Cyclohexanone

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: comparable to guideline study with GLP

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexanone

EC Number:

203-631-1

EC Name:

Cyclohexanone

Cas Number:

108-94-1

Molecular formula:

C6H10O

IUPAC Name:

cyclohexanone

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Each animal was assigned a unique identification number. A tag displaying this number was affixed to the, ear of each animal and a card listing the number was placed on the cage. A label displaying this number was attached to all jars/bags containing any tissues retained.
The P-C males and all females were acclimated for at least 12 days after their arrival. Only apparently healthy animals were used in this study. During the acclimation period, the animals were housed in individual stainless steel, suspended, wire-bottom cages. The females were housed in individual, stainless steel, suspended, wire-bottom cages after completion of their mating trial.
The acclimation and study rooms were cleaned daily. The rooms were well ventilated and air-conditioned. The temperature and humidity were monitored daily and generally ranged from 73 + 5°F and 50 + 20% respectively. The temperature and humidity minimum/maximums were 64 to 79°F and 32 to 80%, respectively . A 12-hour light/dark cycle was maintained.
Purina Certified Rodent Chowand filtered tap water were provided ad libitum throughout the acclimation and study periods. The automatic watering system of each rack was flushed with fresh water on a daily basis. The water was assayed periodically for pathogens and potential chemical, heavy metal, or pesticide contamination. The water quality was deemed acceptable.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Details on mating procedure:

The mating trials for the "a" litters were initiated following the pre-mating period exposure to the test article and were continued for 15 consecutive days. Monogamous cohabitation, whenever possible, was used (1 male : 1 female) with the animal parings conducted randomly employing computer-generated male/female assignments within treatment groups. Following a 5-day cohabitation period, males were rotated among the unbred females in their treatment group and an additional 5 days of cohabitation was allowed. This procedure was rrpeated for a third 5 day male/fernale pairing period for females which remained unbred.
Following discussion with the sponsor and evaluation of the F2a litter data, it was decided to conduct an additional mating trial to obtdain F2b litters. The mating trial tor the F2b litter was initiated approx. 2 weeks after the weaning of the F2a litter and the same procodure used during the "a" litter mating trial were followed. All surviving animals were paired, however, males were not paired with females they had been exposed to during the F2a litter mating trial. In addition, sibling pairings were avoided during the F1 generation mating trials.
Each female was examined daily during the period of cohabitation to detect evidence or breeding. The observation of a copulatory plug in the vagina asperm-positive result of vaginal smears was defined as evidence of copulation. Females for which breeding was confirmed were weighed (gestation day 0) and were housed individually terminating their mating trial.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

2 generations

Frequency of treatment:

6 h/d

Doses / concentrations

Remarks:

Doses / Concentrations:
250; 500; 1000 (resp. 1400) ppm (ca. 1.02; 2.04; 4.1 mg/l)
Basis:
nominal conc.

No. of animals per sex per dose:

30

Control animals:

yes, concurrent no treatment

Details on study design:

Groups of 30 males/30 females were exposed to either 0, 250, 500, or 1,000 ppm during the first (F0) generation. Thirty males/30 females were selected from the F1a litters of each group to continue the test as second (F1) generation animals. The F1 generation animals were exposed to 0, 250, 500, or 1,400 ppm cyclohexanone. Assessments for potential neurotoxicologic/neuropathologic effects were conducted pre-weaning and post-weaning in each F1a litter.

Positive control:

triethylenemelamine

Examinations

Parental animals: Observations and examinations:

All parental animals were weighed weekly during the premating period. Weekly body weights were obtained for all surviving parental males following completion of the matring trials, for all females which did not retain a litter and for all unbred females until their sacrifice. The parental females were weighed on gestation days 0, 6, 15 and 20 and lactation days 0, 5, 7, 14, 21 and 28. Final body weights were obtained for each animal at sacrifice or death.
During all phases of the study, food consumption was monitored visually.
All animals were observed at least wice each day for mortality, morbidity and overt signs of toxicity. At least once each week each animal was removed ferm its cage and thoroughly examined.

