Cyclohexanone

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a two-generation study performed by the American Biogenics Corp. (1986), groups of 30 male and female SD rats per concentration were exposed by inhalation to vapor concentrations of 0, 250, 500, or 1000 (F0)/1400 (F1) ppm (corresponding to ca. 0, 1020, 2040, 4080 and 5712 mg/m³) cyclohexanone for 6 hours a day and 5 (males) and 7 (females) days a week. All animals were exposed for 2,5 months prior to mating. The total duration of exposure of the F0 generation was about 4 months. Groups of 30 animals per sex and dose group from the resulting litters of the F0 generation (= F1 animals) were further exposed from the 29th day of life to concentrations of 0, 250, 500 and 1400 ppm (1020, 2040 and 5712 mg/m³) cyclohexanone. The 15‐day mating period was preceded by exposure for 4 months. The total duration of exposure of the F1 generation was about 8 months.

In the F0 generation there were slight clinical symptoms such as lacrimation, irregular breathing and ataxia only at the beginning in the high concentration group. The offspring were not different from the controls in any of the exposure groups. In a behavioural study no effects of the exposure on the offspring were detected either before or after weaning. Pathological/anatomical and histological investigation of the F0 generation after the end of the experiment revealed no changes relative to the control animals in the sexual organs or the nervous system.

For the F1 generation the high cyclohexanone concentration was increased as no effects had been observed among the parental animals. Six animals died as a result of exposure to this increased concentration. Delays in body weight gain were observed mainly in the males. Clinical symptoms seen during the whole experimental period were lacrimation, irregular breathing, ataxia and fur contaminated with urine. Although in the low and middle concentration groups neither clinical symptoms nor effects on fertility were observed, in the high concentration group there was a reduction in the number of offspring, which was interpreted by the authors as a decrease in male fertility, and reduced survival of the offspring together with retardation in body weight gain. Gross pathological investigation of the F1 and F2 generations did not yield any conspicuous findings. There were also no histopathological changes in the sexual organs or nervous system of the high dose group animals relative to the control group (American Biogenics Corp. 1986). To evaluate the potential reversibility of the treatment-related depression in the fertility of male mice exposed to 1400 ppm cyclohexanone, a study was conducted during a post-exposure recovery period (American Biogenics Corp. 1986). Ten males were selected from each treatment group of F1 generation males based on known reproductive performance during the F2b litter mating trials. As a positive control, fertile males of the same strain and of a similar age were obtained from the same source as the parents of the control and test group males and injected, intraperitoneally, with 0.5 mg/kg triethylenemelamine. It could be shown that the effects on male fertility seen in the high-dose group were reversible. In summary, the cyclohexanone concentration without effects on reproductive toxicological parameters in the F0 generation was about 1000 ppm (4080 mg/m3), for continuous exposure of two generations (about 12 months) 500 ppm (2040 mg/m3).

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep 1984 - Nov 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

other: Add-on study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Sllied Fiber and Plastics Company, Hopewell, VA 23860
- Lot No.of test material: 403102, 506241
- Appearance: Clear, colorless to pale yellow
- Odor: Peppermint-like
- Purity: 99.9% (0.01% water, 0.02% cyclohexanol, 0.058% esters (as cyclohexylformate), and 0.003% acidity (as formic acid)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc
- Age at study initiation: 21 days (F0 generation)
- Housing: group
- Diet: withheld during exposure
- Water: ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-78
- Humidity (%): 30-70
- Air changes (per hr): nd
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Details on exposure:

The cyclohexanone inhalation exposures were conducted in 8m³ stainless steel and glass chambers. Each chamber was supplied
with conditioned air (HEPA and charcoal filtered, 67-77°F, humidity 30-70%), operated dynamically with airflow rates sufficient for at least 12 air changes per hour, and operated under a slight (0.3 in H2O) negative pressure to prevent contamination of the surrounding area. Three concentrations of cyclohexanone, 250, 500, and 1,000 ppm plus conditioned air only control were used . On February 11, 1985 (end of F1 lactation period and day of selection of F1 parental animals), the 1,000 ppm exposure was increased to 1,400 ppm. The exposures were for 6 hours per day on each exposure day. Parental males were exposed 5 days per week . The parental females were exposed 5 days per week pre-mating and 7 days per week for 3 weeks prior to the mating trials. Females continued to be exposed 7 days per week during the mating trials, on gestation days 0 through 20, and on lactation days 5 through 28. Starting on gestation day .21 through lactation day 4, dams remained in the nesting cages unexposed . Females that did not conceive litters or females that did not have viable progeny were exposed 5 days a week .

Airflow rates through the flasks: 80-100 liters/min

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 repeated 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 obtain F2b litters. The mating trial for 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 (F2a and F2b litters).
Each female was examined daily during the period of cohabitation to detect evidence or breeding. The observation of a copulatory plug in the vagina or sperm-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

Details on analytical verification of doses or concentrations:

The concentration of cyclohexanone in the breathing zone within each chamber was analyzed hourly using 'scrub samples' with the trapping liquid being 20 ml of denatured ethyl alcohol. The chamber atmosphere was pulled through the scrubber with a vacuum pump at a rate of 1 to 2 liters per minute for 5 minutes. All exposure levels were thoroughly checked for scrubber 'break through' prior to initiation of the study and it was concluded a single scrub sample was adequate. The scrub samples were quantitatively transferred into volumetric flasks and the appropriate dilutions made with denatured ethyl alcohol.
Samples of chamber atmospheres for analytical determinations were obtained at least hourly during each exposure. The nominal concentration for each level was determined daily by measuring the weight change of each level's reservoir and the corresponding total airflow. Nominal to analytical concentration ratios were determined daily. Once each month during the study, cyclohexanone distribution within each chamber was determined by measuring concentrations at six points in each chamber at the breathing level of the animals. From the start of the study, September 24, 1984, until April 12, 1985 (during the pre-mating period of F1 animals), the presence or absence of aerosol in the exposure chambers was determined by visual inspection of a light beam directed across the chamber interior. This was done once per week during this time.
Beginning on may 7, 1985, a Cascade Impactor was used to monitor aerosol in the exposure chambers. Measurements with this instrument were made twice a week. Chamber supply air temperature and humidity were determined hourly in the untreated control chamber with a Taylor hygrometer.

