Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral route:

no reliable information available

Inhalation route:

Rats: NOEC = 500 ppm (based on cyclohexanone), corresponds to 2007 mg/m3

Dermal route:

no reliable information available

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 September 1984 to 19 November 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with GLP
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
Throughout the first generation of this study, all parent animals were exposed to 0, 250, 500 or 1000 ppm cyclohexanone (30 per sex per group). Thirty males and 30 females were selected from the F1a litters of each group to continue the test as second (F1) generation animals. The F1a progeny selected as potential F1 generation animals were exposed to 0, 250, 500 or 1000 ppm. After weaning of the last F1a litter, the F1 parental animals were selected and the 1000 ppm exposure level was increased to 1400 ppm; the 250 and 500 ppm levels remained unchanged. Assessments for potential neurotoxicologic/neuropathologic effects were conducted pre-weaning and post-weaning in each F1a litter.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Portage, MI facilities of Charles River Breeding Laboratories, Inc.
- Diet: Purina Certified Rodent Chow #5002
- Age at study initiation: F0 Generation: 40 days; F1Generation: 29 to 43 days
- Average weight at study initiation: F0 Males: 156.6 g; F0 Females: 129.9 g; F1 Males: 50.1 g; F1 Females: 52.6 g
- Fasting period before study: No
- Housing: Animals were housed in one of 2 types of cages. Stainless steel, open mesh cage bank units, each containing 10 individual cubicles, were used during acclimation and all study phases, excluding mating, gestating, and lactating periods. Hanging, wire-bottom, galvanized steel caging was used during the mating trials. These cages, equipped with solid-bottom, stainless steel floorplates and nesting material (Bed-O-Cobs, Maumee, OB) were used during gestation and lactation periods. The floorplates and nesting material were supplied to gestating females on approximately the fifteenth day of gestation and were removed from the cages of lactating females when the progeny were approximately 7 days of age. During exposure, animals were individually housed in stainless steel cage bank units. Each cage bank unit contained 10 individual cubicles. During the study phase, when both F0 and potential F1 generation animals were treated, exposures were run with animals housed in 2 layers of cage bank units. All other exposures were run with animals housed in a single layer of 6 cage bank units. The cage bank units were rotated counter-clockwise, one cage bank per week.- Diet: ad libitum- Water: Filtered tap water was provided ad libitum via demand operated valves- Acclimation period: F0 animals were acclimated for 19 days

ENVIRONMENTAL CONDITIONS
- Temperature: 68 to 78 °F
- Humidity: 30 to 70%
- Photoperiod: A 12-hour light/dark cycle was maintained

IN-LIFE DATES
From: 05 September 1984
To: The last parental sacrifice occurred on 18 October 1985. Males used for the post-exposure fertility assessment study were sacrificed on 19 November 1985.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: conditioned air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The cyclohexanone inhalation exposures were conducted in 8 m³ stainless steel and glass chambers.
- Source and rate of air: Each chamber was supplied with conditioned air (HEPA and charcoal filtered) operated dynamically under a slight (0.3 in. H2O) negative pressure to prevent contamination of the surrounding area.
- System of generating test material atmosphere: The generation system for each exposure chamber consisted of an air flow meter, glass column (30 cm) with glass beads (4 mm) and heat tape, thermometer, round bottomed flask with heating mantle, glass vapour delivery tube with heat tape, Teflon delivery tube, FMI pump, test material reservoir and vent.Column and flask temperatures were maintained below 105 °C (flask, column and chamber temperatures were recorded hourly). Flask airflow, column and flask temperatures and FMI pump rate were adjusted to achieve the target concentrations. Exposures started when the test material reached the top of the heated glass bead column and ended when test material flow to the column was stopped. No accumulation of test material occurred in the flasks. All chambers were operated for at least one-half hour after the test material flow ceased. Chamber airflows were proportional to the pressure drop across an orifice placed in the chamber exhaust line. The pressure drop was measured by a minihelic® gauge that was calibrated against a mass flowmeter.
- Temperature, humidity, pressure in air chamber: Conditioned air 67-77 °F, humidity 30-70%. Chamber supply air temperature and humidity were determined hourly in the untreated control chamber with a Taylor 5522 hygrometer.
- Air flow rate: Airflow rates through the flasks were 80-100 litres per minute. Chamber airflows were recorded hourly.
- Air change rate: Airflow rates sufficient for at least 12 air changes per hour

TEST ATMOSPHERE
- Samples taken from breathing zone: Yes
- Brief description of analytical method used: Test material AnalysisThe concentration of the test material in the breathing zone within each chamber was analysed 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 litres 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. Appropriate volumes were then injected into a gas chromatograph (Hewlett Packard 5710A) operated under the following conditions:
- Column: 20 x 0.125 inch stainless steel packed with 10% UCW-982 on 80-100 WAW DMCS
- Temperatures: Detector: 160 °C; Injection Port: 200 °C; and Oven: 950 °C
- Lamp Intensity: 4
- Nitrogen Flow: approximately 30 mL/minute
- Detector: HNU PID (photoionization)
- Chamber monitoring: 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, test material distribution within each chamber was determined by measuring concentrations at six points in each chamber at the breathing level of the animals.From the first part of the study, 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. During the second part of the study, a California Measurements, Inc. Piezoelectric QCM Cascade Impactor was used to monitor aerosol in the exposure chambers. Measurements with this instrument were made twice per week at designated times.
Details on mating procedure:
- M/F ratio per cage: 1:1. Monogamous cohabitation, whenever possible, was used (1 male: 1 female) with the animal pairings conducted randomly employing computer-generated male/female assignments within treatment groups- Length of cohabitation: Maximum of 15 days
- Proof of pregnancy: The observation of a copulatory plug in the vagina or sperm-positive results of vaginal smears was defined as evidence of copulation.- After successful mating each pregnant female was caged: Females for which breeding was confirmed were weighed (gestation day 0) and were housed individually, terminating their mating trial.The mating trials for the "a" litters were initiated following the pre-mating period exposure to the test material and were continued for 15 consecutive days. 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/female pairing period for females which remained unbred.Following 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 approximately 2 weeks after the weaning of the F2a litter and the same procedure 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.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
2 generations. In the parent (F0) generation, animals were exposed for 10 weeks prior to the mating period. Mating was a maximum of 15 days. Males were dosed until initiation of the F1 weanlings while females were dosed until day 28 of lactation. Unbred females were dosed for 28 days. In the F1 generation, animals were exposed for 15 weeks prior to the 15 day mating period and then were dosed until sacrifice. Unbred females were dosed for 28 days post F2b mating trials.
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.
Details on study schedule:
Two male and 2 female weanlings were randomly selected at weaning from each of the F1a litters, when possible, of each group to continue on test as potential F1 parental animals. After all the F1a litters were weaned, 33 males and 33 females were chosen from each treatment group population of retained progeny for F1 parental animals.In order to assess recovery of reproductive performance, ten males were selected from each treatment group of F1 generation males based on known reproductive performance during the F2b litter mating trials. All males were rested/untreated 2 days following the last exposure/treatment with the appropriate test or control material. 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.
Dose / conc.:
0 ppm
Remarks:
F0 and F1 generation
Dose / conc.:
250 ppm (nominal)
Remarks:
F0 and F1 generation
Dose / conc.:
500 ppm (nominal)
Remarks:
F0 and F1 generation
Dose / conc.:
1 000 ppm (nominal)
Remarks:
F0 generation
Dose / conc.:
1 400 ppm (nominal)
Remarks:
F1 generation
No. of animals per sex per dose:
30
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: All parental animals were assigned to treatment groups randomly by computer program. The method used by this program is documented by Carnahan, Luther, and Wilkes, Applied Numerical Methods, Wiley, 1969. The method of selection was also used for the selection of F1a litter progeny for neurotoxicological assessment and as F1 parental animals.
Positive control:
The positive control group males received a single intraperitoneal dose at 1.0 mL/kg of a 0.05% (w/v) solution of triethylenemelamine in deionised water.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice each day for mortality, morbidity and overt signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once each week each animal was removed from its cage and thoroughly examined.

BODY WEIGHT: Yes
- Time schedule for examinations: All parental animals were weighed weekly during the premating period. Weekly body weights were obtained for all surviving parental males following completion of the mating trials, for all females which did not retain a litter and for all unbred females until their sacrifice. The parental females were weighed on gestation days 0, 6, 15 and 20 and lactation days 0, 5, 7, 14, 21 and 28. Final body weights were obtained for each animal at sacrifice or death.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: During all phases of the study, food consumption was monitored visually.

GESTATION AND LACTATION
- Each female was observed daily through gestation day 18. Gravid animals were supplied with nesting material at approximately 15 days of gestation. 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.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.Litters in excess of 8 pups were reduced to that number on lactation day 4 using computer-generated random selections. When possible, 4 males and 4 females in each litter were retained, however if less than 4 of either sex were surviving at day 4, all survivors of that sex were retained along with the appropriate number of pups of the other sex to obtain a total of 8 progeny.

PARAMETERS EXAMINED
- The following parameters were examined in F1 offspring:
- PopulationThe sexes and numbers of pups delivered, delivered viable, delivered stillborn, and found partially cannibalised were recorded for each litter of progeny on the day of parturition. The numbers of pups surviving to lactation days 1, 4, 7, 14, 21 and 28 were determined.
- Progeny were weaned on lactation day 28. In addition, any progeny found dead during the lactation periods, along with those sacrificed on lactation day 4, were preserved, by litter, in 70% ethyl alcohol.- Body WeightsIndividual pup weights and sexes were determined on lactation days 0, 4, 7, 14, 21 and 28 for all surviving progeny.-ObservationsEach litter of progeny was examined daily for mortality and behavioural anomalies. Each pup was also examined thoroughly for developmental anomalies at birth, at each body weight interval, and again at weaning.

NEUROTOXICOLOGICAL ASSESSMENT
- Assessments for neurotoxicological effects were conducted on 1 pup from each F1a litter delivered (survival permitting). One male or female was randomly selected from alternating litters for these procedures. This selection occurred on lactation day 4 following appropriate litter reductions. 1. Suckling Young Test ProceduresTesting 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. 2. Post Weaning Test ProceduresTen males and 10 females per treatment group (11 males and 9 females were retained from the 500 ppm group) were randomly selected from the population of F1a progeny chosen for pre-weaning neurotoxicologic testing and were evaluated at 28, 35, 42 and 49 days of age for: lacrimation; salivation; pupil size; pupil response; corneal response; visual placing; finger approach; respiration; touch escape; toe pinch; tail pinch; grasp irritability; body tone; abdominal tone; limb rotation; limb tone; body position; locomotor activity; spatial locomotion; hypotonic gait; impaired gait; pelvic elevation; tail elevation; positional passivity; righting reflex; wire manoeuvre; grip strength: startle response; vocalisation; tremors; twitches; convulsions; piloerection; hypothermia; urination-defecation; diarrhoea; and acute death.3.

