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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from September 10, 2002 to May 25, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted July 27, 1995
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
-Physical apperance: Clear colourless liquid
-Storage: in metal containers (4kg) at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS:
-Strain: Crl: CD®(SD) IGS Br strain
-Source: Charles River Laboratories, Stone Ridge (Kingston), NY, USA
-Sex: male and female
-Age at study initiation: males - 64 days, females - 67 days
-Acclimation period: 5 days
-Weight at study receipt: males 317.64 ± 11.95 g; females 232.72 ± 10.44 g
-Housing: Animals were housed singly in suspended stainless steel wire mesh cages for the duration of the study except during the matling phase during which time animals were housed in pairs. Male and female cages were housed and racked separately except during mating. Soild bottom pans containing bedding material were placed in the cages of female rats on the 19th day of gestation. Cages and racks were washed approximately once a week. Absorbant paper used to collect excreta, was changed daily throughout the study for males and daily for female rats during the pre-mating, mating and gestation phases of the study. During the lactation phase of the study bedding material in the solid bottom pans was changed only if necessary.
Due to a detectable odour from the test substance each dose group was housed in a separate animal room to reduce the potential of airborne cross-contamination. No other studies were housed in these rooms during this study.
-Diet: Certified Rodent Diet (Purina Rodent Chow #5002, meal (PMI Feed, Inc. Richmond, IN USA) available ad libitum
-Water: local ad libitum
-Method of animal identification: unique eartag
-Method of animal distribution: Stratified randomisation program

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

IN-LIFE DATES:
-Study Initiation Date: September 10, 2002
-Experimental Start Date: September 16, 2002
-Experimental Completion Date: May 25, 2003

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Test formulations:
The test substance was mixed with the ground food to test concentrations of 15.0, 4.5 and 1.5 mg test substance per gram of feed. The mixtures were prepared five or six times and used within 14 days based on the stability of the mixture. The control animals were given the basal diet.

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

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

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

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

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

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

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

Homegeneiy of the test mixtures was evauated by measuring the concentration of the test substance in Batch 1 at three levels (top, middle and bottom of the container) for all three dose levels. It was determined that at room temperature the cis isomer is a liquid but the trans isomer is a solid. Therefore container #2 of the test material was heated slightly to ensure the isomers were mixed and then this was used to prepare all the test mixtures for the studies. Purity of the test substance was analysed at the beginning and end of the study.
Duration of treatment / exposure:
Females: 38 to 57 days
Males: 50 days
Frequency of treatment:
Daily
Details on study schedule:
In-Life Study Dates:
-Study Initiation Date: September 10, 2002
-Experimental Start Date: September 16, 2002
-Experimental Completion Date: May 25, 2003
Doses / concentrationsopen allclose all
Dose / conc.:
1 500 mg/kg diet
Remarks:
Dose level of approximately 92 mg/kg bw/day males and 111 mg/kg bw females
Dose / conc.:
4 500 mg/kg diet
Remarks:
Dose level of approximately 276 mg/kg bw/day males and 351 mg/kg bw females
Dose / conc.:
15 000 mg/kg diet
Remarks:
Dose level of approximately 888 mg/kg bw/day males and 1124 mg/kg bw females
No. of animals per sex per dose:
12 rats/sex/group
Control animals:
yes, plain diet
Details on study design:
Dose selection rationale:
Doses were selected in consultation with the sponsor based on the data from a previous repeated-dose oral toxicity study.

Doses received:
Males: 0, 92, 276 and 888 mg/kg bw/day
Females: 0, 111, 351 and 1124 mg/kg bw/day (time-weight average of dose levels for all phases of the study)
Positive control:
none

Examinations

Parental animals: Observations and examinations:
Clinical Observations:
Clinical observations were conducted daily. Animals were also observed for moribundity/mortality each weekday afternoon. Clinical observations included but were not limited to: examination of hair, skin, eyes, mucous membranes, motor activity, feaces, urine, respiratory system, circulatory system, autonomic nervous system, central nervous system and behaviour patterns.