Duration of treatment / exposure:

2 generations

Frequency of treatment:

The exposures were for 6 hours per day on each exposure day. Parental males were exposed 5 days per week . The parental females were exposed 5 days per week pre-mating and 7 days per week for 3 weeks prior to the mating trials. Females continued to be exposed 7 days per week during the mating trials, on gestation days 0 through 20, and on lactation days 5 through 28. Starting on gestation day 21 through lactation day 4, dams remained in the nesting cages unexposed. Females that did not conceive litters or females that did not have viable progeny were exposed 5 days a week .

Dose / conc.:

250 ppm (nominal)

Remarks:

253.2 ppm/249.8 ppm (actual); approx. 1.02 mg/L

Dose / conc.:

500 ppm (nominal)

Remarks:

499.2 ppm/469.9 ppm (actual); approx. 2.04 mg/L

Dose / conc.:

1 000 ppm (nominal)

Remarks:

1008.9 ppm (actual); approx. 4.1 mg/L

Dose / conc.:

1 400 ppm (nominal)

Remarks:

1387.2 ppm (actual); approx. 5.71 mg/L; F1 generation only

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.

Parental animals: Observations and examinations:

All parental animals were weighed weekly during the premating period. Ten weekly weights for the F0 generation and at least 15 weekly weights for the F1 generation. 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, 4, 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.
Each female was observed daily through gestation 18. Starting on gestation day 19, pregnant females were examined twice daily for signs of parturition. Conception was confirmed by the observation of a vascular membrane and/or the detection of progeny by palpation. The females were allowed to deliver their litters, and daily
observations of the females and young were conducted throughout lactation. Litters were weaned at 28 days of age. Any unusual observations during the periods of
gestation and lactation were recorded.

- Urinalysis from 5 lactating F0 females per group (total of 20)

F1 GENERATION
- 40 F1 parental males (10/test group) were selected for fertility assessment (non-exposure recovery period)

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.

PARAMETERS EXAMINED
The following parameters were examined in [F1, F2] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, body weights, physical or behavioural abnormalities, ophthalmologic examination (F1a, F2a and F2b weanings)

GROSS EXAMINATION OF DEAD PUPS:
- yes, for F1a, F2a and F2b weanlings. The necropsy included an examination of the external surface; all orifices; cranial cavity; carcass ; 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.

HISTOPATHOLOGY:
Microscopic examinations were conducted upon the eyes of the sacrificed F1a progeny and F1a progeny chosen for neurotoxicologic testing. Tissues retained from the control and high-dose group animals were subjected to microscopic evaluation;
Brain: forebrain, center of cerebrum, midbrain, cerebellum and pons, medulla oblongata, and Gasserias ganglia.
Spinal Cord : At cervical (C3-C6), lumbar (L1-L4) swellings, dorsal root ganglia ( C3-C6, L1-L4), and dorsal and ventral root fibers ( C3-C6, L1-L4) .
Sciatic Nerve : Midthigh and sciatic notch, aural nerve (at knee), and tibial nerve ( at knee).


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Conducted on 1 pup from each F1a litter. One male or female were randomly selected from alternating litters for these procedures. This selection occurred on lactation day 4 following appropriate litter reductions. Testing began at 4 days of age and continued until the physical change tested for was present. The tests included in the suckling young evaluations were incisor eruption, eye opening, pinna detachment, surface righting, air righting, and auditory startle. The F1a progeny chosen for neurotoxicological assessment were sacrificed at days 49 of age. The cranium and vertebral column were removed without damage to brain and cord, the brain was then measured (length and width), its weight recorded, and any abnormal coloration or discoloration noted and recorded. The proximal sciatic nerve, sural nerve, tibial nerve and gastrocnemius muscle were taken.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: F0 males were sacrificed at the completion of the F1a litter weanings.
- Maternal animals: F0 females/F1 females that failed to breed were sacrificed 20 days following completion of the F1a/F2b mating trials. F0/F1 females that bred but failed to deliver viable progeny were sacrificed 26 days post copulation. Females that conceived and delivered progeny were sacrificed after completion of the F1a/F2b lactation period.

GROSS NECROPSY
- Gross necropsy consisted of all F0 and F1 parental animals of the external body surface and all orifices; cranial cavity; external and cut surfaces of the brain and spinal cord; thoracic, abdominal and pelvic cavities and their viscera; cervical tissues and organsi and the carcass. Additionally the number of uterine implantation scars
was noted and recorded for all dams. 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 F1 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 0 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.


HISTOPATHOLOGY / ORGAN WEIGHTS
- Yes, 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 (Tukey's of Scheffe's, dependent upon 'N' values). 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.
Untreated control group was used for comparison. Differences were considered significant at p < 0.05 and p < 0.01 confidence levels.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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 F1a litters.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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.
Maternal body weight data were comparable for treated dams and untreated control dams during both generations

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:

effects observed, non-treatment-related

Description (incidence and severity):

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 that correspond to this increase. All other urine parameters obtained for treated females were comparable to the untreated control females.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No microscopic changes were seen in the reproductive organs from the 1,000 ppm animals and the untreated control animals.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Statistical analyses of the mating indices calculated for the treated animals during the F1a mating trials revealed no significant differences. Mating indices and male fertility indices calculated for the 500 and 1000 ppm groups ranged from 13 to 20% less than the untreated control group, however, no statistical significance was reached. 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.

