Cyclohexanone

Toxicity to reproduction

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

other: Add-on study

Reference

Endpoint:

fertility, other

Remarks:

Reproductive recovery study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

A study was conducted during a post-exposure recovery period, to evaluate potential reversibility of treatment-related depressions in the fertility of male rats exposed via inhalation to 1,400 ppm of the test substance. The animals tested were second (F1) generation parental males from the two-generation study (450-1587).

GLP compliance:

yes

Specific details on test material used for the study:

No test article was administered during the study.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Lab., Portage, MI
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 -10 months (positive control males); 66-75 days (females)
- Weight at study initiation: not specified
- Fasting period before study:
- Housing: individual (acclimation period), more than 1 animal (mating), females were housed individual after completion of mating trial
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 12 days (females and PC males)

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 73 +/- 5 (min, max: 64, 79)
- Humidity (%): 50 +/- 20 (min, max: 32, 80)
- Air changes (per hr): nd
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: no administration

Details on exposure:

No test article was administered to the control and test group males. These males were obtained from the second (F1) generation of a two-generation reproduction study using the test substance via inhalation (Study no. 450-1587).
Prior to assignment to this study, control and test group males had received exposures to 0, 250, 500, or 1000 ppm for 6 h/d 5 d/w for a total of 160 to 168 exposures.

Details on mating procedure:

Males:females 1:2. Mating weeks: 1 through 4, 6, and 8. Following up to one full week of cohabitation, females were removed from the breeding cage and replaced with 2 additional untreated virgin females. This procedure was followed for four consecutive weeks. Evidence of of positive copulation was copulatory plug or sperm positive vaginal smears. Upon confirmation of copulation, females were housed individually.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

not applicable

Duration of treatment / exposure:

Positive control males received a single i.p. injection of TEM. Untreated control and treated animals were obtained from the two-generation study, thus, no administration of the test substance was performed.

Frequency of treatment:

Single (PC)

Details on study schedule:

Males of each treatment group were divided into 3 subgroups:
- males that sired F2b litters
- males that did not sire F2b litters but had been paired with females that conceived F2b litters as a result of a subsequent mating
- males that did not sire F2b litters and had been paired with females that did not conceive F2b litters by subsequent pairings with other males.
Ten males were selected from each treatment group, based upon reproductive performance during the second litter of the second generation .

Dose / conc.:

0.5 other: mg/kg

Remarks:

triethylenemelamine

No. of animals per sex per dose:

10

Control animals:

yes

Positive control:

PC group males received a single intraperitoneal dose at 1.0 ml/kg of a 0.05% (w/v) solution of triethylenemelamine (TEM) in deionized water.
Thirteen males were obtained, all were dosed with TEM, and ten were selected to continue as PC animals; the remaining 3 males were euthanized and discarded.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes, visually
- No feed intake measurements were made

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring:
weight, sex

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; 1/2 of the fetuses of each litter were retained in alcohol for potential examination of the skeletal system. Remaining fetuses of each litter were fixed in Bouin's solution for potential internal examination

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All females that bred on gestation day 20. Females that did not show signs of breeding were sacrificed approx. 14 days following completion of the mating trials.

GROSS NECROPSY
- Gross necropsy consisted of all male 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.
Females: Gravid uteri were examined to determine number of corpora lutea, implantation sites, viable fetuses, early fetal deaths, late fetal deaths, and any abnormal condition of the uterus and/or ovaries.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Yes

Statistics:

- Ci-square/Kruskal-Wallis: Fertility indices, pre-implantaton loss, post-implantation loss

Reproductive indices:

Male fertility, female fertility, pre-implantation loss, post-implantation loss
- ANOVA with Tukey's/Scheffe's: Body weight data and numbers of corpora lutea, implantation sites, resorption sites (early and late resorption sites), and viable fetuses

Untreated control group basis for comparison. Differences considered significant at p<0.05 and p<0.01 confidence level

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One 1400 ppm male was found dead on the day of scheduled sacrifice. Necropsy revealed a diffuse red exudate in the nasal region .

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant differences in the mean body weights for the untreated control males and the test group males.
Although no statistical differences were seen, males receiving 1400 ppm weighed less (9%) than the untreated control males at the start of the mating trials (a treatment-related effect seen for the 1,400 ppm males in the reproduction study) and gained twice as much weight during this 8-week non-exposure period than the untreated control males (108.2 g for the 1400 ppm males versus 53.7 g for the untreated contro males).

