained in the Study Records.

Details on mating procedure:

The F0 animals were paired on a 1:1 basis within each treatment group after a minimum of 70 days of treatment. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Method
Analyses were performed by a high performance liquid chromatography method with charged aerosol detection using a validated analytical procedure.

Concentration Analysis
Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Homogeneity Analysis
Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was ≤ 10% and if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Stability Analysis
Test substance formulations have been previously shown to be stable over the range of concentrations used on this study for at least 10 days under refrigerated (2°C to 8°C) conditions.1 Therefore, stability of test substance formulations was not assessed on this study.

Duration of treatment / exposure:

F0: 129-132 days of treatment
F1: Sacrifices at days 4, 21, 91 and 149-161 based on cohort assignment
F2: PND 4

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until ~ 13 weeks (90 days) after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: ~13 weeks (90 days)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

350 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

700 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

Route and Dose Selection
The route of administration was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
The dosage levels were selected from the results of a previous oral (gavage) range-finding reproductive toxicity study in rats. In that study, the test substance was administered at dosage levels of 800 and 1000 mg/kg/day. No effects on maternal body weights or food consumption were noted during gestation or lactation. Lower body weight gains, absolute body weights, and slightly lower food consumption were noted in male and female pups in the F1 generation at both dosage levels. Therefore, dosage levels of 150, 350, and 700 mg/kg/day were selected for the current study for the determination of a NOAEL and for the evaluation of a potential dose response.


Animal group selection
F0 animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals at extremes of body weight range were not assigned to groups.
To reduce variability among the litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups.
For the F1 generation, 2 F1 pups/sex/litter from all available litters (≥20 litters/group) were randomly selected prior to weaning and were assigned to the following cohorts for subsequent reproductive/developmental toxicity testing. Assignment of same-sex littermates to a particular cohort was avoided whenever possible. In addition, if there were an insufficient number of pups to fill both cohorts, Cohort 1A was given priority.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

Clinical Observations
The animals were removed from the cage and a clinical observation was performed once daily throughout the study (see Appendix 1 - Study Protocol and Deviations). During the dosing period, these observations were performed prior to dosing. On dosing days, clinical observations were also recorded approximately 1 hour postdose (see Appendix 1 - Study Protocol and Deviations).
During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

Detailed Physical Examinations
The animals were removed from the cage, and a detailed physical examination was performed weekly throughout the study (see Appendix 1 - Study Protocol and Deviations). In addition, detailed physical examinations were conducted on Gestation Days 0, 7, 14, and 20 for all females with evidence of mating and on Lactation Days 1, 7, 14, and 21 (see Appendix 1 - Study Protocol and Deviations).

Body Weights
Animals were weighed individually weekly throughout the study and prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 0 (when possible), 1, 4, 7, 10, 14, 17, and 21 (see Appendix 1 - Study Protocol and Deviations). A fasted weight was recorded on the day of necropsy. Terminal body weights were not collected from animals found dead or euthanized moribund.

Food Consumption
Food consumption was quantitatively measured weekly throughout the study, except during the mating period. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, 14, 17, and 21.

Food Evaluation
Food efficiency (body weight gained as a percentage of food consumed) was also calculated and reported for each interval.

Oestrous cyclicity (parental animals):

Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each F0 female for 14 days prior to cohabitation and continuing until evidence of mating was observed or until the end of the mating period. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P], beginning 14 days prior to initiation of the mating period and continuing until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.
At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data. The following definitions were used for determination of regular cycling, irregular cycling, non-cycling, and insufficient data. Note that for a “complete cycle”, an animal must exhibit at minimum an E from the previous cycle, followed by one cycle (D to E), and a D from the start of the next cycle:

Sperm parameters (parental animals):

Immediately upon euthanasia, the reproductive tract of each male was exposed via a ventral mid line incision. The right cauda epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis, and it was then placed in Dulbecco's phosphate buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a minimum 10 minute incubation period, a sample of sperm was loaded onto a slide with a 100 µm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all pipettes, slides, and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported.

The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.
The left testis and cauda epididymis from all males were weighed, stored frozen, homogenized, and analyzed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each sample was added to a solution containing a DNA specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20 µm chamber depth. Illumination from a xenon lamp within the analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample.