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.

Dose descriptor:

NOAEC

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Remarks on result:

other: corresponding to approx. 4.1 mg/L

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1400 ppm: ataxia, lacrimation, irregular breathing, and urine soaked fur following treatment (continuing for approx. 3 months). During week 16, lethargy was the predominant post-exposure observation. The pre-exposure examinations revealed 27/60 of the 1400 ppm animals with yellow/brown stained fur, as compared to 3/60 of the untreated control. In addition, starting at week 30 of the F1 generation and continuing through termination, two sibling 1,400 ppm males exhibited a staggering gait prior to test article exposure.
1000 ppm: Ataxia, urine-soaked fur, irregular breathing, and lacrimation were the predominant observations noted post-exposure (prior to initiation of the F1 generation and the subsequent concentration increase).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

1400 ppm: 6 animals died (2 males and 1 female during the first week of exposre, 1 male during 15th week of pre-mating period, 1 male found dead during F2b mating trials, 1 male died following completion of F2b litters)
500 ppm: During the first week of exposure, a significant weight reduction was noted for males
250 ppm: One male was sacrificed moribund prior to F2b mating trials

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1400 ppm: Reduced body weight for males
1000 ppm: significant weight reduction for females during the week of 1000 ppm exposure. Body weights for these females were reduced at the first week of 1400 ppm exposure and these depressions continued during weeks 3 and 4. Week 5, no significant differences were noted.
500 ppm: During the first week, significant weight reduction noted for males
Maternal body weight data were comparable for treated dams and untreated control dams during both generations

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy examinations of parental animals revealed no lesions which appear to be related to the test substance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Statistical analyses 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 to 20.8 percent less than the untreated control males during the F2a and F2b litters, respectively . Male fertility indices calculated using only those males that were paired with females that conceived litters revealed a 24.3 to 28.6 percent reduction for the 1,400 ppm males compared to the untreated control males during the F2a and F2b mating trials, respectively.
Additionally, evaluation of these data for intergroup differences revealed significant depressions ( p<0 .05) for the 1,400 ppm male
fertility index when compared to the 250 ppm males during the F2a and F2b mating trials and compared to the 500 ppm males during
the F2b mating trials. 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.

Dose descriptor:

NOAEC

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

reproductive performance

Remarks on result:

other: reversible effect on fertility

Remarks:

obtained from add-on study (450-2326)

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Progeny delivery and population data were similar for treated groups and the control

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Progeny survival was not altered by maternal exposure.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Statistical reductions were noted for the 250 ppm, 500 pmm and 1000 ppm on lactation days 14, 21, 28; 7, 14, 21, 28; and 4, 14, 21, respectively. However, analysis of the mean litter weights did not reveal consistent results with individual weights, thus, reductions appear to be a result of the litter size. In addition, no dose-response relationship was seen. Therefore, body weight reductions were not considered to be affected by maternal exposure.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Findings were within the range of type and incidence expected in the number of animals examined.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy examination of sacrificed F1a progeny revealed no treatment-related lesions.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Evaluation for behavioral/neurotoxicologic development of selected F1 progeny revealed no consistent differences between treated groups and the untreated control group.

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.

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F2a and F2b:
1400 ppm: Significant reduction in the mean number of progeny viable during the lactation periods. Dams delivered 23 to 24% fewer viable progeny than the control dams; however, no statistical significance was noted.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

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, 1400 ppm progeny survival to lactation days 1 and 4 was significantly less (p<0.01) than that of the untreated control progeny. 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 weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights obtained for the 1,400 ppm F2a and F2b progeny were depressed when compared to the untreated control progeny. Analyses of the 250 and 500 ppm F2a progeny body weights resulted in statistical reductions. However, because the statistical weight differences noted for the 250 and 500 ppm F2a progeny were minimal (5-17% less than control) and were not seen for the F2b progeny, maternal exposure was not considered to adversely affect pup body weight at 250 and 500 ppm.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Findings were within the range of type and incidence expected in the number of animals examined.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Histopathological findings:

no effects observed

Description (incidence and severity):

Microscopic examination of the reproductive organs from the untreated control and 1,400 ppm parental animals revealed no evidence of treatment-related effects.
Neuropathologic examination of tissues from the sibling males that were ataxic revealed no morphologic abnormalities.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEC

Generation:

F2

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 400 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

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 effects such as male body weight depressions, reduced male fertility, reduced progeny survival, and progeny body weight depressions.
In a follow-up study, the effects on the male fertility were investigated in a reproductive recovery study and reversibility could be demonstrated.

Executive summary:

A reproduction study was conducted to ascertain the potential effects of inhalation exposure to cyclohexanone vapor upon reproductive performance, growth, and development of 2 consecutive generations of CD Sprague-Dawley albino rats. Groups of 30 males/30 females were exposed to either 0, 250, 500, or 1,000 ppm during the first (FO) generation. Thirty males/30 females were selected from the Fla litters of each group to continue on 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 Fla-litter.

 

The daily time-weighted average concentrations during the FO generation were 0, 253.2, 499.2 and 1,008.9 ppm and during the F1 generation the daily time-weighted average concentrations were 0, 249.8, 496.9, and 1,387.2 ppm of cyclohexanone. Inhalation exposure to 1,0000 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 the CD Sprague-Dawley derived albino rats. Evaluation for behavioral/neurotoxicologic development of selected Fla progeny revealed no consistent differences between treated groups and the control group. 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.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

4 080 mg/m³

Study duration:

subchronic

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

An OECD guideline study (No. 414) carried out at BASF (1994) with gavage administration to rabbits in doses of 50 - 500 mg/kg showed a NOAEL of 250 mg/kg for maternal toxicity and a NOAEL of 500 mg/kg for teratogenicity. At all dose levels no indications for substance-induced teratogenic effects were observed.