OPHTHALMOLOGIC EXAMINATIONS
- The lenses of all F1a progeny with open eyelids and all surviving F2a and F2b weanlings were examined by a board certified veterinary ophthalmologist.

GROSS EXAMINATION OF DEAD PUPS: Yes
Postmortem examinations (parental animals):
TERMINAL PROCEDURES
- The F0 parental males were sacrificed at the completion of the F1a litter weanings. The F0 parental females that failed to breed were sacrificed 20 days following completion of the F1a mating trials; F0 females that bred but failed to deliver viable progeny (i.e. not gravid or resorbed) were sacrificed 26 days post copulation. Females that conceived and delivered progeny were sacrificed after completion of the F1a litters.All animals that were sacrificed or died prior to final sacrifice were necropsied. In addition, overnight urine samples were collected from 5 lactating F0 females per group, a total of 20. After the last exposure (on lactation day 28) the females were placed in urine collection cages until the following morning. They were then anaesthetised with ether and necropsied after their blood was obtained from the dorsal aorta.The volume of the overnight urine samples was measured and the urine was tested for glucose, pH, protein, ketone, bilirubin, occult blood, and urobilinogen using a dipstick procedure. The urine samples were then stored frozen at -20 °C. The serum was separated from the red cells and stored frozen at -20 °C; the cellular portion of the blood samples was discarded.

PATHOLOGY
- All F0 and F1 parental animals, sacrificed and found dead, were subjected to gross necropsy examination. With the exception of the F0 parental females that were bled and the F1 parental males that were perfused in situ, the sacrificed animals were rendered unconscious by carbon dioxide and exsanguinated. The F0 females that were bled were rendered unconscious using ether anaesthesia prior to blood collection and sacrifice.The necropsy included examination 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 organs; and the carcass. Additionally the number or 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, labelled jars containing 10% buffered formalin. In addition, the eyes were retained from all F1 parental animals. The liver, kidney(s) (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. These tissues were frozen using liquid nitrogen and stored at approximately -80 °C. Additionally, as a result of clinical observations noted for 2 of the 1400 ppm F1 parental generation sibling males (AG3824, AG3025), these males along with 2 males chosen randomly from the remaining 1400 ppm males and 4 of the 0 ppm males were anaesthetised and perfused in situ.Microscopic examinations were conducted upon the above listed tissues from the sacrificed untreated control and high dose parental animals from both generations.
Postmortem examinations (offspring):
TERMINAL PROCEDURES
- 1. F1a ProgenyTwo male and 2 female weanlings, when possible, were selected from each of the F1a litters weaned of each treatment group to continue on test as potential F1 parental animals. Progeny selected for neurotoxicological testing were not included in these selections. After weaning of all F1a litters, 30 males and 30 females were selected from each treatment group to serve as F1 parental animals. Those progeny not selected as F1 parental animals or for neurotoxicologic testing were sacrificed and subjected to pathologic examination.
- 2. F2a and F2b progenyAll surviving F2a and F2b weanlings were sacrificed and subjected to gross pathologic examinations.

PATHOLOGY
- 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; 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. Microscopic examinations were conducted upon the eyes of the sacrificed F1a progeny and F1a progeny chosen for neurotoxicologic testing.

NEUROPATHOLOGY
- The F1a progeny chosen for neurotoxicological assessments were sacrificed at 49 days of age, following completion of the in-life testing. These animals were anaesthetised using an intraperitoneal injection of sodium pentobarbital and perfused in situ, initially with heparinized phosphate buffered saline followed by perfusion with 10% neutral buffered formalin for fixation. The cranium and vertebral column were exposed and the animals stored in fixative at 4 °C for at least 12 hours. 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. These tissues were further fixed, stored for at least 48 hours, prior to further processing.The volume of fixative versus the volume of tissues in a specimen jar was no less than 25:1. Tissues listed below retained from the control and high-dose group animals were subjected to microscopic evaluation:- Brain: forebrain, centre of cerebrum, midbrain, cerebellum and pons, medulla oblongata, and Gasserian ganglia.- Spinal Cord: At cervical (C3-C6), lumbar (L1-L4) swellings, dorsal root ganglia (C3-C6 , L1 -L4), and dorsal and ventral root fibres (C3-C6, L1 -L4).- Sciatic Nerve: Mid-thigh and sciatic notch, sural nerve (at knee), and tibial nerve (at knee).
Statistics:
Quantitative continuous variables, i.e., body weights and food consumption, were analysed by Analysis of Variance with significant differences described by that treatment further studied by multiple comparison (Tukey’s or Scheffe’s, dependent upon ‘N’ values). Progeny body weight data were additionally studied using Analysis of Covariance (with the litter size as the covariate) and Dunnett's T-test. Reproductive data and neurotoxicologic data were analysed using Chi-square analysis and Fisher's Exact test.Unless indicated otherwise, all statistical analyses were interpreted using the untreated control for comparison. Differences were considered significant at the p<0.05 and p<0.01 confidence levels.
Reproductive indices:
Mating Index = (Number of Number of copulations / Number of oestrus cycles* utilised) x 100Fertility Index = (Number of pregnancies / Number of Number of copulations) x 100Gestation Index = (Number of parturitions / Number of pregnancies) x 100Female Fertility Index = (Number of pregnancies / Number of females mated) x 100Male Fertility Index = (Number of sires / Number of males mated) x 100*Five days equals 1 oestrus cycle
Offspring viability indices:
Born Viable = (Number of pups delivered alive / Total number of pups delivered) x 100Born Dead = (Number of stillbirths / Total number of pups delivered) x 100Born and cannibalised = (Number of pups found partially cannibalised / Total number of pups delivered) x 1001 Day = (Number of pups viable at lactation day 1 / Number of pups born alive) x 1004 Day = (Number of pups viable at lactation day 4 / Number of pups born alive) x 1007 Day = (Number of pups viable at lactation day 7 / Number of pups born alive) x 10014 Day = (Number of pups viable at lactation day 14 / Number of pups born alive) x 10021 Day = (Number of pups viable at lactation day 21 / Number of pups born alive) x 10028 Day = (Number of pups viable at lactation day 28 / Number of pups born alive) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
No noteworthy observations were seen for F0 animals pre-exposure. Clinical reactions, such as lacrimation, ataxia and irregular breathing, were noted for the 1000 ppm animals following the first 2 exposures. Starting with the third exposure, these animals appeared to acclimate to the test material and no consistent, recurring observations were noted post-exposure for the 1000 ppm animals through the remainder of the exposure period. No post-exposure reactions were seen for the 250 or 500 ppm animals.
Mortality:
no mortality observed
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 the test material were comparable to the untreated control animals.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis determinations of 5 F0 females per treatment group post-lactation revealed increased volume from the 1000 ppm animals; however, no qualitative differences were noted in glucose, pH, protein, ketone, bilirubin, occult blood or urobilinogen. All other urine parameters for treated females were comparable to the untreated control females.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No microscopic changes were seen in the reproductive organs from the 1000 ppm animals and the untreated control animals.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
REPRODUCTIVE PERFORMANCE
- 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 for the 500 and 1000 ppm dose groups ranged from 13 to 20 % less than the control group, but these were not statistically significant. All other parameters were comparable to the control.