Litter observations:

Viability
Litters were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. A daily record of litter size was maintained. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings. Total litter loss was determined when the last pup in the litter was found dead or euthanized in extremis prior to the scheduled necropsy. Litters that were euthanized prior to scheduled necropsy due to reasons unrelated to test substance administration (e.g., drowning, mechanical injury, inadvertent removal from room, early group termination, or death of the dam) were not considered to be a total litter loss on the data tables and were not included in the pup viability calculations.

Observations
A detailed physical examination was performed for each pup on PND 1, 4, 7, 14, and 21 (see Appendix 1 - Study Protocol and Deviations). Any abnormalities in nesting and nursing behavior were recorded.

Sex Determination
Pups were individually sexed on PND 0, 4, 14, and 21.

Anogenital Distance
The anogenital distance of all pups was measured on PND 1. Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle.

Body Weights
Pups were weighed individually on PND 1, 4, 7, 14, and 21.

Assessment of Areolas/Nipple Anlagen
On PND 13, all male pups were evaluated for the presence of thoracic nipples/areola. The number of nipples was recorded.

Thyroid Hormone Analysis

Sample Collection
Blood samples for thyroid hormone analysis were collected (prior to 1200 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels) via cardiac puncture of animals anesthetized with isoflurane (PND 4 culled pups) or via the vena cava following euthanasia by carbon dioxide inhalation (PND 21 nonselected pups) into tubes without anticoagulants.

Postmortem examinations (parental animals):

Thyroid Hormone Analysis
Sample Collection
Blood samples for thyroid hormone analyses were collected (prior to 1200 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels) from the jugular vein into tubes without anticoagulants. Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored at approximately -65°C to 85°C.

Clinical Pathology
Animals were fasted overnight prior to blood collection. Urine was collected overnight using metabolism cages. Blood samples for hematology and serum chemistry were collected from the jugular vein. Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation. K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was used for samples collected for clotting determinations. Samples for serum chemistry were collected without anticoagulants. In addition, blood smears were prepared, stained with Wright Giemsa stain, cover-slipped, and retained for possible future evaluation.

Scheduled Euthanasia
All surviving animals, including females that failed to deliver or with total litter loss, were euthanized by carbon dioxide inhalation following the selection of the F1 generation.

Necropsy
All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. The numbers former implantation sites were recorded for females that delivered. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.

Organ Weights
The organs identified in Text Table 18 were weighed at necropsy for all scheduled euthanasia animals (see Appendix 1 - Study Protocol and Deviations). Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

Representative samples of the tissues identified in Text Table 19 were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated (see Appendix 1 - Study Protocol and Deviations).

Ovaries were processed at PAI Durham. All other histology procedures were performed at the Testing Facility. Tissues identified in Text Table 19 from all animals in the control and high dose groups and from all animals found dead or euthanized in extremis, as well as gross lesions from all animals in all groups and reproductive organs of all F0 animals suspected of reduced fertility, were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Processing of the testes, epididymides, and ovaries were performed as noted below.
Sections of 2–4 microns of the testis (transverse) and epididymis (longitudinal) were stained with PAS and hematoxylin staining in addition to the routine hematoxylin and eosin (H&E) staining. The following regions of the epididymis were embedded in paraffin: caput, corpus, and cauda; the vas deferens was examined when possible.
Five (5) sections were taken approximately 100 µm apart from the inner third of each ovary from any F0 females suspected of reduced fertility. In addition, a single section was taken from all F0 females (with no signs of reduced fertility) for a qualitative bilateral evaluation of each ovary.

Histopathology
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified in Text Table 19 for microscopic examination were evaluated from all animals in the control and high dose groups and from all animals found dead and euthanized in extremis. Gross lesions were examined from all animals in all groups (see Appendix 1 - Study Protocol and Deviations). In addition, reproductive organs of all animals suspected of reduced fertility were examined.
Histopathological examination of the testis included a qualitative assessment of the stages of spermatogenesis. For males that survived to the scheduled necropsy, microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules. The progression of these cellular associations defines the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).
For any F0 female suspected of reduced fertility, a quantitative histopathologic evaluation of multiple sections was conducted. This examination included enumeration of the total number of primordial follicles according to the methods of Bolon et al., Bucci et al., and Picut et al. Uterine and ovarian histopathology were considered in light of the terminal estrous stage.