Biodynamics Inc. reported 1984 that cyclohexanone administered to pregnant rats by the inhalation route at exposure levels of 300 and 650 ppm was not considered maternally toxic, embryotoxic or teratogenic. At the highest dose level (1400 ppm), maternal toxic effects were evident and embryotoxicity; however, no teratogenicity was evident at this same dose level.

Biodynamic Inc. (1984) reported that cyclohexanone administered to pregnant mice by the inhalation route at an exposure level of 1400 ppm (= about 5.6 mg/l) was maternally toxic, embryotoxic and fetotoxic, but was not considered teratogenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Limit test:

no

Specific details on test material used for the study:

purity: > 99 .9 %

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

Sexually mature, virgin Himalayan rabbits, which were free from clinical signs of disease, were used for the investigations. This strain was selected since extensive experience is available on Himalayan rabbits and this strain has been proved to be sensitive to substances with a teratogenic potential. Unique identification of the rabbits by ear tattoo had already been carried out by the breeder.
During the acclimatization and the study periods, the rabbits were housed singly. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
The animals were accommodated in fully airconditioned rooms in which central air conditioning guaranteed a range of temperature of 20 - 24°C and a range of relative humidity of 30 - 70%. The day/night rhythm was 12 hours (12 hours light from 6.00 to 18.00 hours and 12 hours darkness from 18.00 to 6 .00 hours). The food used was pelleted Kliba maintenance diet, which was available to the animals ad libitum throughout the study (from the day of supply to the day of necropsy), as was drinking water of tap water quality from water bottles.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance was administered to the animals orally (by gavage) once a day during the period of major organogenesis (day 7 to day 19 p.i.) always at approx. the same time of day (in the morning). The volume administered each day was 10 ml/kg body weight. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 7 p.i.). The control group was dosed with the vehicle only (doubly distilled water). Each day the test substance solutions were freshly prepared shortly before the test substance was administered. For the preparation of the solutions, an appropriate amount of test substance was weighed in a volumetric flask, subsequently topped up with doubly distilled water and intensively shaken.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in doubly distilled water for a period of 4 hours at room temperature were carried out in a range-finding maternal toxicity study in Himalayan rabbits.

Details on mating procedure:

After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination. This implied that 0.2 ml of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe were injected intramuscularly to the female rabbits about 1 hour before insemination. The pooled ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.
During the acclimatization period the animals were assigned to the different test groups according to a randomization plan and on the basis of their body weights. The day of insemination was designated as day 0 (beginning of the study) and the following day as day 1 post insemination (p.i.). At the beginning of the study (day 0) the does were between 22 and 37 weeks old. Their mean body weight was approx. 2,641 g.
On day 29 p.i., all surviving females were sacrificed in randomized order and examined macroscopically. The fetuses were removed from the uterus and further investigated with different methods.

Duration of treatment / exposure:

day 7 through day 19 post insemination (p.i.)

Frequency of treatment:

daily

Duration of test:

until day 29 p.i.

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

The selection of doses for the present examination was based on the results of a preceding range-finding study in Himalayan rabbits. From the results of this preliminary study it could be seen, that cyclohexanone caused slight signs of maternal toxicity at 450 mg/kg body weight, but induced no substance-related effects on the does of the low and the intermediate dose groups (50 and 150 mg/kg body weight/day). There were no substance-related effects on the gestational parameters in any of the groups and the fetuses showed no malformations.

Maternal examinations:

Food consumption, body weight data, corrected body weight gain (net maternal body weight change), clinical symptoms, mortality

Ovaries and uterine content:

Weight of uterus before it was opened, number of corpora lutea, number and distribution of implantation sites classified as live fetuses and dead implantations, conception rate, preimplantation loss, postimplantation loss.

Fetal examinations:

Examination of the fetuses after dissection from the uterus, soft tissue examination of the fetuses, skeletal examination of the fetuses, malformations, variations, retardations, unclassified observations.

Statistics:

The DUNNETT-Test was used for a simultaneous comparison of several dose groups with the control. The hypothesis of equal means was tested. This test was performed two-sided and was used for the statistical evaluation of the following parameters: Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses; proportion of preimplantation loss, postimplantation loss, resorptions and live fetuses in each litter; litter mean fetal body weight and litter mean placental weight.
FISHER's Exact Test was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions. This test was performed one-sided and was used for female mortality, females pregnant at terminal sacrifice and the number of litters with fetal findings.
The WILCOXON Test was used for a comparison of each dose group with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardations and/or unclassified observations in each litter.
If the results of these tests were significant, labels (* for p < 0.05, ** for p < 0.01) were printed in summary tables.