PROGENY
- 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.The observations of gestating and/or lactating dams for untoward reactions revealed no effects which were considered to be related to the treatment with the test material.
Key result
Dose descriptor:
NOAEC
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
System:
male reproductive system
Organ:
seminal vesicle
testes
other: epididymides
Key result
Critical effects observed:
no
System:
female reproductive system
Organ:
ovary
uterus
vagina
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Observations recorded prior to exposure revealed 27/60 of the 1400 ppm animals had yellow/brown stained fur in comparison to 3/60 of the untreated control group. In addition, starting at week 30 of the F1 generation and continuing through termination, two sibling 1400 ppm males exhibited a staggering gait prior to test material exposure.Exposure of F1 parental animals to 1000/1400 ppm resulted in noteworthy pharmacotoxic reactions. The F1a progeny exposed to 1000 ppm (post-weaning, prior to the selection of the F1 parental animals and subsequent increase to 1400 ppm) exhibited clinical signs such as ataxia, lacrimation, irregular breathing, and urine soaked fur following treatment. After the increase to 1400 ppm, and continuing for approximately 3 months, these reactions (along with prostration in the first week of 1400 ppm exposure) continued to occur. Starting at week 16 of the F1 generation, the 1400 ppm animals appeared to adapt to treatment with lethargy being the predominant post-exposure reaction. No observations were noted post-exposure during the final 3 weeks of the F1 generation. During the first 3 weeks of exposure, urine soaked fur was noted post-exposure for 3 to 37% of the animals exposed to 500 ppm cyclohexanone. No other noteworthy reactions were seen among the 500 ppm animals. No untoward reactions were seen for the F1 generation animals exposed to 250 ppm cyclohexanone.
Mortality:
mortality observed, treatment-related
Description (incidence):
Six of the F1 generation animals exposed to 1400 ppm cyclohexanone died. Two males and 1 female died during the first week of exposure. One male died during the fifteenth week of the pre-mating period and one male died during the F2b mating trials. One of the males used for the post-exposure assessment of fertility was found dead on the scheduled day of sacrifice. A 250 ppm male was sacrificed moribund prior to the F2b mating trials; no other deaths occurred among the 250 and 500 ppm animals or the untreated control animals during the second generation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Starting with the first week of exposure to 1400 ppm, statistically significant (p<0.01, p<0.05) weight depressions were noted for the 1400 ppm males when compared to the untreated control males. These depressions were seen at 29 of the subsequent 33 weeks of 1400 ppm exposure. Females from this exposure level weighed less (p<0.05) than the untreated control females during the week of exposure to 1000 ppm. Body weights for these females were reduced (p<0.01) at the first week of 1400 ppm exposure and these depressions (p<0.05) continued during weeks 3 and 4. Starting with the fourth week of 1400 ppm exposure through final sacrifice, no significant body weight differences were seen for the 1400 ppm females when compared to the untreated control females.During the first week of exposure, a significant weight depression (p<0.05) was seen for the 500 ppm males when compared to the untreated control males. All other body weight data obtained for the 250 and 500 ppm animals were similar to the untreated control animals. In addition, body weight data recorded for gestating and lactating dams were similar for the treated groups and the untreated control group during both generations.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Ophthalmologic examinations of the F1a progeny revealed lens opacities for 2/296 of the 250 ppm progeny (unilateral), 2/174 of the 500 ppm progeny (unilateral), and 1/174 of the 1400 ppm progeny (bilateral). The 250 and 500 ppm F1a progeny were retained, unexposed, to determine if the findings would reverse. Approximately 3 months following the initial examination, these animals were re-examined. One 250 ppm male which initially had a thread-like white opacity (unilateral) was normal at the subsequent examination; one 250 ppm male with a cloudy anterior lens capsule (unilateral) had an anterior cortical cataract at the subsequent exam; a 500 ppm male that earlier had a lens capsule which appeared cloudy (unilateral) had a roughened cornea and normal lens at the subsequent examination; and a 500 ppm male with an incipient cataract involving the nucleus of the lens (unilateral) had a granular corneal surface and nuclear opacity in lens of the previously affected eye and pinpoint opacities on the posterior lens cap of the eye that appeared normal at the initial examination. Due to the low incidence and the minimal nature of these effects, the pathologist concluded that they were not related to treatment.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Evaluation of behavioural/neurotoxicologic development of selected F1a progeny revealed no consistent statistical differences between treated and control groups. On lactation day 15, 31 to 56 percent fewer test progeny had open eyelids than the untreated control progeny; however no dose-response pattern was apparent.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross pathologic examinations of all F1 parental animals revealed no consistent lesions which were considered to be treatment-related.
Neuropathological findings:
no effects observed
Description (incidence and severity):
Neuropathologic examination of tissues from the sibling males that were ataxic revealed no morphologic abnormalities. Examination of the specified areas of the nervous system of the untreated control and 1000 ppm F1a progeny chosen for neurotoxicologic evaluation did not reveal lesions in any of the tissues. Microscopic examination of the eyes from the F1a progeny revealed lenticular vacuolation (vacuolation of a few outer cortical fibres in the lens) for 2/115 of the 500 ppm progeny and 3/114 of the 1000 ppm progeny. The examining pathologist concluded that due to the low incidence and minimal nature of these changes, they were not treatment-related.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination of the reproductive organs from the untreated control and 1400 ppm parent animals revealed no evidence of treatment-related effects.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
REPRODUCTIVE PERFORMANCE
- Statistical analyses of the reproductive indices showed no statistical depressions for the test groups when compared to the untreated control group. However, the fertility indices for male rats exposed to 1400 ppm calculated using all males paired were 19.8 to 20.8% less than the untreated control males during the F2a and F2b litters, respectively. The male fertility calculated using only the animals that were paired with females conceiving litters showed a 24.3 to 28.6% reduction for the males exposed to 1400 ppm compared to the untreated control males during the F2a and F2b mating trials, respectively. Evaluation of these data for intergroup differences revealed significant depressions (p <0.05) for the 1400 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 in the F2b mating trials. Furthermore, mating indices calculated for the 1400 ppm group were significantly less than those in the 250 ppm group during both the F2a (p <0.01) and F2b (p <0.05) mating trials.
Key result
Dose descriptor:
NOAEC
Effect level:
500 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
System:
male reproductive system
Organ:
seminal vesicle
testes
other: epididymides
Key result
Critical effects observed:
no
System:
female reproductive system
Organ:
ovary
uterus
vagina
Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Statistical analyses of the F2a and F2b progeny data revealed significant (p <0.01, p<0.05) reductions for the mean number of 1400 ppm progeny viable during lactation periods. Furthermore, the 1400 ppm dams delivered 23 and 24% fewer viable progeny than the untreated control dams during the F2a and F2b litters; however, no statistically significant differences to the controls were noted. Progeny delivery and population data for the 250 and 500 ppm groups were comparable to the data of the untreated control group.The percentage of 1400 ppm F2a progeny delivered viable and surviving to lactation days 1 and 4 were significantly less (p <0.01) than in the untreated control. Progeny survival of the 1400 ppm F2a progeny at lactation days 14, 21 and 28 was not statistically different from the untreated control group; the survival indices on these days were 14 to 22% less than that of the untreated control progeny. During the F2b litter, 1400 ppm progeny survival to lactation days 1 and 4 was statistically less (p <0.01) than that of the untreated control progeny. Survival of 1400 ppm F2b progeny after lactation day 4 (lactation days 7, 14, 21 and 28) was comparable to the untreated control progeny survival. Progeny survival indices calculated for the 250 and 500 ppm groups during the F2a and F2b litters were similar to the untreated control group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistical analyses of the 1400 ppm F2a and F2b progeny body weights revealed significant reductions (p <0.05, p <0.01) when compared to the untreated control progeny using both the Analyses of Variance of the individual pup weights and Analyses of Covariance of the mean litter weight data. Analyses of the 250 and 500 ppm F2a progeny body weights resulted in statistical reductions (p <0.05) in individual weights at lactation days 0, 14, 21 and 28. The mean litter weights for 250 ppm progeny were reduced (p <0.05) at lactation day 21. Statistical depressions were noted for the 500 ppm mean litter weights at lactation day 21 (p <0.01) and mean male litter weights at lactation day 28 (p <0.05). The F2b progeny from these dams exhibited significant (p <0.05, p <0.01) weight increases on lactation days 0 and 4 when analysed using individual body weight data. Because the statistical weight differences noted for the 250 and 500 ppm F2a progeny were minimal (5 to 17% less than the untreated control progeny) and were not seen for the F2b progeny, maternal exposure to 250 or 500 ppm cyclohexanone was not considered to adversely affect pup body weights.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Examination of the F2a progeny, post-weaning, revealed lens opacities or cloudiness for 3/151 of the 250 ppm progeny (unilateral) and 1/60 of the 1400 ppm progeny (unilateral). Additionally, 1/159 of the 500 ppm progeny had a corneal opacity. Ophthalmologic examination of the F2b progeny, post-weaning, for lens opacities or cloudiness revealed no finding for treated or control groups. The ophthalmologist's interpretation of the examinations was that the test substance did not increase the incidence of cataracts in the progeny. The lens and other ocular abnormalities appeared to be within the range of type and incidence expected in the number of animals expected.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy examinations of progeny revealed no lesions which appeared to be related to cyclohexanone.
Histopathological findings:
not examined
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
500 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Reproductive effects observed:
yes
Lowest effective dose / conc.:
4 100 mg/m³ air (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

Table 1: Reproductive Performance

Dose Group (ppm)

Mating Index (percent)

Fertility Index (percent)

Gestation Index (percent)

Female Fertility Index (percent)

Male Fertility Index (percent)

Average Gestation Length (days)

F0 Generation - F1a Litter

0

88.2

96.7

96.6

96.7 (100.0)

83.3 (86.2)

22

250

88.2

93.3

96.4

93.3 (100.0)

80.0 (85.7)

22

500

73.2

96.7

100.0

96.7 (100.0)

66.7 (69.0)

22

1000

70.7

93.1

92.6

90.0 (96.4)

70.0 (75.0)

22

F1 Generation - F2a Litter

0

46.9

91.3

95.2

70.0 (80.8)

60.0 (81.8)

22

250

71.1*

85.2

100.0

76.7 (88.5)

73.3 (91.7)

22

500

57.8

88.5

95.6

76.7 (85.2)

56.7 (73.9)

22

1400

44.4***

75.0

94.4

62.1 (72.0)

48.1** (61.9)

22

F1 Generation - F2b Litter

0

44.0

77.3

100.0

56.7 (77.3)

46.7 (82.4)

22

250

56.8

84.0

95.2

70.0 (84.0)

62.1 (90.0)

22

500

82.2

75.0

100.0

60.0 (81.8)

53.3 (88.9)

22

1400

35.7**

85.0

94.1

58.6 (70.8)

37.0† (58.8)

22

 NB: values in parentheses represent the indices omitting either females that were paired only with males that failed to sire an F1a litter or males that were only paired with females that failed to conceive an F1a litter.

*Statistically significantly different from the control group at the 95 % confidence level

**Statistically significantly different from the 250 ppm group at the 95 % confidence level

***Statistically significantly different from the 250 ppm group at the 99 % confidence level

† Statistically significantly different from the 250 and 500 ppm groups at the 95 % confidence level

 

Table 2: Total Progeny Survival

Dose Group (ppm)

Percent of Progeny Born

Progeny Survival (percent) on Lactation Day

Viable

Dead

Cannibalised

1

4

7

14

21

28

F0 Generation - F1a Litter

0

98.7

1.3

0.0

99.2

95.5

93.7

82.0

77.0

77.0

250

98.9

1.1

0.0

99.2

96.2

99.5

98.6

95.4

95.4

500

97.8

2.0

0.2

98.5

94.4

99.1

86.6

79.0

78.1

1000

100.0

0.0

0.0

95.7

93.7

100.0

97.4

92.6

92.1

F1 Generation - F2a Litter

0

96.7

3.3

0.0

99.6

97.4

98.7

93.5

92.9

92.9

250

99.3

0.4

0.4

98.9

97.1

98.8

91.3

87.9

87.9

500

95.2

4.4

0.4

95.4

92.7

99.4

99.4

98.8

98.8

1400

85.6*

12.8

1.7

77.9*

75.3*

96.4

79.5

79.5

72.3

F1 Generation - F2b Litter

0

98.1

1.9

0.0

100.0

99.0

99.2

99.2

99.2

98.5

250

99.6

0.4

0.0

94.5

94.1

99.3

94.6

94.6

94.6

500

99.5

0.5

0.0

99.0

94.9

96.9

95.3

95.3

95.3

1400

97.4

2.0

0.7

75.2*

69.1*

96.9

92.3

92.3

92.3

Data in this table reflect the progeny survival as a group

*Statistically significantly different at the 99 % confidence level

 

Table 3: Progeny Body Weight Data (g)

Dose Group (ppm)

 