Postmortem examinations (offspring):

Scheduled Euthanasia
On PND 4, culled pups were euthanized by exsanguination (bilateral thoracotomy, if necessary; those pups used for blood/thyroid collection) or an intraperitoneal injection of sodium pentobarbital.
On PND 21, nonselected pups were euthanized by carbon dioxide inhalation.
Necropsy
On PND 4, all culled pups were subjected to a complete necropsy examination. Pups were necropsied using a fresh dissection technique, which included examination of the heart and major vessels.
On PND 21, nonselected pups were subjected to a complete necropsy examination, which included evaluation of the cranial, thoracic, abdominal, and pelvic cavities, with emphasis on developmental morphology and organs of the reproductive system.
Organ Weights
The organs identified were weighed at necropsy from 10 nonselected F1 pup/sex/group on PND 21. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

Tissue Collection and Preservation
Representative specimens with malformations and gross lesions from offspring found dead or euthanized for humane reasons were preserved in 10% neutral buffered formalin.
Representative samples of the tissues identified were collected from all PND 4 culled pups and preserved in 10% neutral buffered formalin.

Unscheduled Deaths
A necropsy was conducted for animals that died on study, and specified tissues were saved.
If necessary for humane reasons, animals were euthanized as per Testing Facility SOPs. These animals underwent necropsy, and specified tissues were retained.

Scheduled Euthanasia
All surviving animals, were euthanized by carbon dioxide inhalation.

Spermatogenic Evaluations (Cohort 1A)
Immediately upon euthanasia, the reproductive tract of each male was exposed via a ventral mid line incision. The right cauda epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis, and it was then placed in Dulbecco's phosphate buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a minimum 10 minute incubation period, a sample of sperm was loaded onto a slide with a 100 µm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all pipettes, slides, and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported.
The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.
The left testis and cauda epididymis from all males were weighed, stored frozen, homogenized, and analyzed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each sample was added to a solution containing a DNA specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20 µm chamber depth. Illumination from a xenon lamp within the analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample.

Necropsy
All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. The numbers of former implantation sites were recorded for females that delivered. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.

Organ Weights
The organs identified were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

Tissue Collection and Preservation
Representative samples of the tissues identified were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated.

Histology (Cohort 1A)
Ovaries were processed at PAI Durham. All other histology procedures were performed at the Testing Facility. Tissues identified from all animals in the control and high dose groups and from all animals found dead or euthanized in extremis, as well as gross lesions form all animals in all groups and reproductive organs of all animals suspected of reduced fertility, were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Processing of the testes, epididymides, and ovaries were performed as noted below.
Sections of 2–4 microns of the testis (transverse) and epididymis (longitudinal) were stained with PAS and hematoxylin staining in addition to the routine hematoxylin and eosin (H&E) staining. Testes and epididymides from males that were found dead or euthanized in extremis were stained with H&E only. The following regions of the epididymis were embedded in paraffin: caput, corpus, and cauda; the vas deferens was examined when possible.
Five (5) sections were taken approximately 100 µm apart from the inner third of each ovary from all F1 Cohort 1A females at scheduled termination. In addition, a single section was taken from all F1 Cohort 1A females for a qualitative bilateral evaluation of each ovary.
The coagulating glands, ovaries, pituitary gland, prostate gland, seminal vesicles, testes with epididymides, uterus with cervix and vagina, and gross lesions from Cohort 1B animals were processed to the block stage (in paraffin).

Histopathology (Cohort 1A)
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified for microscopic examination were evaluated from all animals in the control and high dose groups and from all animals found dead and euthanized in extremis. Gross lesions were examined from all animals in all groups. In addition, reproductive organs of all animals suspected of reduced fertility were examined.
Histopathological examination of the testis included a qualitative assessment of the stages of spermatogenesis.13 For males that survived to the scheduled necropsy, microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules. The progression of these cellular associations defines the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). When possible, sections of the rete testis were examined.
For F1 females in the control and high-dose groups, a quantitative histopathologic evaluation of multiple sections was conducted. This examination included enumeration of the total number of primordial follicles according to the methods of Bolon et al., Bucci et al., and Picut et al. Uterine and ovarian histopathology were considered in light of the terminal estrous stage.