Indices:

-

Historical control data:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg body weight/day: unsteady gait in all animals about 2 - 4 hours after the daily gavaging during the first treatment days

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control animal died due to gavage error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg - body weight/day: body weight loss at the beginning of the treatment period; weight gain during the treatment period was only 25% of the control animals. This corresponds to the reduced food intake of the high dose dams.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg - body weight/day: statistically significantly reduced food consumption (about 15% less than the controls) during the treatment period

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Basis for effect level:

body weight and weight gain

clinical signs

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One control and one intermediate dose fetus each showed external malformations. For the control fetus cleft palate and kinky tail were observed, while the
intermediate dose fetus showed cheiloschisis. Only one type of external variation (pseudoankylosis) was found and it was seen in 4 out of 75 control fetuses (in 2 out of 13 litters), in one out of 105 fetuses from the 50 mg/kg group (in one out of 15 litters) and in one out of 81 fetuses of the 250 mg/kg group (in one out of 15 litters).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations of the fetal skeletons were noted for 2 out of 75 control fetuses (= 2 .7%) (in 2 out of 13 litters (= 15%)) and in 2 out of 81 intermediate dose fetuses (= 2 .5%) (in 2 out of 15 litters (= 13%)). These malformations were related to the vertebral column (cervical/lumbar vertebrae fused and/or of irregular shape), the scapula(e) (deformed), the sternum (sternebrae severely fused (bony plate)) and the hindlimbs (bent with involvement of the bones). None of the fetuses of the low and the high dose group showed skeletal malformations.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of the organs of the fetuses revealed several types of soft tissue malformations in fetuses of the control and the intermediate dose group . In the control group one out of 75 examined fetuses 1.3%) from one out of 13 litters (= 7 .7%) showed hydrocephaly . In the 250 mg/kg group in total 3 out
of 81 examined fetuses (= 3 .7%) from 3 out of 15 litters (= 20%) were malformed . All of them had a septal defect ; one of these fetuses (No . 7 of doe No .
38) had in addition a hypoplastic thymus and another one (No . 3 of doe No . 43) showed additionally malformations of the great vessels, agenesia of gallbladder and hypoplastic kidneys (associated with other kidney findings) (see also Tab . B 034 , Volume II). No soft tissue malformations occurred at 50 or 500 mg/kg body weight/day.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

No substance-induced signs of embryo-/fetotoxicity, especially no teratogenic effects were observed in the present full-scale prenatal toxicity study up to
and including 500 mg/kg body weight/day.

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Basis for effect level:

other: no teratogenic effects up to and including the high dose

Developmental effects observed:

no

Conclusions:

Under the conditions of this study, the test substance caused some overt signs of maternal toxicity at 500 mg/kg body weight/day (reduced food consumption, impaired body weight gain and unsteady gait), but was not toxic to the does at 50 and 250 mg/kg body weight/day.
There occurred no substance-related adverse effects on the gestational parameters and on the fetuses up to and including the highest dose level (500 mg/kg body weight/day); at all dose levels no indications for substance-induced teratogenic effects were observed.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Guideline:

other: in accordance with the U.S. EPA "Health Effects Test Guidelines" (Teratogenicity Study, August 1982)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Limit test:

no

Specific details on test material used for the study:

concentration: 99.9% active ingredient

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Age at receipt: 69 days old
Animals were acclimated for 14 days prior to mating. Animals were examined for suitability to initiate mating by the staff veterinarian. These examinations were performed at completion of the acclimation period.
Mated female rat was identified with a metal ear tag bearing its animal number. This individual animal number plus Bio/dynamics' project number comprised a unique identification.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

clean air

Details on exposure:

Glass and stainless steel chambers in which the animals were exposed had a total volume of 1 cubic meter. The chambers for Groups I through IV were operated dynamically at airflow rates of 237, 236, 230 and 224 liters per minute. These flow rates provided one complete air change every 4.2, 4.2, 4.3 and 4.5 minutes and 99% equilibrium times of 19.4, 19.5, 20.0 and 20.6 minutes, respectively.
Group I: Room air was supplied to a 1m3 glass and stainless steel exposure chamber which housed the control animals. Groups II, III and IV: A modified Laskin nebulizer (one, two and three barrel for Groups II, III and IV, respectively) was placed in the center of a 1000 ml three-necked flask. For Groups I, II and IV, the nebulizer was connected to a FMI lab pump which was equipped with a 1/8" piston and metered cyclohexanone from a 1000 ml graduated cylinder reservoir. The setting of 80-100% was used on the metering pump. The nebulizer was pressurized with houseline air at approximately 9 psi back back pressure for Group II; 8 psi for Group III ; and 19 psi for Group IV using a Matheson pressure gauge.
In addition, a flow meter was used to assure the nebulizer was not clogged. The resultant aerosol was directed from the flask via a glass "T" connecting tube to a point where room air was added. The diluted stream of test material, which after dilution appeared to be only a vapor, was drawn into the top inlet port of the 1m3 inhalation chamber. Chamber air flow was monitored using a magnehelic gauge (1.0 cm of water equalled 236 lpm for Group II and 230 lpm for Group III; 0.8 cm of water equalled 224 lpm for Group IV) which was attached to an in-line calibrated orifice. At the end of the exposure
the residual cyclohexanone in the flask was removed via a FMI pump to determine the nominal concentration. Animals remained in the chamber approximately 1/2 hour following exposure to allow chamber clearing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of chamber atmospheres were obtained at least four times per exposure at approximate hourly intervals during the exposure. The nominal concentration was determined by measuring the volume of test material before and after each exposure for all exposure groups.
The difference in volumes represented the total amount of the test material delivered into the chamber. This value was corrected for the density and was divided by the total volume of air, which yielded the nominal exposure concentration.
In addition, concentration distribution was monitored at four locations in all test chambers once per week. The presence of particulate was assessed once daily throughout the exposure using a Portable Particle Monitor. An Airguide humidity indicator was used to monitor relative humidity and a Weston thermometer was used to monitor air temperature in the chamber once daily throughout the treatment period.

Analysis of chamber distribution samples in the exposure chambers showed a uniform distribution of the test material throughout the chambers including the locus of the normal sampling ports. Chamber temperature ranged from 61 to 70°F and relative humidity ranged from 31 to 60% during the series of exposures. Temperatures in all exposure chamber were approximately the same. No particulate was found to be present in any test chamber.