Mean Values on Lactation Day

0

4

7

14

21

28

Male

Female

F0 Generation - F1a Litter

0

Mean

SD

N

5.4

0.8

372

7.8

1.5

357

10.8

2.7

208

19.0

4.5

182

31.6

7.3

171

61.6

13.1

86

55.7

11.3

85

250

Mean

SD

N

5.4

0.7

371

7.9

1.3

359

10.3

2.1

215

17.8*

3.1

213

28.2**

5.4

206

55.0**

9.8

101

52.7

9.8

105

500

Mean

SD

N

5.4

0.8

395

7.6

1.6

373

9.7**

2.5

222

16.0**

4.7

194

26.9**

7.0

177

50.9**†

14.0

87

48.8**

13.9

88

1000

Mean

SD

N

5.5

1.0

294

8.3**

1.6

283

10.0

2.3

189

17.6*

4.0

184

28.9**

5.9

175

56.5

11.7

86

53.8

10.2

88

F1 Generation - F2a Litter

0

Mean

SD

N

5.9

0.9

235

7.9

1.5

229

11.7

2.4

125

19.7

0.7

144

32.2

6.2

143

62.7

10.4

70

66.0

11.4

73

250

Mean

SD

N

5.6**

0.8

274

8.2

1.8

267

11.4

2.4

171

17.8**

3.9

158

28.4**‡

6.0

152

53.1**

13.1

78

51.0

12.2

74

500

Mean

SD

N

5.5**

0.8

259

8.1

1.6

240

11.3

2.1

161

17.9**

3.5

161

27.2**†

5.7

160

52.2**‡

12.8

78

49.5**

10.7

82

1400

Mean

SD

N

5.0**†

0.8

150

7.8

1.1

116

9.1**†

2.0

80

14.3**†

2.0

66

20.8**†

4.6

66

38.8**†

9.3

34

37.5**†

9.9

26

F1 Generation - F2b Litter

0

Mean

SD

N

5.9

0.8

209

8.9

1.4

207

13.1

1.5

129

22.5

3.7

129

35.5

6.9

129

67.8

11.0

63

64.6

9.3

65

250

Mean

SD

N

6.2*

0.9

255

9.3*

1.3

240

12.9

2.2

147

23.3

2.8

140

36.8

5.2

140

69.2

9.0

67

56.2

7.9

73

500

Mean

SD

N

6.2*

1.0

197

9.5**

1.5

187

13.5

1.8

124

22.0

3.4

122

34.6

6.5

122

69.2

11.3

59

62.1

10.9

63

1400

Mean

SD

N

5.8

1.2

142

8.3*

1.7

103

10.8**†

2.3

63

17.3**†

4.0

60

26.0**†

6.3

60

51.9**

15.7

30

47.9**†

11.5

30

SD = Standard deviation

N = Number of animals

*Statistically significantly different at the 95 % confidence level (individual progeny body weight data) using ANOVA and Scheffe’s multiple comparison

** Statistically significantly different at the 99 % confidence level (individual progeny body weight data) using ANOVA and Scheffe’s multiple comparison

‡ Statistically significantly different at the 95 % confidence level (mean litter weight data) using ANCOVA and Dunnett’s t-test

† Statistically significantly different at the 99 % confidence level (mean litter weight data) using ANCOVA and Dunnett’s t-test

Conclusions:
Inhalation exposure to 1000 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 rat. Evaluation for behavioral/neurotoxicologic development of selected progeny revealed no consistent differences between treated groups and the control group.The NOAEC values were therefore 1000 ppm (4.01 mg/L) for the F0 generation and 500 ppm (2.01 mg/L) for the F1 and F2 generations.
Executive summary:

A reproduction toxicity study was conducted to ascertain the potential effects of inhalation exposure to cyclohexanone vapour upon growth, development, and reproductive performance of 2 consecutive generations of CD® Sprague Dawley derived albino rats. The method was broadly equivalent to that of the standardised guideline OECD 416 and the study was conducted under GLP conditions.

Groups of 30 males and 30 females were exposed by inhalation to 0, 250, 500 or 1000 ppm during the first parent (F0) generation. Thirty males and 30 females were selected from the F1a litters of each treatment group to continue on test as second parent (F1) generation animals. The F1 generation animals were exposed to 0, 250, 500 or 1400 ppm cyclohexanone (increased to 1400 ppm after 1 week of exposure to 1000 ppm). Assessments for neurotoxicologic effects were conducted pre-weaning on one pup from each F1a litter. Twenty-eight of the 0 ppm progeny, 27 of the 250 ppm progeny, 29 of the 500 ppm progeny and 25 of the 1000 ppm progeny were selected for pre-weaning testing. Post-weaning neurologic testing and neuropathologic evaluations were conducted on 20 (10 males and 10 females, survival permitting) F1a progeny per treatment group chosen from those tested pre-weaning.

There were no treatment related effects during the first generation on parental animals, reproduction, or on the F1a pups.

Six of the F1 generation animals exposed to 1400 ppm died (5 males and 1 female). In addition reduced body weight gains were noted in 1400 ppm animals during some periods. All other body weight data obtained for the 250 and 500 ppm animals were similar to the untreated control animals. Furthermore, body weight data recorded for gestating and lactating dams were similar for the treated groups and the untreated control group.

The F1a progeny exposed to 1000 ppm (post-weaning, prior to the selection of the F1 parental animals and subsequent increase to 1400 ppm) exhibited clinical signs such as ataxia, lacrimation, irregular breathing and urine soaked fur following treatment. After the increase to 1400 ppm, and continuing for approximately 3 months, these reactions continued to occur. Starting at week 16 of the F1 generation, the 1400 ppm animals appeared to adapt to treatment with lethargy being the predominant post-exposure reaction. No observations were noted post-exposure during the final 3 weeks of the F1 generation.

During the first 3 weeks of exposure, urine soaked fur was noted post-exposure for 3 to 37% of the animals exposed to 500 ppm. No other noteworthy reactions were seen among the 500 ppm animals. No untoward reactions were seen for the F1 generation animals exposed to 250 ppm.

Statistical analysis of the reproductive indices in the F2a and F2b mating trials revealed no statistical depressions for the test groups when compared to the untreated control group. However, the 1400 ppm male fertility indices, calculated using all males paired were 19.8 and 20.8 percent less than the untreated control males during the F2a and F2b litters, respectively. Also, male fertility calculated including only males which were paired with fertile females (females that conceived litters) were 24.3 to 28.6 percent less than the untreated control males during the F2a and F2b mating trials.

Statistical analyses of the progeny population data revealed significant depressions in the mean numbers of 1400 ppm viable progeny during the F2a and F2b lactation periods. The mean number of progeny born viable by 1400 ppm dams was not statistically reduced; however, in comparison to the untreated control dams, the 1400 ppm dams delivered 23 and 24% fewer viable progeny during the F2a and F2b litters, respectively. Progeny delivery and population data for the 250 and 500 ppm groups during the F2a and F2b litters were similar to the untreated control group.

The percent of 1400 ppm F2a progeny born viable and surviving to lactation days 1 and 4 were significantly less than in the untreated control group. The percentag of F2a progeny surviving during lactation up to day 21 was 14 to 22% less than that of the untreated control, although statistical significance was not achieved. During the F2b litter, progeny survival was significantly less than that of the untreated control progeny at lactation days 1 and 4. Survival of 1400 ppm F2b progeny after lactation day 4 was comparable to that of the untreated control. Survival of the 250 and 500 ppm progeny during the F2a and F2b litters was not altered by maternal exposure to cyclohexanone.

Body weights obtained for the 1400 ppm F2a and F2b progeny were depressed when compared to the untreated control progeny. No body weight reductions were noted for the 250 and 500 ppm F2a and F2b progeny which were considered to be treatment-related. Examination for progeny external morphologic changes revealed no anomalies attributable to maternal cyclohexanone exposure.

Gross and microscopic pathologic examinations of all F1 parental animals and F2a and F2b progeny revealed no treatment-related effects.

In conclusion, inhalation exposure to 1000 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 rat. Evaluation for behavioural/neurotoxicologic development of selected F1a progeny revealed no consistent differences between treated groups and the control group.

The NOAEC values were therefore 1000 ppm (4.01 mg/L) for the F0 generation and 500 ppm (2.01 mg/L) for the F1 and F2 generations.

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
READ-ACROSS CONSIDERATIONS
Cyclohexanol is structurally very similar to cyclohexanone, differing only in the oxidation level of the functional group (secondary alcohol vs. ketone). As expected the similarity in chemical structure is reflected in the similarity of the physico-chemical properties. The low melting points show that both substances could be toxicologically tested as liquids at temperatures slightly above ambient conditions. As expected due to the higher polarity of the hydroxyl group, the boiling point of cyclohexanol is a little higher than that of cyclohexanone and the vapour pressure a little lower. Both substances are slightly soluble in water, which in turn leads to relatively low partition coefficients. The similarity in chemical structure and physico-chemical properties would suggest that the substances should have a similar toxicity profile, and that is supported by studies reported in the literature common to both Cyclohexanone and Cyclohexanol. Both Cyclohexanone and Cyclohexanol are readily absorbed and are reversibly interconvertible in vitro and in vivo. In animals, Cyclohexanol is the major metabolite of Cyclohexanone, which itself clears rapidly from circulation. Both the administration of Cyclohexanone and of Cyclohexanol lead to the excretion of the glucuronic acid conjugate of cyclohexanol. In humans, cyclohexanol is the primary metabolite of cyclohexanone (which is short-lived in circulation), but it is further metabolized to 1,2- and 1,4-cyclohexanediols. The excretion of the latter metabolites is qualitatively and quantitatively independent of the parent compound administered, be it ketone or alcohol. Although it is highly likely that all toxic effects seen upon treatment with Cyclohexanone are in fact caused by its rapidly formed metabolite Cyclohexanol, it cannot be fully excluded that either substance has some additional intrinsic mode of action. If this were the case for Cyclohexanone, read-across from the ketone to the alcohol would lead to an overestimation of the toxicity of the latter. However, it was shown that the ketone is actively formed from Cyclohexanol administered to rabbits, so that it could exert its intrinsic effect even in Cyclohexanol studies. The opposite possibility, specific effects of Cyclohexanol, is fully covered by toxicity studies on Cyclohexanone, since it was shown that peak plasma concentrations of Cyclohexanol are similar, irrespective whether the ketone or the alcohol were administered. Given the reversible conversion of Cyclohexanone into Cyclohexanol, their consistent pattern of toxicokinetics and their identical metabolic fate, it is concluded that the endpoint data on the source substance, Cyclohexanone, are relevant to the human risk assessment on Cyclohexanol, and that the proposed read-across for this endpoint is justified (see read across document attached to Section 13 of the dossier).

Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Dose descriptor:
NOAEC
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
System:
male reproductive system
Organ:
seminiferous tubules
testes
other: epididymides
Key result
Critical effects observed:
no
System:
female reproductive system
Organ:
ovary
uterus
vagina
Key result
Dose descriptor:
NOAEC
Effect level:
500 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
System:
male reproductive system
Organ:
seminiferous tubules
testes
other: epididymides
Key result
Critical effects observed:
no
System:
female reproductive system
Organ:
ovary
uterus
vagina
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
500 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Reproductive effects observed:
yes
Lowest effective dose / conc.:
4 100 mg/m³ air (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Conclusions:
Read-across from cyclohexanone to cyclohexanol is considered valid for the reproductive toxicity endpoints as well as classification and labelling. Inhalation exposure to 1000 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 rat. Evaluation for behavioral/neurotoxicologic development of selected progeny revealed no consistent differences between treated groups and the control group. The NOAEC values were therefore 1000 ppm (4.1 mg/L) for the F0 generation and 500 ppm (2.04 mg/L) for the F1 and F2 generations.
Executive summary:

A reproduction toxicity study was conducted to ascertain the potential effects of inhalation exposure to cyclohexanone vapour upon growth, development, and reproductive performance of 2 consecutive generations of CD® Sprague Dawley derived albino rats. The method was broadly equivalent to that of the standardised guideline OECD 416 and the study was conducted under GLP conditions.

There were no treatment related effects during the first generation on parental animals, reproduction, or on the F1a pups. Six of the F1 generation animals exposed to 1400 ppm died (5 males and 1 female). In addition reduced body weight gains were noted in 1400 ppm animals during some periods. All other body weight data obtained for the 250 and 500 ppm animals were similar to the untreated control animals. Furthermore, body weight data recorded for gestating and lactating dams were similar for the treated groups and the untreated control group.

The F1a progeny exposed to 1000 ppm (post-weaning, prior to the selection of the F1 parental animals and subsequent increase to 1400 ppm) exhibited clinical signs such as ataxia, lacrimation, irregular breathing and urine soaked fur following treatment. After the increase to 1400 ppm, and continuing for approximately 3 months, these reactions continued to occur. Starting at week 16 of the F1 generation, the 1400 ppm animals appeared to adapt to treatment with lethargy being the predominant post-exposure reaction. No observations were noted post-exposure during the final 3 weeks of the F1 generation.

During the first 3 weeks of exposure, urine soaked fur was noted post-exposure for 3 to 37% of the animals exposed to 500 ppm. No other noteworthy reactions were seen among the 500 ppm animals. No untoward reactions were seen for the F1 generation animals exposed to 250 ppm.

Statistical analysis of the reproductive indices in the F2a and F2b mating trials revealed no statistical depressions for the test groups when compared to the untreated control group. However, the 1400 ppm male fertility indices, calculated using all males paired were 19.8 and 20.8 percent less than the untreated control males during the F2a and F2b litters, respectively. Also, male fertility calculated including only males which were paired with fertile females (females that conceived litters) were 24.3 to 28.6 percent less than the untreated control males during the F2a and F2b mating trials.

Statistical analyses of the progeny population data revealed significant depressions in the mean numbers of 1400 ppm viable progeny during the F2a and F2b lactation periods. The mean number of progeny born viable by 1400 ppm dams was not statistically reduced; however, in comparison to the untreated control dams, the 1400 ppm dams delivered 23 and 24% fewer viable progeny during the F2a and F2b litters, respectively. Progeny delivery and population data for the 250 and 500 ppm groups during the F2a and F2b litters were similar to the untreated control group.

The percent of 1400 ppm F2a progeny born viable and surviving to lactation days 1 and 4 were significantly less than in the untreated control group. The percentag of F2a progeny surviving during lactation up to day 21 was 14 to 22% less than that of the untreated control, although statistical significance was not achieved. During the F2b litter, progeny survival was significantly less than that of the untreated control progeny at lactation days 1 and 4. Survival of 1400 ppm F2b progeny after lactation day 4 was comparable to that of the untreated control. Survival of the 250 and 500 ppm progeny during the F2a and F2b litters was not altered by maternal exposure to cyclohexanone.

Body weights obtained for the 1400 ppm F2a and F2b progeny were depressed when compared to the untreated control progeny. No body weight reductions were noted for the 250 and 500 ppm F2a and F2b progeny which were considered to be treatment-related. Examination for progeny external morphologic changes revealed no anomalies attributable to maternal cyclohexanone exposure.

Gross and microscopic pathologic examinations of all F1 parental animals and F2a and F2b progeny revealed no treatment-related effects.

In conclusion, inhalation exposure to 1000 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 rat. Evaluation for behavioural/neurotoxicologic development of selected F1a progeny revealed no consistent differences between treated groups and the control group.

The NOAEC values were therefore 1000 ppm (4.1 mg/L) for the F0 generation and 500 ppm (2.04 mg/L) for the F1 and F2 generations.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Quality of whole database:
No reliable information on the effect on fertility via the oral route is available for cyclohexanol or cyclohexanone.
Effect on fertility: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
2 007 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Reliable information on the effect on fertility via the inhalation route is available for cyclohexanone that is appropriate for hazard and risk characterisation and the purpose of classification and labelling.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
No reliable information on the effect on fertility via the dermal route is available for cyclohexanol or cyclohexanone.
Additional information

DESCRIPTION OF KEY INFORMATION

Relevant and reliable information on potential effects of repeated inhalation exposure to cyclohexanone on the fertility in the rat from a 2-generation reproductive toxicity study is used to cover the endpoint of reproductive toxicity of cyclohexanol, based on a robust read-across argumentation. The inhalation route is anticipated to be most relevant with regards to human exposure to the substance based on the physical-chemical properties (vapour pressure of about 82 Pa) and resulting from its identified uses (e.g. as a co-formulant in plant protection products used by spray application).

Inhalation route of exposure

Information on the effects of cyclohexanone on the fertility of rats is available from a reliable 2-generation inhalation reproductive toxicity study in the rat (Mayhew 1986). Male and female rats were exposed to vapours of cyclohexanone by inhalation for 6 hours per day for 5 days per week (females were exposed for 7 days per week during certain study periods, female exposure was interrupted for a short period during late gestation and early lactation phase) over a period of about 16 weeks. Inhalation exposure at the highest tested dose of 1400 ppm (5620 mg/m3) through one generation resulted in exposure related pharmacokinetic reactions, decreased male body weights, reduced male reproductive performance, reduced progeny survival and progeny body weight reductions. Inhalation exposure to 1000 ppm (4014 mg/m3) through one generation and to 250 or 500 ppm (1004 or 2007 mg/m3) through two consecutive generations did not adversely affect the reproductive performance, sexual, behavioural and neurotoxicologic development of exposed rats or their progeny. Microscopic histological examinations of the male and female reproductive organs did not reveal any dose-related changes in the high-dose groups (1000 ppm in the first parent generation, 1400 ppm in the second parent generation). An assessment of male reproductive performance during a post exposure recovery period of males from the second generation revealed full reversibility of the depressions in the fertility index of the males previously exposed to 1400 ppm. The no-effect concentration for structural changes in the male and female reproductive organs of 1400 ppm (5620 mg/m3) can be converted for purposes of comparison, into an equivalent oral dose of 1630 mg/kg bw/day following the approach given in ECHA guidance R.8 (ECHA 2012, version 2.1) and assuming 100% availability via inhalation and oral routes and the physiological parameters for the rat given in Table R.8-2 in the ECHA guidance.

The reduction in the male reproductive performance may have resulted from the systemic toxicity of cyclohexanone at the highest dose, which consisted of ataxia, lacrimation, irregular breathing and urine soaked fur following treatment and persisted as lethargy as the predominant post-exposure reaction after adaptation to exposure. Lethargy is likely to be a sign of a narcotic effect of cyclohexanone, which was seen also in other toxicological studies and may have negatively affected the mating behaviour leading to reduced male reproductive performance.

The effects described in the 2-generation study with cyclohexanone were similar to the effects observed in an unpublished study with cyclohexanol conducted to the OECD TG 422 (Newton 2005). A summary of the report has been published in the US HPV Programme, but the registrant has no access to the full study report. No robust study summary has been written and included in the registration dossier for this reason. However, a short description of the study derived from the published HPV summary has been included here for completeness and to provide all relevant data, in accordance with Annex I requirements for Chemical Safety Report. The study is not used to conclude on the endpoint of reproductive toxicity of cyclohexanol. The screening study investigated the effects of cyclohexanol administered by inhalation to rats at doses of 0, 50, 150 and 450 ppm (0, 205, 614, 1843 mg/m3) for a period of approximately 16 weeks. At the highest dose level of 450 ppm, two of eleven pregnancies (18.2%) did not result in viable pups at parturition. Mean pup body weights were reduced at birth and 4 days after birth in the high-dose group. Male rats in the high-dose group showed a reduction in testicular sperm count at the completion of the exposure period, which was not statistically significant and within the range of historical controls for the tested rat strain. No adverse dose-related effects were seen during premating, gestation or lactation on reproductive and fertility parameters, litter size, pup sex ratios and pup survival to lactation day 4. Furthermore, histopathological, microscopic examination of male and female organs and tissues did not reveal any dose-related changes in the group exposed to 450 ppm. The overall NOEL in this reproductive toxicity screening study with cyclohexanol was considered to be 150 ppm (614 mg/m3) based on the slightly increased incidence of pregnancies with no viable pups at parturition. The no-effect concentration for structural changes in the male and female reproductive organs of 450 ppm (1843 mg/m3) can be converted for purposes of comparison, into an equivalent oral dose of 534 mg/kg bw/day following the approach given in ECHA guidance R.8 (ECHA 2012, version 2.1) and assuming 100% availability via inhalation and oral routes and the physiological parameters for the rat given in Table R.8-2 in the ECHA guidance.

Conclusion for the inhalation route: The inhalation route of exposure is regarded as most important with regards to human exposure to cyclohexanol. Relevant and reliable information on cyclohexanone is used to cover the endpoint of reproductive toxicity, based on a robust read-across. Repeated inhalation exposure of rats to cyclohexanone over a period of several months did not cause adverse effects in the reproductive organs of males or females up to the highest dose of 5620 mg/m3 in a 2-generation study (Mayhew 1986). The reduced male reproductive performance in animals exposed to 5620 mg/m3 is likely to be due to a narcotic effect of cyclohexanone at this high dose. Signs of toxicity in the high-dose group consisted of exposure related pharmacokinetic reactions, lethargy, decreased male body weights, reduced progeny survival and progeny body weight reductions and the NOEL for these effects over two generations in this study was 2007 mg/m3.