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

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

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

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

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

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

All adult animals were subjected to necropsy and a gross examination with special attention to the reproductive organs.The uteri from all adult females were examined for implantation sites, Corpora lutea were also counted.
The following tissues from adult animals were fixed in 10% buffered formalin: ovaries, uterus, vagina, Fallopian tubes, male accessory sex glands and gross lesions. After mortality analysis the right testis and right epididymis were fixed in Bouin's fixative; after approximately 24 hours, these tissues were rinsed twice with 50% ethyl alcohol and then stored in 70% ethyl alcohol.
For long term storage, the right testis and right epididymis were stored in 10% buffered formalin. The left testis and left epididyis were placed into individual containers, frozen with dry ice and stored at -70°C.

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

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

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

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

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

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

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


Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Minimal reductions in the amount of faeces or softened faeces, which were observed for five rats in the high-dose group for one to two days of the study. Other abnormalities observed were considered to be incidental to exposure.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean feed consumption values and body weights for males and females in the high-dose groups were lower than the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Mean feed consumption values and body weights for males and females in the high-dose groups were lower than the control group.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Clinical observations and mortality:
No mortality was observed during the study. There were no clinical observations that could be attributed to treatment with the test substance.

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

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

Overall mean bodyweight gains during gestation were comparable amongst groups.

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

Test substance intake:
No effects.

Reproductive performance:

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

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

Histopathology:
No treatment-related changes.

Assessment of sperm morphology and development
No treatment-related effects.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 124 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose tested in females was selected as pregnant females are considered to be more sensitive than males and as feed consumption patterns in males appears to be food refusal.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

The duration of the gestation phase was shorter (p≤0.05) for female rats from the mid-dose group when compared with the control group. All other parameters were comparable amongst the groups.

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

Body weight:
Mean pup weight change and percent pup weight change for days 0 to 4 were significantly (p≤0.05) higher for pups from the low-dose group when compared with the control group.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 124 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect at the highest dose tested

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:
The no-observed-effect-level for reproductive and neonatal toxicity was considered to be 1124 mg/kg bw/day (females), the highest concentration level tested.
Executive summary:

In a reproductive/developmental toxicity screening study performed according to the OECD test guideline No. 421 and in compliance with GLP, 12 rats/sex/group were exposed ad libitum in the diet to dimethyl 1,4-cyclohexanedicarboxylate at dose concentrations of 0, 0.15% ( approximately 92 mg/kg bw/day males and 111 mg/kg bw females), 0.45% (approximately 276 mg/kg bw/day males and 351 mg/kg bw females) and 1.5% (888 mg/kg bw/day males and 1124 mg/kg bw female)test substance in feed for 38 - 57days (females) or 50 days (males). 

There were no treatment-related effects on male or female fertility, pregnancy performance, or pup development in this reproductive and developmental toxicity screening study. There were no adverse effects on duration of gestation, gestation index, mean number of implant sites per pregnancy, mean number of pups born per litter, mean number of live pups per litter, live birth index, fertility and fecundity indexes, and group mean litter weights. No treatment-related abnormalities were observed in the pups. Although the duration of geatation phase was shorter for female rats in the mid-dose group there was no apparent effect on pup viability. The increased mean pup weight change and percentage pup weight change from days 0 to 4 of the low-dose group in comparison to the control group were not considered to be biologically significant. The no-observed-effect-level for reproductive and neonatal toxicity was considered to be 1124 mg/kg bw/day (females), the highest concentration level tested.

Under the conditions of the study, there was no clear evidence of test substance-related effects on reproduction of parental males and females or on survival and development of F1 pups. Based on this information, dimethyl 1,4-cyclohexanedicarboxylate is not a reproductive toxicant and is not selectively toxic to the developing fetus. Dimethyl 1,4-cyclohexanedicarboxylate is not classified for Developmental or Reproductive Toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).