Details on mating procedure:

Females selected for mating were placed with male rats nightly in a 2:1 reatio (females:males). Vaginal smears were taken early in the morning following intervals of cohabitation and females were considered to have mated if sperm was observed at microsopic evaluation of the vaginal smear. Day on which evidence of mating was observed was defined as Day 0 of gestation.
Females which mated were assigned to groups daily in such a way as to most nearly equalize the Day 0 mean group body weights. A manual sorting procedure was used.
Each mated female rat was identified with a metal ear tag bearing its animal number. This individual animal number plus Bio/dynamics' project number comprised a unique identification.

Duration of treatment / exposure:

g.d. 6 - 19

Frequency of treatment:

6 hours/day

Duration of test:

until day 20 of gestation

Dose / conc.:

300 ppm (nominal)

Remarks:

approximately 1.2 mg/L

Dose / conc.:

650 ppm (nominal)

Remarks:

approximately 2.6 mg/L

Dose / conc.:

1 400 ppm (nominal)

Remarks:

approximately 5.6 mg/L

No. of animals per sex per dose:

26 females/group

Control animals:

yes, concurrent vehicle

Details on study design:

The inhalation route is one of the expected routes of human exposure for this test substance.

Maternal examinations:

Mortality, pregnancy rates, body weight data - gestation period, observation of animals in-chamber, physical in-life evaluation data, corpora lutea and uterine implantation data, gross postmortem observation data

Ovaries and uterine content:

The ovaries and uterine content was examined after termination.

Fetal examinations:

Fetal weight data, fetal sex distribution data, fetal external examination data, fetal visceral evaluation data, fetal skeletal evaluation data, malformation data, ossification variation data

Statistics:

Statistical evaluation of equality of means was made by the appropriate one way analysis of variance technique, followed by a multiple comparison procedure if needed. First, Bartlett's test was performed to determine if groups had equal variance. If the variances were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric procedures were the standard one way ANOVA using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett's test was used to determine which means were significantly different from the control. If a nonparametric procedure for testing equality of means was needed, the Kruskal-Wallis test was used, and if differences were indicated a summed rank test (Dunn) was used to determine which treatments differed from control. A statistical test for trend in the dose levels was also performed. In the parametric case (i .e ., equal variance) standard regression techniques with a test for trend and lack of fit were used. In the nonparametric case Jonckheere's test for monotonic trend was used. The test for equal variance (Bartlett's) was conducted at the 1%, two-sided risk level. All other statistical tests were conducted at the 5% and 1%, two-sided risk level.
Statistical analysis of incidence data was performed using contingency tables. First, a standard chi-square analysis was performed to determine if the proportion of incidences differed between the groups tested . Next, each treatment group was compared to the control group using a 2x2 Fisher Exact test ; the significance level was corrected via the Bonferroni inequality to assure an overall test of the stated significance level. Thirdly, Armitage's test for linear trend in the dosage groups was performed. All tests were reported at the 5 % and 1 % level of significance.

Historical control data:

The laboratory has historical control data for the reproductive performance of this strain of rat and data that this strain is responsive to a known animal teratogen (acetylsalicylic acid).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No adverse effect of treatment at the low- or mid-dose exposure levels was evident in physical in-life evaluation data. At the high-dose level, all of the females were noted with lacrimation following exposure on Day 6; however, this observation was noted with considerably less frequency for the remaining evaluation intervals. Other observations noted for the high-dose animals following the exposure intervals were: lethargy; nasal discharge and observation of a red/brown vaginal discharge noted for several females on Day 15 of gestation.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the control or treated groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No adverse effect of treatment was evident in body weight data during gestation in the low-dose group. In the mid-dose group, mean weight data during gestation were comparable to control; however, mean weight gain during the Day 6-20 gestation interval utilizing corrected Day 20 weights (actual Day 20 weight corrected by subtracting the weight of the intact gravid uterus) was slightly lower than control (this latter difference from control data was not statistically significant). All other weight change data during gestation for the mid-dose group were comparable to control data. In the high-dose group, mean body weights at Day 15 and 20 of gestation were significantly lower than control and mean weight gain during the Day 6-20 gestation interval, using both actual and corrected Day 20 weights, was significantly lower than control.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Description (incidence and severity):

Animals were also evaluated as a group in the chambers during the exposure intervals. In the low-dose group, animals were noted as responding to the startle stimulus during the exposures and only in a few instances were some animals noted to be sluggish to this response. In the mid-dose group, animals still were responding to stimulus but a greater number were noted as lethargic or sluggish in their response and this was noted as early as one hour into exposures. In the high-dose group, animals were sluggish in their response after an hour of exposure and were not responding at later intervals (at three and six hours of exposure).

Immunological findings:

not examined

Description (incidence and severity):

No adverse effect of treatment was evident in uterine implantation data (e.g., mean numbers of implantations, viable fetuses and resorption; mean percentage of resorptions to implants). The incidence of females with at least one resorption site was increased in the mid- and high-dose group; however, this difference was slight and not statistically significant.

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Pregnancy rates for the control, low-, mid- and high-dose groups were 92.3%, 88.5 %, 92.3% and 88.5%, respectively.

Dose descriptor:

NOAEC

Effect level:

650 ppm

Basis for effect level:

other: maternal toxicity

Description (incidence and severity):

Mean fetal weight data, distinguished by sex, were comparable between the control, low- and mid-dose groups and significantly lower than control at the high-dose level.

Description (incidence and severity):

No adverse effect of treatment was evident in fetal sex distribution data.

Description (incidence and severity):

No increase in type or incidence of malformation was evident in fetuses recovered from treated females during either the external, visceral or skeletal evaluations.

Description (incidence and severity):

No increase in type or incidence of malformation was evident in fetuses recovered from treated females during either the external, visceral or skeletal evaluations. In the high-dose group, the incidence of fetuses with at least one ossification variation was increased and fetuses recovered from high-dose females were noted with an increased incidence of incompletely ossified cranial bones (nasals, parietals, frontals, interparietals), unossified or incompletely ossified sternebrae and unossified metatarsals and phalanges (fore-limbs).