Oral route of exposure

A non-guideline, non-GLP 2-generation study investigating the potential reproductive effects of oral exposure to cyclohexanol administered to mice at a concentration of 1% in the diet was published (Gondry 1972). This concentration corresponds to an oral dose of approximately 1200-1300 mg/kg bw/day, using the parameters for dose calculation given in Table R.8-17 in ECHA guidance R.8 (ECHA 2012, version 2.1). The study is of limited quality and rated as unreliable due to missing characterisation of the test substance (no source or purity given) and a low reporting level concerning methods and results. No adverse effects on the testes or the reproductive performance were reported. A statistically significant increase in the mortality and reduction in the body weight of the offspring in the first and second generations occurred during the 21 days after birth. The reduced body weight gain persisted when cyclohexanol was administered to five or six successive generations, but as soon as administration was ceased, the following generations experienced normal growth.

Inconsistent and contradicting information on potential effects of oral exposure to cyclohexanol on the male reproductive organs is available from two published repeated-dose toxicity studies (Lake et al. 1982, Dixit et al. 1980), for details refer to the section on repeated dose toxicity. Both studies are non-guideline, non-GLP and rated as unreliable due to missing characterisation of the test substance (no purity given, source missing in the Dixit et al. publication) and poor documentation of test conditions and results (e.g. only one dose tested, number of exposure days per week not given). In the first study, potential effects of oral exposure to a dose of 455 mg cyclohexanol/day over a period of 7 days were studied in the rat (Lake et al. 1982). Information on mortality or clinical signs is lacking. The weight of the testes of male rats was not significantly changed, whereas the relative weight of the liver was significantly increased. Histological examinations under the microscope were performed on fixed slices of the testes, but no results of this examination or adverse effects resulting from oral exposure to cyclohexanol were reported.

A second publication by Dixit et al. (1980) studied the effects of exposure to cyclohexanol in five male rabbits receiving a repeated oral dose of 25 mg/kg body weight/day over a period of 40 days. Information on mortality or clinical signs is lacking. The paper describes a significant reduction in the relative weights of testes and epididymides and an inhibition of the spermatogenesis in the exposed male rabbits. A microscopic evaluation of the reproductive organs of the treated animals in this study revealed that the Leydig cells were shrunken and the diameters of their nuclei were reduced. Spermatogonia, spermatocytes and spermatids were missing in the seminiferous tubules, and changes in certain testicular and epididymal biochemical parameters were noted (such as protein and RNA concentration). The majority of measured parameters were restored and spermatogenesis was normal in a second group of male rabbits after a recovery period of 70 days without exposure to cyclohexanol.

Conclusion for the oral route: The oral route of exposure is considered to be of less relevance for human exposure to cyclohexanol. The published non-reliable 2-generation study (Gondry 1972) did not reveal adverse effects on the testes or reproductive performance of treated mice receiving cyclohexanol in their diet at a concentration of 1% (about 1200 to 1300 mg/kg bw/day). Inconsistent and contradicting information on potential adverse effects of oral exposure to cyclohexanol on the male reproductive organs from two published non-guideline repeated dose toxicity studies (Lake et al. 1982, Dixit et al. 1980) is available. This information is unreliable and will not be used in the chemical safety assessment.

Dermal route of exposure

No information available

Other routes of exposure

Literature data on the potential effects on male fertility via subcutaneous injection are available (Tyagi et al. 1979) coming from the same work group studying effects resulting from oral exposure to cyclohexanol (Dixit et al. 1980). The subcutaneous route of test material administration is not appropriate for assessing the toxicity of a chemical in a standardised way and the study was disregarded. A daily subcutaneous dose of 15 mg/kg bw/day for a period of 21 days was administered to male gerbils, and male rats were treated in a similar way for 37 days. The observed effects in both species resemble very much the effects observed in the study by Dixit et al. (1980) on oral effects of cyclohexanol, in that relative weights of testes and epididymides were significantly reduced and spermatogenesis was impaired.

Effects on developmental toxicity

Description of key information

Oral route:

Rabbits: NOAEL (development) = 500 mg/kg bw/day (based on cyclohexanone)

Rabbits: NOAEL (maternal) = 250 mg/kg bw/day (based on cyclohexanone)

Inhalation route:

Rats: NOAEC (development) = 1400 ppm (based on cyclohexanone), corresponds to 5620 mg/m3

Rats: NOAEC (foetal) = 650 ppm (based on cyclohexanone), corresponds to 2609 mg/m3

Rats: NOAEC (maternal) = 650 ppm (based on cyclohexanone), corresponds to 2609 mg/m3

Dermal route:

no information available

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 March 1993 to 05 July 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
Himalayan
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Karl Thomae, Biberach an der Riss, Federal Republic of Germany
- Strain: Chbb:HM (outbred strain)
- Age at study initiation: At Day 0, the does were between 22 and 37 weeks old (sexually mature).- Weight at study initiation: At Day 0 mean body weight was approximately 2641 g.
- Housing: Singly in type 12.2395.C stainless steel wire mesh cages (Draht Bremer GmbH, Marktheidenfeld, Federal Republic of Germany) with floor area of about 3000 cm². The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet: pelleted Kliba maintenance diet type 23-341-4 for rabbits, supplied by Klingentalmühle AG, Kaiseraugst, Switzerland, ad libitum throughout the study from the day of supply to the day of necropsy.
- Water: Water of tap water quality was provided in water bottles available ad libitum throughout the study from the day of supply to the day of necropsy.- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: Central air conditioning guaranteed a range of temperature of 20 - 24 °C. There were no deviations from these limits.
- Humidity: Relative humidity of 30 - 70%. There were no deviations from these limits.
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours of light from 06:00 to 18:00 hours and 12 hours of darkness from 18:00 to 06:00 hours).

IN-LIFE DATES
- From: 22 March 1993
- To: 01 June 1993
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(doubly distilled)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Each day the test material solutions were freshly prepared shortly before the test material was administered. For the preparation of the solutions, an appropriate amount of test material was weighed in a volumetric flask, subsequently topped up with doubly distilled water and intensively shaken.

VEHICLE
- Concentration in vehicle: 0, 500, 2500 and 5000 mg/100 mL for the 0, 50, 250 and 500 mg/kg dose levels, respectively.
- Amount of vehicle: The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 7 post insemination (p.i.)).
- Dose volume: 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSES
- All analyses were carried out at the analytical department of the testing facility.

ANALYSES OF THE TEST MATERIAL
- Analytical determinations of the purity and the homogeneity of the test material itself were carried out before the beginning of the study by GC. The stability of the test material over the study period has been proven by reanalysis.The homogeneity of the test material was proven by visual inspection.

ANALYSES OF THE SOLUTIONS OF TEST MATERIAL
- Analytical verifications of the stability of the test material 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. The test material solutions were analysed by GC. As the test material preparations were true solutions, the homogeneity did not need to be proven analytically.
Details on mating procedure:
- Impregnation procedure: Artificial insemination after an acclimatisation period of at least 5 days, whereby 0.2 mL of a synthetic hormone (Receptal®) 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.The day of insemination was designated as day 0 (beginning of the study) and the following day as day 1 p.i.
Duration of treatment / exposure:
Day 7 to Day 19 post insemination (p.i.)
Frequency of treatment:
Once daily during the period of major organogenesis
Duration of test:
29 days
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control receiving distilled water.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The selection of doses for the present examination was based on the results of a preceding range-finding study in Himalayan rabbits in which the test material was administered as an aqueous solution to 4 - 5 pregnant female rabbits/group by stomach tube in doses of 50, 150 and 450 mg/kg body weight on day 7 through day 19 post insemination. A standard dose volume of 10 mL/kg body weight was used. The control group was dosed with the vehicle only (doubly distilled water). In this preliminary study, the test material caused slight signs of maternal toxicity at 450 mg/kg body weight, but induced no material-related effects on the does of the low and the intermediate dose groups (50 and 150 mg/kg body weight/day). There were no test material-related effects on the gestational parameters in any of the groups and the foetuses showed no malformations. Taking this into consideration, the doses were chosen for the full-scale prenatal toxicity study in Himalayan rabbits.- Rationale for animal assignment: Animals were assigned to the different test groups according to a randomisation plan and on the basis of their body weights.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check was made twice daily on working days or once daily (Saturday, Sunday or on public holidays) on days 0 - 29 p.i.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0- 29 p.i.).

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 2, 4, 7, 9, 11, 14, 16, 19, 21, 23, 25 and 29 p.i. The body weight change of the animals was calculated from these results.Corrected body weight gain (net maternal body weight change) was calculated after terminal sacrifice (terminal body weight on day 29 p.i. minus weight of the uterus before it was opened minus body weight on day 7 p.i.).