Litter observations:

Individual pup weights and sexes were determined an lactation days 0, 4, 7, 14, 21 and 28 for all surviving progeny. In addition to the population counts and body weight data collection specified above, each litter of progeny was examined daily for mortality and behavioral anomalies. Each pup was also examined thoroughly tor devologmental anomalies at birth, at each body weight interval, and again at weaning.

Postmortem examinations (parental animals):

The vagina, uterus and ovaries or testes (with epididymides, seminal vesicles and prostate and any masses or gross lesions were retained in individual labeled jars containing 10t buffered formalin. In addition, the eyes were retained from all P1 parental animals.
The liver, kidneys (at least one or one-half of each), brain (at least one fourth) and ovarie(s) (one) or testes (one) were retained from 2 F1 parental generation males and 2 F1 parental generation females from each exposure group.
Additionally, as a result of clinical observations noted for 2 of the 1400 ppm F1 parental generation sibling males, these males along with 2 males chosen randomly from the remaining 1400 ppm males and 4 of the 8 ppm males were anesthetized and perfused in situ. Microscopic examinations were conducted upon the above listed tissues from the sacrificed untreated control and high dose parental animals from both generations.

Postmortem examinations (offspring):

All F1a progeny in excess of those chosen as F1 parental animals, or for neuropathology and all F2a and F2b progeny were sacrificed using CO2 asphyxiation, exsanguinated, and subjected to gross pathologic examination. The necropsy included an examination of the external surface; all orifices; cranial cavity; carcas, external and cut surfaces of the brain and spinal cord; the thoracic abdominal, and pelvic cavities and their viscera; and the cervical tissues and organs. The eyes and any gross lesions were retained in 10% buffered formalin.
Microscopic examinations were conducted upon the eyes of the sacrificed F1a progeny and F1a progeny chosen for neurotoxicologic testing.

Statistics:

Quantitative continuous variables, i.e., body weights, food consumption, were analyzed by Analysis of Variance with significant differences described by that treatment further studied by multiple comparison Progeny body weight data were additionally studied using Analysis of Covariance with the litter size as the covariante and Dunnett's T-test. Reproductive data and neurotoxicologic data were analyzed using Chi-square analysis and Fisher's Exact test.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

During the first generation, no deaths occurred among the treated animals. Two untreated control females died prior to final sacrifice. One dam was sacrificed moribund and one dam was found dead following completion of their respective Fla litters. Body weight data for the-F0 parent animals exposed to cyclohexanone were comparable to the untreated control animals. No noteworthy observations were seen for F0 animals pre-exposure. Clinical reactions, such as lacrimation, ataxia, and irregular breathing, were noted for the 1,000 ppm animals following the first 2 exposures. Starting with the third exposure, these animals appeared to acclimate to the test article and no consistent, recurring observations were noted post-exposure for the 1,000 ppm animals through the remainder of the exposure period. No post-exposure reactions were seen for the 250 or 500 ppm animals. Statistical analyses of the mating indices calculated for the treated animals during the F1a mating trials revealed no significant differences. Progeny data (delivery and population data, survival, and body weight data) obtained during the F1a litter were not considered to be altered by maternal exposure to cyclohexanone.
Evaluation for behavioral/neurotoxicologic development of selected F1 progeny revealed no consistent differences between treated groups and the untreated control group. One of 174 of the 500 ppm progeny and 1/174 of the 1,000 ppm progeny had grossly apparent eye opacities. Ophthalmic examination of all F1a progeny with open eyelids revealed lens opacities for 2/206 of the 250 ppm pups, 2/174 of the 500 pups, and 1/174 of the 1,000 ppm pups. The 250 and 500 ppm progeny were retained untreated and examined approximately 3 months after trhe initial examination. At this time, lens opacities were noted for one of the 250 ppm progeny and one of the 500 ppm progeny; the other 2 animals did not exhibit lens opacities.
Urinalysis determinations of 5 F0 females per treatment group post lactation revealed increased volume from the 1,000 ppm animals; however, no qualitative differences were noted in glucose, pH, protein, ketone, bilirubin, occult blood or urobilinogen. All other urine parameters for treated females were comparable to the untreated control females.
Necropsy examination of sacrificed F0 parental animals and F1a progeny revealed no treatment-related lesions. No microscopic changes were seen in the reproductive organs from the 1,000 ppm animals and the untreated control animals. Examination of the specified areas of the nervous system of the untreated control and 1,000 ppm F1a progeny chosen for neurotoxicologic evaluation did not reveal lesions in any of the tissues. Microscopic examination of the eyes from the F1a progeny revealed lenticular vacuolation (vacuolation of a few outer cortical fibers in the lens) for 2/115 of the 500 ppm progeny and 3/114 of the 1,000 ppm progeny. The examining pathologist concluded that due to the low incidence and minimal nature of these changes, they were not treatment-related.
Based on the in-life data obtained during the F0 generation, which revealed that exposure to 250, 500, or 1,000 ppm cyclohexanone did not result in noteworthy signs of toxicity or effects upon growth and reproductive parameters, and discussions with the Sponsor, the 1,000 ppm level was increased to 1,400 ppm. The F1a progeny selected as potential F1 generation animals began exposure to cyclohexanone at 29 days of age and were exposed to either 0, 250, 500, or 1,000 ppm through completion of the last F1a litter. After all F1a litters were weaned, 30 males and 30 females/treatment group were selected from the group of potential F1 generation animals to continue as F1 generation animals. This selection occurred at the start of week 2 of the F1 generation, and the 1,000 ppm level was increased to 1,400 ppm.