Description (incidence and severity):

No increase in type or incidence of malformation was evident in fetuses recovered from treated females during either the external, visceral or skeletal evaluations.

Dose descriptor:

NOAEC

Effect level:

1 400 ppm

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Cyclohexanone administered to rats by the inhalation route at exposure levels of 300 and 650 ppm during the Day 6-19 gestation interval (6 hours/day) was not considered maternally toxic, embryotoxic or teratogenic. At the highest exposure level (1400 ppm), maternal toxic effects were evident (reduced body weight data, and at physical in-life evaluation an increased incidence of lacrimation, lethargy and nasal discharge) and embryotoxicity (reduced fetal weight data; increase in ossification variation data); however, no teratogenicity was evident at this same dose level.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Guideline:

other: in accordance with the U.S. EPA "Health Effects Test Guidelines" (Teratogenicity Study, August 1982), however, only one dose level was evaluated.

Deviations:

yes

Remarks:

only one dose level evaluated

Principles of method if other than guideline:

The test substance was administered via inhalation during the Day 6-17 gestation interval at a dose level of 1400 ppm. Included in this study was a sham-air control group. Females were sacrificed on Day 18 of gestation and corpora lutea/uterine implantation data were recorded. Fetuses recovered at this time were weighed and evaluated for external malformations ; approximately one-half of the fetuses in each litter were further evaluated for skeletal malformations, and the remaining fetuses in each litter were processed for soft tissue examination.

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Specific details on test material used for the study:

Concentration: 99.9% active ingredient

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

Age at receipt: 29 days old - females; 36 days old - males
Animals were acclimated for 15 days prior to mating. Animals were examined for suitability to initiate mating by the staff veterinarian. These examinations were performed at completion of the acclimation period. Each mated female mouse was identified with a metal ear tag bearing its animal number. This individual animal number plus Bio/dynamics' project number comprised a unique identification.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Details on exposure:

Glass and stainless steel chambers in which the animals were exposed had a total volume of 1 cubic meter (1m3). The chambers for Groups I and II were operated dynamically at airflow rates of 237 and 224 liters per minute (lpm), respectively. These flow rates provided one complete air change every 4.2 and 4.5 minutes and 99% equilibrium times of 19.4 and 20.6 minutes, respectively.
Group I: Room air was supplied to a 1m3 glass and stainless steel exposure chamber which housed the control animals. Group II: A modified three barrel Laskin nebulizer was placed in the center of a 1000 ml glass three-necked flask. The nebul i zer was connected to a FMI lab pump which was equipped with a G-20 1/8" piston and metered cyclohexanone from a 1000 ml graduated cylinder reservoir. The setting of 100% was used on the metering pump . The nebulizer was pressurized with house-line air at a range of 19-30 psi back pressure.
In addition, a Dwyer flow meter was used to assure the nebulizer was not clogged. The resultant aerosol was directed from the flask via a glass "T" connecting tube to a point where room air was added. The diluted stream of test material, which after dilution appeared to be only a vapor, was drawn into the top inlet port of the 1m3 inhalation chamber. Chamber air flow was monitored using a magnehelic gauge (0.8 cm of water equalled 224 lpm) which was attached to an in-line calibrated orifice. At the end of the exposure the residual cyclohexanone in the flask was removed via a FMI pump to determine the nominal concentration.
Animals remained in the chamber approximately 1/2 hour following exposure to allow chamber clearing.

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

Females selected for mating were placed with male mice nightly in a 1:1 ratio (females:male). In the morning following intervals of cohabitation, females were evaluated for the presence of a vaginal plug. Day on which evidence of mating was observed was defined as Day 0 of gestation.
Females which mated were assigned to groups daily in such a way as to most nearly equalize the Day 0 mean group body weights. A manual sorting procedure was used. Each mated female mouse was identified with a metal ear tag bearing its animal number. This individual animal number plus Bio/dynamics' project number comprised a unique identification.

Duration of treatment / exposure:

day 6-17 of gestation

Frequency of treatment:

6 h daily

Duration of test:

dams were sacrificed on day 18 of gestation

Dose / conc.:

1 400 ppm (nominal)

Remarks:

approximately 5.6 mg/L

No. of animals per sex per dose:

30

Control animals:

yes, concurrent vehicle

Maternal examinations:

Mortality, pregnancy rates, body weight data - gestation period, observation of animals in-chamber, physical in-life evaluation data, corpora lutea and uterine implantation data, gross post-mortem observation data

Fetal examinations:

Fetal weight data, fetal sex distribution data, fetal external examination data, fetal visceral evaluation data, fetal skeletal evaluation data, malformation data, ossification variation data.

Statistics:

Data were analyzed between the control and treated group; statistically significant differences from control are presented in mean tables of appendices.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the detailed physical in-life evaluations, lacrimation, inactivity (lethargy) and very shallow breathing following exposures on Day 6 were exhibited by the treated animals and these same observations were noted at lower incidence among the treated females at remaining intervals during the treatment period. Additionally, several females in the treated group were noted at some intervals during the treatment period to have a white material in the anterior chamber of the eye (this observation was usually unilateral in occurrence).

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the control or treated group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean actual Day 18 body weights were significantly lower in the treated group when compared to the control. Likewise, mean body weight gain during the Day 6-18 interval was significantly lower than in the control group. Mean uterine weight at the Day 18 sacrifice was significantly lower in the treated group and using the corrected Day 18 body weights (actual Day 18 body weight corrected by subtracting the weight of the gravid uterus), mean weight gain during the Day 6-18 gestation interval was significantly higher in the treated group.