FOOD CONSUMPTION: Yes
-The consumption of food was determined daily during the entire study period.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 29 p.i.- Organs examined: The surviving dams were sacrificed in randomised order by intravenous injection of a pentobarbital and the foetuses were removed from the uterus.Dams that died intercurrently as well as the contents of the uterus from these animals were examined, if possible, in the same way as at terminal sacrifice (with the exception of uterus weight). After the dams had been sacrificed, they were necropsied and assessed by gross pathology.
Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
- Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes; only decidual or placental tissues visible or according to Salewski from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy.
- Number of late resorptions: Yes; embryonic or foetal tissue in addition to placental tissue visible.
- Other: Dead foetuses; hypoxemic foetuses which did not breathe spontaneously after the uterus had been opened.
Fetal examinations:
- External examinations: Yes: Each foetus was weighed and examined macroscopically for any external findings. The viability of the foetuses and the condition of the placentae, umbilical cords, foetal membranes and fluids were examined. Individual placental weights were recorded.
- Soft tissue examinations: Yes: After the foetuses had been sacrificed by CO2, the abdomen and thorax were opened in order to be able to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to assess the internal structure. The sex of the foetuses was determined by internal examination of the gonads.
- Skeletal examinations: Yes: After fixation in ethyl alcohol the skeletons, with the possible exception of the skulls, were stained according to a modified method of Dawson. The stained skeletons were placed on an illuminated plate and examined, evaluated and assessed. After the examination the stained skeletons were retained by litter.
- Head examinations: Yes: After skinning, all foetuses (if required without heads) were fixed in ethyl alcohol. After fixation for approx. 1 - 5 days, the foetuses were removed from the fixative for a short while and a cross section of the heads from all intact foetuses was made in the parietal bone area using a scalpel. The two halves of the heads were carefully bent to allow a thorough examination of the brain. Subsequently, the foetuses were placed back into the fixative for further fixation.If heads of foetuses revealed severe findings (e.g. anophthalmia, microphthalmia, hydrocephalus or cleft palate), the heads of these foetuses were severed from the trunk, fixed in Bouin's solution and later processed and assessed according to Wilson's method.About 10 transverse sections were prepared per head. After the examination the heads treated in this way were discarded.
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 various parameters.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 foetal 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 foetuses with malformations, variations, retardations and/or unclassified observations in each litter.For the parameter “food consumption”, the “mean of means” was calculated. The “mean of means” values allow a rough estimation of the total food consumption during the different time intervals (pre-treatment, treatment and post-treatment period); they are not exactly precise values because the size of the intervals taken for calculation differs. For the “mean of means” values no statistical analysis was performed.
Indices:
Calculation of conception rate and pre- and post-implantation losses were carried out.Conception rate (%) = (number of pregnant animals / number of fertilised animals) x 100Pre-implantation loss (%) = ((number of corpora lutea – number of implantations) / number of corpora lutea) x 100Post-implantation loss (%) = ((number of implantations – number of live foetuses) / number of implantations) x 100
Historical control data:
Yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All high dose animals showed unsteady gait about 2 - 4 hours after the daily gavaging during the first 3 days of the treatment period. On the following days (days 10 and 11 p.i.) unsteady gait occurred only in some of the high dose females. This finding has to be related to the test material administration. No defecation was recorded for high dose female No. 55 on days 19 and 20 p.i.; this doe consumed only minimal amounts of food the days before.
Mortality:
no mortality observed
Description (incidence):
One mis-gavaged doe of the control group (No. 10) was found dead on day 21 p.i.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically significant differences between the controls and the test material-treated does concerning mean body weights. If the mean body weight gain over the whole treatment period (Days 7 - 19 p.i.) is calculated, the high dose females reached only about 25% of the body weight gain of the control group. This corresponds to the reduced food intake of the high dose dams and is therefore assessed as a test material-induced effect. During the post-treatment period (Days 19 - 29 p.i.) the increased food consumption of the 500 mg/kg dams resulted in a statistically significantly increased weight gain.The weight gains of the dams of test groups 1 and 2 (50 or 250 mg/kg body weight/day) did not show any statistically significant differences in comparison to the controls. In the absence of test material-related effects on the food consumption of these dams, all differences in respect to body weight change are considered to be spontaneous in nature.The results of the corrected body weight gain (terminal body weight on day 29 p.i. minus weight of the uterus before it was opened minus body weight on day 7 p.i.) do not show any differences of biological relevance between the groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption of the 500 mg/kg dams was statistically significantly reduced during the first days of the treatment period. The consumption of approximately 15% less of the diet in the high dose group in comparison to the controls during the treatment period is assessed as a clear substance-related effect.Food consumption of dams of test groups 1 and 2 (50 and 250 mg/kg body weight/day) was uninfluenced by the test substance administration.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no substantial differences concerning the uterus weights between the controls and test groups 1, 2 or 3 (50, 250 and 500 mg/kg body weight/day). All these values lie within the range of biological variation. No other organ weights were reported.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy none of the does exposed to the test substance showed any substance-induced lesions. Only some spontaneous necropsy findings were recorded for single animals of all dose groups, including the controls.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
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:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
Details on maternal toxic effects: Only pregnant dams were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant dams with scheduled sacrifice (day 29 p.i.) were taken for the calculation of mean gravid uterine weights, mean net maternal body weight change (corrected body weight gain) and summary of reproduction data.In this study, the following females were partially or totally excluded from the above mentioned calculations:
- Test group 0 (Control):
- Female No. 11790 (6) - not pregnant
- Female No. 11886 (10) - died intercurrently

- Test group 3 (500 mg/kg body weight/day):
- Female No. 11300 (49) - not pregnant
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
not specified
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
One control and one intermediate dose foetus each showed external malformations. The control foetus showed kleft palate and kinky tail; the intermediate dose foetus showed cheiloschisis. These external malformations occur sporadically in control foetuses. Furthermore, no dose-response relationship was given. The findings in this study are considered to be spontaneous in nature.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Malformations of the foetal skeletons were noted for 2.7% of control foetuses (occurring in 15% of the litters) and 2.5% of the foetuses of the intermediate dose group (occurring in 13% of the litters). Skeletal malformations were related to the vertebral column (cervical and lumbar vertebrae fused and/or of irregular shape), deformed scapulae, severely fused sternebrae (bony plate) and the hindlimbs (bent with involvement of bones). None of the low and the high dose groupss showed skeletal malformations. Skeletal variations were observed, which included splitting of skull bones, epactal bone between nasal and frontal bones, accessory 13th ribs, shortened or absent 12th ribs, rudimentary cervical ribs, accessory lumbar vertebra and sternebrae of irregular shape, fused or accessory sternebra or bipartite sternebra. All of the described skeletal variations appeared without any relation to dosing and/or without statistically significant differences between the control group and the substance-treated groups.Skeletal retardations (incomplete or missing ossification of skull bones, vertebral column, sternebrae and talus) occurred in all groups, including the controls, at a comparable frequency. All differences between the groups concerning foetal skeletal retardations are withouth biological relevance. This includes the statistically significantly higher litter incidence of incomplete ossified or smaller sternebrae at 50 and 500 mg/kg body weight/day. For this finding, which appears in the historical control data at even higher litter incidence, no clear dose-response relationship was found.
Visceral malformations:
no effects observed
Description (incidence and severity):
The examination of the organs of the foetuses revealed several types of soft tissue malformations in foetuses of the control and the intermediate dose group. Malformations consisted of hydrocephaly, septal defect, hypoplastic thymus, malformations of the great vessels, agenesia of the gallbladder and hypoplastic kidneys. No soft tissue malformations occurred in the 50 or 500 mg/kg body weight/day dose groups. Because no relation to dosing is given and because most soft tissue malformations were also present at low incidence in the historical control data, the recorded visceral findings were considered to be spontaneous in nature.
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
There were no dose-related foetal abnormalities.
Key result
Developmental effects observed:
no

Table 1: Mean Maternal Food Consumption During Gestation (g/animal/day)

Days

Parameter

Test Group 0

Control

Test Group 1

50 mg/kg bw/day

Test Group 2

250 mg/kg bw/day

Test Group 3

500 mg/kg bw/day

0 to 1

Mean

118.4D

124.9

122.2

118.9

1 to 2

Mean

119.2D

131.8

124.9

125.1

2 to 3

Mean

121.8D

133.3

126.9

122.7

3 to 4

Mean

123.7D

135.1

129.0

126.3

4 to 5

Mean

127.2D

135.9

132.6

127.0

5 to 6

Mean

120.7D

126.3

128.3

122.2

6 to 7

Mean

121.4D

131.6

121.4

120.9

7 to 8

Mean

112.3D

131.6*

105.2

60.1**

8 to 9

Mean

110.8D

129.2*

105.9

75.0**

9 to 10

Mean

110.5D

122.8

111.6

89.1**

10 to 11

Mean

111.2D

119.4

109.6

97.6

11 to 12

Mean

109.5D

118.4

108.4

95.8

12 to 13

Mean

107.4D

117.6

106.9

94.2

13 to 14

Mean

103.0D

112.4

105.6

92.5

14 to 15

Mean

96.4D

115.4

96.1

86.1

15 to 16

Mean

98.3D

113.5

94.8

89.7

16 to 17

Mean

93.0D

120.2

98.8

91.2

17 to 18

Mean

102.9D

124.5

105.4

102.0

18 to 19

Mean

98.8D

122.2

110.0

97.5

19 to 20

Mean

93.2D

126.9*

101.0

101.3

20 to 21

Mean

98.9D

118.5

113.1

112.0

21 to 22

Mean

99.4D

118.9

106.5

112.0

22 to 23

Mean

100.8D

117.5

104.2

118.0

23 to 24

Mean

102.9D

117.4

105.6

119.3

24 to 25

Mean

113.4D

119.5

112.0

121.8

25 to 26

Mean

117.0D

120.2

114.2

122.3

26 to 27

Mean

118.9D

123.6

115.7

127.5

27 to 28

Mean

113.7D

111.2

113.8

121.1

28 to 29

Mean

118.7D

124.4

107.9

130.6

D = Dunnett-test (two-sided)

*p0.05

**p0.01

Table 2: Mean Maternal Body Weight Change During Gestation (g)

Days

Parameter

Test Group 0

Control

Test Group 1

50 mg/kg bw/day

Test Group 2

250 mg/kg bw/day

Test Group 3

500 mg/kg bw/day

0 to 7

Mean

63.4D

68.2

68.7

56.5

7 to 19

Mean

70.9D

99.6

33.1

18.0

19 to 29

Mean

132.8D

190.3

147.5

198.9*

0 to 29

Mean

268.9D

358.1

249.2

273.4

D = Dunnett-test (two-sided)

*p0.05

Conclusions:
Under the conditions of this study, the NOAEL for maternal toxicity was determined to be 250 mg/kg/day based on systemic effects and the NOAEL for teratogenicity/developmental toxicity was 500 mg/kg/day as no test material-related effects were observed.
Executive summary:

A study was conducted to investigate the potential developmental toxicity of cyclohexanone to rabbits via the oral route. The study was conducted in accordance with the standardised guidelines OECD 414 and EU Method B.31 under GLP conditions.

The test material was administered as an aqueous solution to 15 artificially inseminated female Himalayan rabbits/group (Chbb:HM (outbred strain)) by oral gavage at doses of 0, 50, 250 and 500 mg/kg body weight on Day 7 through to Day 19 post-insemination (p.i.). A standard dose volume of 10 mL/kg body weight was used. The control group was dosed with the vehicle only (doubly distilled water).

The state of health of the animals was checked each day. Food consumption and body weights of the animals were recorded regularly throughout the study period. On Day 29 p.i., all surviving females were sacrificed and assessed by gross pathology. The foetuses were dissected from the uterus, sexed, weighed and investigated for any external, soft tissue and skeletal findings.