Effect levels (P0)

Dose descriptor:

NOAEC

Effect level:

1 000 ppm

Sex:

male/female

Basis for effect level:

other: overall effects

Results: F1 generation

Details on results (F1)

F1 Generation: Six of the F1 generation animals exposed to 1,400 ppm cyclohexanone died. Two males and 1 female died during the first week of exposure. One male died during the fifteenth week of the pre-mating period, one male was found dead during the F2b mating trials, and one male died following completion of the F2b litters. A 250 ppm male was sacrificed moribund prior to the F2b mating trials; no other deaths occurred among the 250 and 500 ppm animals or the untreated control animals during the second generation.
Starting with the first week of exposure to 1,400 ppm, statistically significant (p<0.01, p<0.05) weight depressions were noted for the 1,400 ppm males when compared to the untreated control males. These depressions were seen at 31 of the 34 weeks of 1,400 ppm exposure . Females from this exposure level weighed less (p<0.05) than the untreated control females during the week of exposure to 1,000 ppm. This weight depression (p<0.05, p<0 .01) continued through the first 3 weeks of 1,400 ppm exposure. Starting with the fourth week of 1,400 ppm exposure through final sacrifice, no significant body weight differences were seen for the 1,400 ppm females when compared to the untreated control females. During the first week of exposure, a significant weight depression (p<0.05) was seen for the 500 ppm males when compared to the untreated control males. All other body weight data obtained for the 250 and 500 ppm animals were similar to the untreated control animals. In addition, body weight data recorded for gestating and lactating dams were similar for the treated groups and the untreated control group during both litters (F2a and F2b).
Exposure of Fl parental animals to 1,000/1,400 ppm resulted in noteworthy pharmacotoxic reactions. The F1a progeny exposed to 1,000 ppm (post-weaning, prior to the selection of the F1 parental animals and subsequent increase to 1,400 ppm) exhibited clinical signs such as ataxia, lacrimation, irregular breathing, and urine soaked fur following treatment. After the increase to 1,400 ppm, and continuing for approximately 3 months, these reactions continued to occur. Starting at week 16 of the F1 generation, the 1,400 ppm animals appeared to adapt to treatment with lethargy being the predominant post-exposure reaction. No observations were noted post-exposure during the final 3 weeks of the F1 generation. Observations recorded prior to exposure revealed 27/60 of the 1,400 ppm animals had yellow/brown stained £ur in comparison to 3/60 of the untreated control group. In addition, starting at week 30 cf the F1 generation and continuing through termination, two sibling 1,400 ppm males exhibited a staggering gait prior to test article exposure.
During the first 3 weeks of exposure, urine soaked fur was noted post-exposure for 3 to 37% of the animals exposed to 588 ppm cyclohexanone. No other noteworthy reactions were seen among the 500 ppm animals. No untoward reactions were seen for the F1 generation animals exposed to 250 ppm cyclohexanone.
Statistical analysis of the F2a and F2b reproductive indices revealed no statistical depressions for the test groups when compared to the untreated control group. However, the 1,400 ppm male fertility indices, calculated using all males paired were 19.8 and 20.8 percent less than the untreated control males during the F2a and F2b litters. Also, male fertility calculated including only males which were paired with fertile females (females that conceived litters) were 24.3 to 28.6 percent less than the untreated control males during the Fla and F2b mating trials. Statistical evaluation of the male fertility data for intergroup differences revealed significant depressions (p<0.05) for the 1,400 ppm males when compared to the 250 ppm males during the F2a and F2b mating trials and the 500 ppm males during the F2b litter. Additionally, mating indices calculated for the 1,400 ppm group were significantly less than the 250 ppm group during both the F2a ( p<0.01) and F2b (p<0.05) mating trials.