Statistics:

See "other information" section. We suggest that ECHA remove the character limit here. There's no reason for it, and there's normally a great deal of detail in this section.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related clinical observations were noted during the generation at the detailed physical examinations, daily examinations, or 1-hour postdosing observations. Findings noted in the test substance treated groups, including hair loss, scabbing, and red material on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no test substance-related effect on survival. There were early deaths in all test substance group females, but none of the deaths were considered test substance-related. Two females each in the 150, 350, and 700 mg/kg/day groups were euthanized in extremis due to adverse clinical findings or found dead around the time of parturition or early lactation. In order to determine if the parturition findings and early deaths in the F0 generation were related to test substance administration, the F1 Cohort 1B animals were bred to obtain an F2 generation. There were no adverse clinical findings during parturition or test substance-related early deaths in the F1 Cohort 1B females, indicating that the early deaths in the F0 females were not reproducible and therefore not related to test substance administration. The incidence of early deaths in the F0 females lacked a clear dose response. In addition, there were no test substance-related alterations in reproductive performance and no organ weight, gross, or microscopic changes in the reproductive or other tissues at the scheduled F0 euthanasia. Therefore, the early deaths in the F0 animals were considered incidental and due to biologic variation and without a relationship to administration of the test substance

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related effects on mean body weights, body weight gains, and cumulative body weight gains were noted in the 150, 350, and 700 mg/kg/day groups. The values in the test substance treated groups were generally comparable to the control group values for the pre-mating period (females) or the entire generation (males). Statistically significant differences in mean body weight gain were sporadically noted in the test substance-treated groups; however, these differences were transient and/or did not affect absolute mean body weights. Other differences from the control group were slight, did not occur in a dose related manner, and/or were not statistically significant.

Mean maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance administration during gestation. Differences between the control, 150, 350, and 700 mg/kg/day groups were slight and not statistically significant.

Mean maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance administration during lactation. Differences between the control, 150, 350, and 700 mg/kg/day groups were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

Food consumption, evaluated as g/animal/day, and food efficiency in the 150, 350, and 700 mg/kg/day groups were unaffected by test substance administration. The values in the test substance treated groups were generally comparable to the control group values for the pre-mating period (females) or the entire generation (males). Statistically significant differences in mean food consumption and food efficiency were sporadically noted in the test substance-treated groups; however, these differences were transient and/or did not affect mean body weight gains. Other differences from the control group were slight, did not occur in a dose related manner, and/or were not statistically significant.

Mean maternal food consumption, evaluated as g/animal/day, and food efficiency were unaffected by test substance administration during gestation. Differences between the control, 150, 350, and 700 mg/kg/day groups were slight and not statistically significant

Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency were unaffected by test substance administration during lactation. Differences between the control, 150, 350, and 700 mg/kg/day groups were slight and not statistically significant.

Food efficiency:

not specified

Description (incidence and severity):

Food consumption, evaluated as g/animal/day, and food efficiency in the 150, 350, and 700 mg/kg/day groups were unaffected by test substance administration. The values in the test substance treated groups were generally comparable to the control group values for the pre-mating period (females) or the entire generation (males). Statistically significant differences in mean food consumption and food efficiency were sporadically noted in the test substance-treated groups; however, these differences were transient and/or did not affect mean body weight gains. Other differences from the control group were slight, did not occur in a dose related manner, and/or were not statistically significant.

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not examined

Description (incidence and severity):

There were no test substance-related effects on hematology parameters. Differences between the control, 150, 350, and 700 mg/kg/day groups were slight and not statistically significant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on serum levels of T3 (triiodothyronine), T4 (thyroxine), or TSH (thyroid stimulating hormone) in F0 males and females. Statistically significantly higher mean T4 values were noted for males and females in the 700 mg/kg/day group and lower (not statistically significant) mean TSH levels were noted for males in this group; however, there were no effects on T3 levels, there was no dose-response at 150 and 350 mg/kg/day, there were no effects on thyroid gland weights, or any histologic correlates in the thyroid glands. Other differences from the control group were slight, not statistically significant, and/or did not occur in an exposure related manner.

There were no test substance-related effects on serum chemistry parameters. Statistically significantly lower mean chloride values were noted for males in the 350 and 700 mg/kg/day groups and statistically significantly lower creatinine values were noted for females in the 150, 350, and 700 mg/kg/day groups. However, the differences in chloride were minimal compared to the control group and the changes in creatinine did not occur in a dose-related manner. Other differences from the control group were slight and not statistically significant.