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:

effects observed, non-treatment-related

Description (incidence and severity):

1400 ppm: Male with tilted head: Necropsy showed a thickened urinary bladder with multiple stones and pale kidneys.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Moderate and moderately severe lymphocytic infiltrate was seen in the seminal vesicle and prostate and minimal tubular atrophy was seen for the testis. However, lymphocytic infiltrate in the prostate was also seen at minimal to slight incidence for 3/10 of the untreated control males and tubular atrophy was seen at minimal to moderate incidence in the testes of 3/10 of the untreated control males and therefore, the presence of the microscopic changes in male (tilted head) are not considered treatment-related.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The fertility of the test group males during the post exposure recovery period was comparable to the untreated control males.
- 1400 ppm: 2/6 males failed to sire litters
- 500 ppm: 1/6 male failed to sire litters
- 250 ppm: 1/6 male failed to sire litters
One of the 1400 ppm males exhibited a tilted head throughout the 8 week recovery period. Necropsy showed a thickened urinary bladder with multiple stones and pale kidneys. Moderate and moderately severe lymphocytic infiltrate was seen in the seminal vesicle and prostate and minimal tubular atrophy was seen for the testis. However, lymphocytic infiltrate in the prostate was also seen at minimal to slight incidence for 3/10 of the untreated control males and tubular atrophy was seen at minimal to moderate incidence in the testes of 3/10 of the untreated control males and therefore, the presence of the microscopic changes in male (tilted head) are not considered treatment-related. No gross findings and no microscopic findings were noted for the other 1400 ppm male. The other three males were the heaviest in the study. They revealed no gross findings. Microscopic examinations were not conducted for these males.

Females revealed no statistical significant differences between test groups and control. Positive control showed expected effects (increase in early resorption sites, significant decrease in viable fetuses, decrease in implantation sites, decrease in corpora lutea).

No significant differences in male or female fertility indices. No increases in pre- and post-implantation losses. Statistical increases in pre-implantation loss seen for the test groups using the totals/week were sporadic in occurrence and did not appear to be treatment-related. Using individual male data/week (Kruskal Wallis), a single statistical increase in the 1400 ppm pre-implantation loss at week 6 was noted. No other statistically significant differences were noted.

Dose descriptor:

other: not applicable

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

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:

not examined

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Statistical analysis of the mean litter weight data using ANOVA, with the litter size as the covariate, revealed no significant differences .
Fetal structural development was not altered by paternal exposure to lthe test substance.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

other: not applicable

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

In conclusion, the exposure of male CD Sprague-Dawley derived albino rats to 250, 500 or 1400 ppm of the test substance pre-natal, post-natal and through sexual maturity did not result in adverse effects on reproductive performance during a post-exposure recovery period. Therefore, treatment-related depressions in male fertility were reversible.

Executive summary:

A study was conducted, during a post-exposure recovery period, to evaluate the potential reversibility of treatment-related depressions in the fertility of male rats exposed via inhalation to 1,400 ppm of the test substance. The animals tested were CD Sprague Dawley-derived males from the second (Fl) parent generation of a two-generation reproduction study with the test substance via inhalation (American Biogenics Corporation Study No . 450-1587). Ten males were selected from each treatment group of F1 generation males based on known reproductive performance during the F2b litter mating trials. Each group of untreated control and treated males consisted of 4 fertile and 6 apparently non-fertile males. The males had received 160 to 168 exposures (6 hours/exposure) to 0 (U-C), 250 (T-I), 500 (T-II) or

1400 (T-III) ppm of the test substance. Fertile males of the same strain and of a similar age were obtained from the same source as the parents of the U-C and test group males and injected, intraperitoneally, with 0.5 mg/kg triethylenemelamine (positive control). All males were rested/untreated 2 days following the last exposure/treatment with the appropriate test or control article. The males were paired weekly, with 2 untreated virgin females for 4 consecutive weeks. The males were rested 1 week, paired during the sixth week of the recovery period, rested during the seventh week, and paired during week 8. The females were sacrificed on gestation day 20. The uterine contents were examined, and findings recorded, and fetuses were sexed, weighed, and examined externally.

The exposure of male CD 0 Sprague-Dawley derived albino rat s to 250, 500 or 1400 ppm of the test substance pre-natal, post-natal and through sexual maturity did not result in adverse effects on

reproductive performance during a post-exposure recovery period. Therefore, treatment-related depressions in the fertility of second-generation males exposed to 1,400 ppm of the test substance were reversible.

Sensitivity of the CD 0 Sprague-Dawley derived albino rat was demonstrated in the positive control group by significant increases in the pre-implantation losses during weeks 1 through 4 and significant increases in the post-implantation losses during weeks 1, 2, 3, 4, and 6.

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.

Limit test:

no

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

Details on test material:

TS-Freetext:
Cyclohexanone
CAS No.: 108-94-1

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)

Test animals

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

Administration / exposure

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 .

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 ppm (nominal)

Remarks:

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

Doses / concentrations 2

Dose / conc.:

500 ppm (nominal)

Remarks:

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

Doses / concentrations 3

Dose / conc.:

1 000 ppm (nominal)

Remarks:

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

Doses / concentrations 4

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.

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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

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

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Effect levels (P1)

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)

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

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.

Effect levels (F1)

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

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

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.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

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

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

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

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 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.