Statistical analyses of the progeny population data revealed significant (p<0.01, p<0.05) depressions in the mean numbers of 1,400 ppm viable progeny during the F2a and F2b lactation periods. The mean number of progeny born viable by 1,400 ppm dams was not statistically reduced; however, in comparison to the untreated control dams, the 1,400 ppm dams delivered 23% and 24% fewer viable progeny during the F2a and F2b litters, respectively. Progeny delivery and population data for the 250 and 500 ppm groups during the F2a and F2b litters were similar to the untreated control group.
The percent of 1,400 ppm F2a progeny born viable and surviving to lactation days 1 and 4 were significantly less (p<0.01) than the untreated control group. Survival of the 1,400 ppm F2a progeny at lactation days 14, 21, and 28 was not statistically different than the untreated control group; however, the percent of progeny surviving on these lactation days was 14 to 22% less than that of the untreated control. During the F2b litter, progeny survival was significantly less (p<0.01) than that of the untreated control progeny at lactation days 1 and 4. Survival of 1,400 ppm F2b progeny after lactation day 4 was comparable to that of the untreated control. Survival of the 250 and 500 ppm progeny during the F2a and F2b litters was not altered by maternal exposure to cyclohexanone.
Body weights obtained for the 1,400 ppm F2a and F2b progeny were depressed when compared to the untreated control progeny. No body weight reductions were noted for the 250 and 500 ppm F2a and F2b progeny which were considered to be treatment-related. Examination for progeny external morphologic changes revealed no anomalies which appeared to be a result of maternal cyclohexanone exposure.
Ophthalmologic examination of the weaned F2a progeny revealed lens opacities/cloudiness for 3/151 of the 250 ppm progeny and 1/60 of the 1,400 ppm progeny. In addition, 1/159 of the 500 ppm progeny had a corneal opacity. No ophthalmolgic findings (lens opacities) were seen for the F2b progeny. The ophthalmologist's interpretation of the findings noted in the eyes of all progeny examined (including F1a) was that the test article did not increase the incidence of opacities in the progeny. The lenticular and other ocular abnormalities appeared to be within the range of type and incidence expected in the number of animals examined.
Gross pathologic examinations of all F1 parental animals and F2a and F2b weaned progeny revealed no consistent lesions which were considered to be treatment-related. Microscopic examination of the reproductive organs from the untreated control and 1,400 parent animals revealed no evidence of treatment-related effects. Neuropathologic examination of tissues from the sibling males that were ataxic revealed no morphologic abnormalities.

Effect levels (F1)

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

500 ppm

Sex:

male/female

Basis for effect level:

other: overall effects

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEC

Generation:

F2

Effect level:

500 ppm

Sex:

male/female

Basis for effect level:

other: overall effects

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, inhalation exposure to 1,000 ppm cyclohexanone through one generation and exposure to 250 or 500 ppm cyclohexanone through two consecutive generations did not adversely affect the growth, development, and reproductive performance of rats.
Inhalation exposure of the CD rat (progeny from parental animals exposed to 1,000 ppm) to 1,400 cyclohexanone through one generation resulted in exposure-related pharmacotoxic reactions, male body weight depressions, reduced male fertility, reduced progeny survival, and progeny body weight depressions.