There were no test substance-related effects on coagulation parameters. Differences between the control, 150, 350, and 700 mg/kg/day groups were slight and not statistically significant.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on urinalysis parameters. Statistically significantly included lower mean pH values for males in all test substance-treated, lower mean specific gravity for females in the 350 mg/kg/day group, and higher total urine volume for females in the 700 mg/kg/day group. However, the differences were slight and/or did not occur in a dose related manner.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related histologic changes, including no test substance-related histologic changes in the thyroid gland. A very low incidence of testicular atrophy was noted in the vehicle control group and 350 mg/kg/day group males. As this change was not observed in the 700 mg/kg/day group males, a clear dose response was not observed; therefore, the testicular atrophy was considered spontaneous and not related to administration of 1,4 cyclohexanedimethanol.
All histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean numbers of days between pairing and coitus in the test substance treated groups were comparable to the control group value. The mean lengths of estrous cycles and cycling statuses in these groups were also similar to the control group value. None of these differences were statistically significant.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test substance related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage level. Differences from the control group were slight and were not statistically significant.

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance related effects on F0 reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance treated groups. Five, 3, 3, and 2 males in the control, 150, 350, and 700 mg/kg/day groups, respectively, did not sire a litter. Five, 3, 3, and 2 females in these same respective groups were determined to be nongravid.
The mean numbers of days between pairing and coitus in the test substance treated groups were comparable to the control group value. The mean lengths of estrous cycles and cycling statuses in these groups were also similar to the control group value. None of these differences were statistically significant.

Details on results (P0)

In this study report, F0, F1 and F2 are used rather than the "P" nomenclature in this form. In order to be consistent with the study report, the language of the report is retained.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

700 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (sperm measures)

reproductive performance

other: No adverse effects at any dose

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance administration during gestation. Differences between the control, 150, 350, and 700 mg/kg/day groups were slight and not statistically significant.

Mean maternal body weights and body weight gains were unaffected by test substance administration during Lactation Days 1-4. Differences between the control, 150, 350, and 700 mg/kg/day groups were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

Mean maternal food consumption, evaluated as g/animal/day, and food efficiency were unaffected by test substance administration during gestation. Differences between the control, 150, 350, and 700 mg/kg/day groups were slight and not statistically significant.

Mean maternal food consumption, evaluated as g/animal/day, and food efficiency were unaffected by test substance administration during Lactation Days 1–4. Differences between the control, 150, 350, and 700 mg/kg/day groups were slight and not statistically significant.

Food efficiency:

no effects observed

Description (incidence and severity):

Mean maternal food consumption, evaluated as g/animal/day, and food efficiency were unaffected by test substance administration during gestation. Differences between the control, 150, 350, and 700 mg/kg/day groups were slight and not statistically significant.

Mean maternal food consumption, evaluated as g/animal/day, and food efficiency were unaffected by test substance administration during Lactation Days 1–4. Differences between the control, 150, 350, and 700 mg/kg/day groups were slight and not statistically significant.

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related effects were noted on mean gestation lengths or the process of parturition at any dosage level. Mean F1 gestation lengths in the test substance treated groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the 150, 350, and 700 mg/kg/day groups were 21.8, 21.5, and 21.9 days, respectively, compared to mean gestation lengths of 21.8 days in the concurrent control group and 21.8 days in the Charles River Ashland historical control data. No signs of dystocia were noted at any dosage level.

Effect levels (P1)

Dose descriptor:

NOAEL

Effect level:

700 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

food efficiency

Remarks on result:

other:

Remarks:

no effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Prior to weaning, the general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by test substance administration.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The mean number of F1 pups born, live litter size, percentage of males per litter at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0–1, 1–4 (pre selection), 4 (post-selection)–7, 7–14, 14–21, and from birth to PND 4 (pre-selection) and PND 4 (post-selection)–21 were unaffected by the test substance at all dosage levels. Differences from the control group were slight, were not statistically significant, and/or did not occur in a dose related manner. The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by test substance administration. Twenty (13), 20(5), 30(7), and 4(4) pups (litters) in the control, 150, 350, and 700 mg/kg/day groups, respectively, were found dead or euthanized in extremis. Seven (6), 13(7), 13(7), and 6(4) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.