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:

effects observed, treatment-related

Description (incidence and severity):

In the treatment-group, several females were noted to have a white material in the anterior chamber of the eye (usually unilateral in occurence). The significance of this finding remained unclear.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

During the exposure intervals, animals were observed for response to a tapping on the chamber wall. When performed, these observations were made prior to exposure initiation and at one, three and six hours into exposure. In the control group, all animals were noted to respond with movement to the tapping stimulus. In the treated group, most of the animals did not respond to stimulus throughout the exposure period and this lack in responsiveness was noted as early as one hour into the exposure.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The pre-implantation loss index of the treated group was lower than the index of the control group. This difference was attributed to the slightly greater ovulation rate in the control group .

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

A total of eight treated females (27.6%) had uterine implantations comprised entirely of resorption sites; none of the control females had complete resorption of a litter. The mean number of viable fetuses per pregnant female was also significantly reduced in the treated group.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

The mean number of resorption sites for the treated group was significantly higher than control data. Likewise, the mean percentage of
resorptions to implants for the treated group was higher than control, however, this parameter was not evaluated statistically.
The overall incidence of females with resorptions among their uterine implants was significantly higher than control in the treated group.
The mean number of viable fetuses per litter for the treated group was significantly lower than control and this difference was statistically
significant .

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Pregnancy rates for the control and treated group were 93.3% and 96.7%, respectively.

Dose descriptor:

LOAEC

Effect level:

1 400 ppm

Basis for effect level:

body weight and weight gain

clinical signs

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal weight data were significantly reduced in the treated group.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

A slight increase in the incidence of male fetuses was noted in the treated group. This change was not considered of significant magnitude to indicate an adverse effect of treatment .

External malformations:

no effects observed

Description (incidence and severity):

No external malformations were seen in the treated fetuses.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of fetuses with at least one ossification variation was significantly increased in the treated group and certain ossification variations including incomplete ossification of the cranial bones, asymmetrical sternebrae, unossified cervical vertebral centra and unossified phalanges (fore- and hindlimbs) were seen with increased incidence in the treated fetuses.
In the treated group, two fetuses had fused ribs. Though not seen in the concurrent control group, fused ribs malformations are seen at low incidence in this laboratory with this strain of mouse. The increased incidence of fused ribs seen in the treated group was not considered indicative of a treatment-related effect. No adverse effect of treatment was indicated from skeletal malformation data.
The incidence of skeletal malformations was comparable between the control and treated group.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus was noted with a cleft palate at soft tissue evaluation. During the soft tissue evaluation, a slight increase in the incidence of distended renal pelvis was seen in the treated group; however, in most instances, this distention was noted as slight and unilateral in occurrence and was not considered treatment related. The overall incidence of visceral malformations in the treated group was 4.4% (4/91 fetuses); this difference from control data was statistically significant. The incidence of occurrence of this observation (i .e ., distended renal pelvis) among treated fetuses was not considered indicative of a treatment-related effect .

Description (incidence and severity):

The incidence of fetuses with at least one ossification variation was significantly increased in the treated group and certain ossification variations including incomplete ossification of the cranial bones, asymmetrical sternebrae, unossified cervical vertebral centra and unossified phalanges (fore- and hindlimbs) were seen with increased incidence in the treated fetuses.
A rudimentary 14th rib structure and short 14th rib were noted with increased incidence in the treated group and 14th rib pair was noted at lower incidence in the treated group . The significance of these latter differences is unclear. Thus, obvious retardation in ossification was evident in the treated group.

Dose descriptor:

LOAEC

Effect level:

1 400 ppm

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

Abnormalities:

effects observed, treatment-related

Conclusions:

Cyclohexanone administered by the inhalation route at the 1400 ppm exposure level to pregnant CD-1 mice during the Day 6-17 gestation period was maternally toxic, embryotoxic and fetotoxic but was not considered teratogenic.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

5 600 mg/m³

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

An OECD guideline study (No. 414) carried out at BASF (1994) with gavage administration to rabbits in doses of 50 - 500 mg/kg showed a NOAEL of 250 mg/kg for maternal toxicity and a NOAEL of 500 mg/kg for teratogenicity. Some overt signs of maternal toxicity were found at 500 mg/kg (reduced food consumption, impaired body weight gain and unsteady gait), but was not toxic to the does at 50 and 250 mg/kg. There occurred no substance-related adverse effects on the gestational parameters and on the fetuses up to and including the highest dose level (500 mg/kg); at all dose levels no indications for substance-induced teratogenic effects were observed.

Biodynamics Inc. reported 1984 that cyclohexanone administered to pregnant rats by the inhalation route at exposure levels of 300 and 650 ppm was not considered maternally toxic, embryotoxic or teratogenic. At the highest dose level (1400 ppm), maternal toxic effects were evident (reduced body weight data, and at physical in-life evaluation an increased incidence of lacrimation, lethargy and nasal discharge) and embryotoxicity (reduced fetal weight data, increase in ossification variation data); however, no teratogenicity was evident at this same dose level.

Biodynamic Inc. (1984) reported that cyclohexanone administered to pregnant mice by the inhalation route at an exposure level of 1400 ppm (= about 5.6 mg/l) was maternally toxic, embryotoxic and fetotoxic, but was not considered teratogenic.

Justification for classification or non-classification

Since the reduced male reproductive performance seen in the high dose (1400 ppm) of a two-generation study was accompanied by severe signs of clinical toxicity (including deaths among high-dose animals), classification for reproduction toxicity according to Regulation EC/1272/2008 is not warranted.

Since no malformations were found in the above described studies and maternal toxicity were only found in high-dosed animals, classification for developmental toxicity according to Regulation EC/1272/2008 is not warranted.