Following weaning, There was no test substance-related effect on survival. Early deaths were noted in the control, 150 and/or 350 mg/kg/day groups, but were not at 700 mg/kg/day group, the highest dose level tested. None of the early deaths were considered test substance-related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean male and female pup body weights and body weight changes in the 150, 350, and 700 mg/kg/day groups were unaffected by test substance administration throughout the postnatal period. A statistically significantly lower mean body weight gain was noted for males in the 150 mg/kg/day group during PND 1–4; however, this change did not occur in a dose-related manner. No other statistically significant differences from the control group were noted.

Following weaning, No test substance related effects on mean body weights, body weight gains, and cumulative body weight gains were noted in the 150, 350, and 700 mg/kg/day groups. The values in the test substance treated groups were generally comparable to the control group values for the pre-mating period (Cohort 1B females) or the entire generation (males and Cohort 1A females). A statistically significantly lower mean body weight gain was noted for males in the 700 mg/kg/day group during PND 84–90 and statistically significantly higher mean body weight gains were noted for females in the 150 and 700 mg/kg/day groups during PND 35–42; however, these changes were transient and did not affect absolute mean body weights. Other differences from the control group were slight, did not occur in a dose related manner, and/or were not statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption, evaluated as g/animal/day, and food efficiency in the 150, 350, and 700 mg/kg/day groups were unaffected by test substance administration. The values in the test substance treated groups were generally comparable to the control group values for the pre-mating period (cohort 1B females) or the entire generation (males and cohort 1A females). Differences from the control group were slight, did not occur in a dose related manner, and/or were not statistically significant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on hematology parameters. Statistically significantly higher absolute and percent leukocyte values were noted for males in the 350 and 700 mg/kg/day groups; however, there were no correlating microscopic findings. Other differences from the control group were slight and not statistically significant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on serum levels of T3 (triiodothyronine) or T4 (thyroxine) in pups that were culled on PND 4. Differences from the control group were slight, not statistically significant, and/or did not occur in a dose related manner. There were no test substance-related effects on serum levels of T3 (triiodothyronine), T4 (thyroxine), or TSH (thyroid stimulating hormone) in nonselected pups on PND 21. Mean T4 levels for males and females in the 150, 350, and 700 mg/kg/day groups were statistically significantly lower than the control group; however, this change did not occur in a dose-related manner and there were no effects on T3 levels or effects on organ weights. The thyroid glands were not examined histopathologically, so a correlation cannot be made at this time. No other statistically significant differences were noted.

There were no test substance-related effects on coagulation parameters. A statistically significantly higher prothrombin time was noted for females in the 700 mg/kg/day group; however, the difference from the control group was minimal, and no other effects on coagulation parameters were observed. Other differences from the control group were slight and not statistically significant.

There were no test substance-related effects on serum chemistry parameters. A statistically significantly lower mean total bilirubin value was noted for females in the 350 mg/kg/day; however, this change did not occur in a dose-related manner and was not in a direction that was toxicologically relevant. Other differences from the control group were slight and not statistically significant.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on urinalysis parameters. Statistically significantly lower mean pH values were noted for males and females in all test substance-treated groups; however, the values were within the range of values in the Charles River Ashland historical control data (Version 3.6). In addition, statistically significantly higher mean specific gravity and lower mean total volume were noted for males in the 350 mg/kg/day group; however, these changes did not occur in a dose-related manner.

Sexual maturation:

no effects observed

Description (incidence and severity):

The mean ages at the first occurrence of estrus in the 150, 350, and 700 mg/kg/day groups (35.8, 35.4, and 35.7 days, respectively) were generally comparable to the control group (34.6 days). In addition, the duration from vaginal opening to first estrus in these same respective groups (3.4, 2.1, and 3.5 days) was generally comparable to the control group (2.4 days). None of the differences were statistically significant.

Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance administration. The mean ages of attainment of balanopreputial separation were 43.6, 44.1, and 43.8 days in the 150, 350, and 700 mg/kg/day groups, respectively, when compared to 43.3 in the control group. Mean body weights at the age of attainment were 224.7 g, 231.9 g, and 227.6 g in the same respective groups compared to 230.9 g in the control group. None of the differences from the control group were statistically significant.

Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance administration. The mean ages of attainment of vaginal patency were 33.4, 34.5, and 33.0 days in the 150, 350, and 700 mg/kg/day groups, respectively, when compared to 33.2 days in the control group. The mean age at attainment of vaginal patency in the 350 mg/kg/day group was statistically significantly higher than the control group; however, this change did not occur in a dose-related manner. Mean body weights at the age of attainment were 118.2 g, 125.0 g, and 117.2 g in the same respective groups compared to 118.2 g in the control group. None of the differences from the control group were statistically significant.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distances (absolute and relative to the cube root of pup body weight) in the 150, 350, and 700 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen in the F1 male pups was not affected by test substance administration. There was no retention of nipples noted in any male pup on study on PND 13.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related alterations in organ weights including no test substance-related organ weight changes in thyroid/parathyroid gland weights. For Cohort 1B, testes weights (absolute and relative to body weight) were slightly higher in the 350 and 700 mg/kg/day group males. These minor perturbations did not exhibit a clear dose response and were considered due to biologic variation rather than the test substance.
Some organ weight differences were statistically significant when compared to the control group but were considered to be a result of final body weight fluctuations which were not dose-dependent and were considered due to biologic variation. Thus, all organ weight differences observed were considered incidental and unrelated to administration of the test substance

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance. All gross changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
No test substance related effects were observed on the number of former implantation sites, the number of corpora lutea, and the number of unaccounted-for sites. The differences between the control and test substance treated groups were slight and not statistically significant.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related histologic changes, including no test substance-related histologic changes in the thyroid gland. A very low incidence of testicular atrophy was noted in the vehicle control group and 350 mg/kg/day group males. As this change was not observed in the 700 mg/kg/day group males, a clear dose response was not observed; therefore, the testicular atrophy was considered spontaneous and not related to administration of 1,4 cyclohexanedimethanol.
All histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

700 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

other: No effects observed

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical findings) of all F2 pups in this study was unaffected by test substance administration. Seven (5), 24(7), 14(8), and 5(5) pups (litters) in the control, 150, 350, and 700 mg/kg/day groups, respectively, were found dead. Five (4), 4(2), 17(5), and 3(2) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. The aforementioned findings did not occur in a dose-related manner and were therefore not considered related to CHDM administration.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The mean number of F2 pups born, live litter size, percentage of males per litter at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0-1, 1-4, and from birth to PND 4 were unaffected by the test substance at all dosage levels. Differences from the control group were slight, were not statistically significant, and/or did not occur in a dose related manner.

The general physical condition (defined as the occurrence and severity of clinical findings) of all F2 pups in this study was unaffected by test substance administration. Seven (5), 24(7), 14(8), and 5(5) pups (litters) in the control, 150, 350, and 700 mg/kg/day groups, respectively, were found dead. Five (4), 4(2), 17(5), and 3(2) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. The aforementioned findings did not occur in a dose-related manner and were therefore not considered related to CHDM administration.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean male and female pup body weights and body weight changes in the 150, 350, and 700 mg/kg/day groups were unaffected by parental test substance from PND 1-4. No statistically significant differences from the control group were noted.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

Pups were sacrificed prior to weaning so there is nothing to measure.

Food efficiency:

not examined

Description (incidence and severity):

Pups were sacrificed prior to weaning so there is nothing to measure.

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

not examined

Description (incidence and severity):

Pups were sacrificed prior to weaning so there is nothing to measure.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Description (incidence and severity):

Pups were sacrificed at PND 4, so the measure is not relevant

Anogenital distance (AGD):

not examined

Description (incidence and severity):

Pups were sacrificed at PND 4, so the measure is not relevant

Nipple retention in male pups:

not examined

Description (incidence and severity):

Pups were sacrificed at PND 4, so the measure is not relevant

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Seven (5), 24(7), 14(8), and 5(5) pups (litters) in the control, 150, 350, and 700 mg/kg/day groups, respectively, were found dead from PND 0 to 4. No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach, the only other internal finding in the test substance-treated groups was a major blood vessel variation for Pup No. 8991 15-06 in the 350 mg/kg/day group; however, this findings was not observed in a dose related manner.

Histopathological findings:

not examined

Other effects:

not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

700 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

Remarks on result:

other:

Remarks:

no adverse effects

Overall reproductive toxicity

Reproductive effects observed:

no

Treatment related:

no

Any other information on results incl. tables

Results of F₀Reproductive Performance

Parameter	Dosage Level (mg/kg/day)				CRL HC^a Mean (Range)
	0	150	350	700
Male Mating Index (%)	100.0	92.0	96.0	96.0	97.9 (83.3–100.0)
Female Mating Index (%)	100.0	92.0	96.0	96.0	97.9 (83.3–100.0)
Male Fertility Index (%)	80.0	88.0	88.0	92.0	94.0 (80.0–100.0)
Female Fertility Index (%)	80.0	88.0	88.0	92.0	94.0 (80.0–100.0)
Male Copulation Index (%)	80.0	95.7	91.7	95.8	95.9 (80.0–100.0)
Female Conception Index (%)	80.0	95.7	91.7	95.8	95.9 (80.0–100.0)
Estrous Cycle Length (days)	4.2	4.0	4.2	4.3	4.2 (3.9–5.2)
Pre-Coital Interval (days)	3.4	3.2	2.2	3.2	2.7 (1.8–4.5)
^a Charles River Ashland historical control data (version 2018.02)

Results of F₁Reproductive Performance

Parameter	Dosage Level (mg/kg/day)				CRL HC^a Mean (Range)
	0	150	350	700
Male Mating Index (%)	100.0	89.5	94.7	100.0	97.9 (83.3–100.0)
Female Mating Index (%)	100.0	90.0	95.0	100.0	97.9 (83.3–100.0)
Male Fertility Index (%)	89.5	84.2	89.5	90.5	94.0 (80.0–100.0)
Female Fertility Index (%)	90.0	85.0	90.0	90.5	94.0 (80.0–100.0)
Male Copulation Index (%)	89.5	94.1	94.4	90.5	95.9 (80.0–100.0)
Female Conception Index (%)	90.0	94.4	94.7	90.5	95.9 (80.0–100.0)
Estrous Cycle Length (days)	4.3	4.2	4.2	4.1	4.2 (3.9–5.2)
Pre-Coital Interval (days)	2.6	2.5	2.6	2.8	2.7 (1.8–4.5)
^a Charles River Ashland historical control data (version 2018.02). None statistically significantly different from the control group.

Applicant's summary and conclusion

Conclusions:

Based on the lack of effects, a dosage level of 700 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for parental systemic toxicity, reproductive toxicity, and neonatal toxicity of 1,4-cyclohexanedimethanol when administered via oral gavage to Crl:CD(SD) rats.

Executive summary:

The objective of this study was to evaluate the potential adverse effects of the test substance, 1,4‑cyclohexanedimethanol, hereafter referred to as CHDM, on reproduction in an extended one-generation study. This included evaluation of life stages not covered by other types of toxicity studies and tested for effects that may occur as a result of pre- and postnatal chemical exposure.

Animals were dosed via oral gavage daily for at least 70 consecutive days prior to mating and continuing through the day prior to euthanasia. The offspring selected to become the F₁parental generation were dosed following weaning.

The following parameters and end points were evaluated in this study: survival, clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen, developmental landmarks, thyroid hormones, clinical pathology, gross necropsy findings, spermatogenic endpoints, organ weights, and histopathologic examinations.

No test substance-related effects were noted at any dosage level for any generation for the following parameters: survival, clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen, developmental landmarks, thyroid hormones, clinical pathology, gross necropsy findings, spermatogenic endpoints, organ weights, and histopathologic examinations. Two F₀females each in the 150, 350, and 700 mg/kg/day groups were euthanized in extremis or found dead around the time of parturition. In order to determine if the early deaths in the F₀generation were related to test substance administration, the F₁Cohort 1B animals were bred to produce an F₂generation. Based on the absence of a clear dose-response and the lack of a reproducible effect on parturition or survival in the bred F₁Cohort 1B generation, these deaths were not considered related to test substance administration.

Based on the lack of effects, a dosage level of 700 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F₀and F₁male and female systemic toxicity, reproductive toxicity, and neonatal toxicity of 1,4‑cyclohexanedimethanol when administered via oral gavage to Crl:CD(SD) rats.