At 50 and 250 mg/kg bw/day there were no test material-related effects on does, gestational parameters or foetuses. At 500 mg/kg bw/day statistically significantly reduced food consumption (about 15% less than the controls) during the treatment period was observed. Body weight loss at the beginning of the treatment period was also observed; weight gain during the treatment period was only 25% of the control animals. All the animals in this group were also observed to have an unsteady gait 2 – 4 hours after daily gavaging during the first treatment days.

Thus, the test material caused some overt signs of maternal toxicity at 500 mg/kg body weight/day, but was not toxic to the does at 50 and 250 mg/kg body weight/day.

There were no test material-related adverse effects on the gestational parameters and on the foetuses up to and including the highest dose level (500 mg/kg body weight/day); at all dose levels no indications for test material-induced teratogenic effects were observed.

Under the conditions of this study, the NOAEL for maternal toxicity was determined to be 250 mg/kg/day based on systemic effects. The NOAEL for teratogenicity and developmental toxicity was 500 mg/kg/day as no test material-related effects were observed. The read-across from cyclohexanone to dyclohexanol is considered to be valid for the characterisation of the developmental/teratogenicity toxicity endpoints. The results of this study with cyclohexanone are considered to be valid for classification and labelling purposes.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
READ-ACROSS CONSIDERATIONS
Cyclohexanol is structurally very similar to cyclohexanone, differing only in the oxidation level of the functional group (secondary alcohol vs. ketone). As expected the similarity in chemical structure is reflected in the similarity of the physico-chemical properties. The low melting points show that both substances could be toxicologically tested as liquids at temperatures slightly above ambient conditions. As expected due to the higher polarity of the hydroxyl group, the boiling point of cyclohexanol is a little higher than that of cyclohexanone and the vapour pressure a little lower. Both substances are slightly soluble in water, which in turn leads to relatively low partition coefficients. The similarity in chemical structure and physico-chemical properties would suggest that the substances should have a similar toxicity profile, and that is supported by studies reported in the literature common to both Cyclohexanone and Cyclohexanol. Both Cyclohexanone and Cyclohexanol are readily absorbed and are reversibly interconvertible in vitro and in vivo. In animals, Cyclohexanol is the major metabolite of Cyclohexanone, which itself clears rapidly from circulation. Both the administration of Cyclohexanone and of Cyclohexanol lead to the excretion of the glucuronic acid conjugate of cyclohexanol. In humans, cyclohexanol is the primary metabolite of cyclohexanone (which is short-lived in circulation), but it is further metabolized to 1,2- and 1,4-cyclohexanediols. The excretion of the latter metabolites is qualitatively and quantitatively independent of the parent compound administered, be it ketone or alcohol. Although it is highly likely that all toxic effects seen upon treatment with Cyclohexanone are in fact caused by its rapidly formed metabolite Cyclohexanol, it cannot be fully excluded that either substance has some additional intrinsic mode of action. If this were the case for Cyclohexanone, read-across from the ketone to the alcohol would lead to an overestimation of the toxicity of the latter. However, it was shown that the ketone is actively formed from Cyclohexanol administered to rabbits, so that it could exert its intrinsic effect even in Cyclohexanol studies. The opposite possibility, specific effects of Cyclohexanol, is fully covered by toxicity studies on Cyclohexanone, since it was shown that peak plasma concentrations of Cyclohexanol are similar, irrespective whether the ketone or the alcohol were administered. Given the reversible conversion of Cyclohexanone into Cyclohexanol, their consistent pattern of toxicokinetics and their identical metabolic fate, it is concluded that the endpoint data on the source substance, Cyclohexanone, are relevant to the human risk assessment on Cyclohexanol, and that the proposed read-across for this endpoint is justified (see read across document attached to Section 13 of the dossier).

Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
There were no dose-related foetal abnormalities.
Key result
Developmental effects observed:
no
Conclusions:
Read-across from cyclohexanone to cyclohexanol is considered valid for the developmental toxicity endpoints as well as classification and labelling. Under the conditions of this study, the NOAEL for maternal toxicity was determined to be 250 mg/kg/day based on systemic effects and the NOAEL for teratogenicity/developmental toxicity was 500 mg/kg/day as no test material-related effects were observed.
Executive summary:

A study was conducted to investigate the potential developmental toxicity of cyclohexanone to rabbits via the oral route. The study was conducted in accordance with the standardised guidelines OECD 414 and EU Method B.31 under GLP conditions.

The test material was administered as an aqueous solution to 15 artificially inseminated female Himalayan rabbits/group (Chbb:HM (outbred strain)) by oral gavage at doses of 0, 50, 250 and 500 mg/kg body weight on Day 7 through to Day 19 post-insemination (p.i.). A standard dose volume of 10 mL/kg body weight was used. The control group was dosed with the vehicle only (doubly distilled water).

At 50 and 250 mg/kg bw/day there were no test material-related effects on does, gestational parameters or foetuses. At 500 mg/kg bw/day statistically significantly reduced food consumption (about 15% less than the controls) during the treatment period was observed. Body weight loss at the beginning of the treatment period was also observed; weight gain during the treatment period was only 25% of the control animals. All the animals in this group were also observed to have an unsteady gait 2 – 4 hours after daily gavaging during the first treatment days.

Thus, the test material caused some overt signs of maternal toxicity at 500 mg/kg body weight/day, but was not toxic to the does at 50 and 250 mg/kg body weight/day.

There were no test material-related adverse effects on the gestational parameters and on the foetuses up to and including the highest dose level (500 mg/kg body weight/day); at all dose levels no indications for test material-induced teratogenic effects were observed.

Under the conditions of this study, the NOAEL for maternal toxicity was determined to be 250 mg/kg/day based on systemic effects. The NOAEL for teratogenicity and developmental toxicity was 500 mg/kg/day as no test material-related effects were observed. The read-across from cyclohexanone to dyclohexanol is considered to be valid for the characterisation of the developmental/teratogenicity toxicity endpoints. The results of this study with cyclohexanone are considered to be valid for classification and labelling purposes.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 November 1983 to 06 August 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was not conducted to GLP. Klimisch 2 rating was considered appropriate as the study was conducted to US EPA Test Guidelines available at the time of conduct and because documentation of the test and the results is very detailed.
Qualifier:
according to
Guideline:
other: U.S. EPA "Health Effects Test Guidelines" (Teratogenicity Study, August 1982)
Deviations:
no
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts, United States of America
- Strain: CD® (Sprague-Dawley derived)
- Age at study initiation:females were 83 days old at initiation of mating
- Mean weight on Day 0 of gestation: 273.5 g
- Housing: During exposure, animals were housed individually in stainless steel wire mesh cages. During the non-exposure periods, females were housed 2/cage during the first week of the acclimation period and individually (except during mating) thereafter in stainless steel, suspended cages with wire mesh floors.- Diet: Purina(r) Certified Rodent Chow No. 5002, mash form, ad libitum
- Water: tap water, Elizabethtown Water Company, presented by automated watering system, ad libitum (not available during exposure)
- Acclimation period: 14 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature: 66 to 76 °F; the temperature in the exposure chambers was specified to be 71 ± 5 °F (19 to 24 °C)
- Humidity: 40 to 60%; the relative humidity in the exposure chambers was specified to be 50 ± 10%
- Photoperiod: 12 hour light/dark cycle (7 a.m. to 7 p.m.) via automatic timer

IN-LIFE DATES
- From: 01 November 1983To: 06 through 09, 12 through 16, 19 and 20 December 1983
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
nose/head only
Remarks:
Administration of substance occurred in the breathing zone
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Glass and stainless steel chambers with a total volume of 1 cubic metre.
- Source and rate of air: Air was drawn from the room.- Method of conditioning air: Air was preconditioned using filters and humidifiers.
- System of generating particulates/aerosols: Room air was supplied to the exposure chamber which housed the control animals. For the 300, 600 and 1400 ppm dose groups a modified Laskin nebuliser (one, two and three barrel, respectively) was placed in the centre of a 1000 mL three-necked flask. The nebulizer was connected to an 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 nebuliser was pressurised with houseline air at approximately 9, 8 and 19 psi back pressure for the 300, 600 and 1400 ppm dose groups, respectively, using a Matheson pressure gauge.In addition, a flow meter was used to assure the nebuliser 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 vapour, was drawn into the top inlet port of the 1 m³ inhalation chamber. Animals remained in the chamber for approximately 1/2 an hour following exposure to allow chamber clearing.- Temperature, humidity, pressure in air chamber: Relative humidity and air temperature were monitored once daily throughout the treatment period. - Air flow rate: The chambers for the 0, 300, 600 and 1400 ppm dose groups were operated dynamically at airflow rates of 237, 236, 230 and 224 litres per minute. Chamber air flow was monitored using a magnehelic gauge (1.0 cm of water equalled 236 and 230 lpm for the 300 and 600 ppm dose groups; 0.8 cm of water equalled 224 lpm for the 1400 ppm dose group) which was attached to an in-line calibrated orifice.- Air change rate: The 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 for the 0, 300, 600 and 1400 ppm dose groups, respectively.- Method of particle size determination: The presence of particulate was assessed once daily throughout the exposure using a Royco (Model #227) Portable Particle Monitor or TSI APS Particle Sizer (Model 3300/3302).

TEST ATMOSPHERE
- Brief description of analytical method used: At the end of the exposure the residual cyclohexanone in the flask was removed via an FMI pump to determine the nominal concentration. 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. - Samples taken from breathing zone: The test material was administered as a vapour in the animals’ breathing zone.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Determination of test material concentration was carried out with a MIRAN IA Gas Analyzer Spectrophotometer. Atmospheric samples were pulled directly from the exposure chamber into a Wilks MIRAN IA Gas Analyzer Spectrophotometer by a Thomas air pump (Model 107CA18-3), or a Dynaflow air pump (Model AB1 PC231). Absorbance was read directly from the MIRAN absorbance .

SELECTION OF MIRAN WAVELENGTH
- Three instrument settings for the units were used (MIRAN 5 replaced MIRAN 4). Some parameters were the same for all three:
- Wavelength (microns): 3.4
- Range: 1A
- Response: 1
- Slit width (mm): 1
- Chart speed (cm/min): 1
- Volts full scale for chart: 1

MIRAN No. 02
- Concentration to be read (ppm): 0, 300
- Pathlength, dial setting: 4.0- Sensitivity: low
- Typical reading at target conc.: 0, 0.65

MIRAN No. 04
- Concentration to be read (ppm): 650, 1400
- Pathlength, dial setting: 1.0
- Sensitivity: high
- Typical reading at target conc.: 0.47, 0.80

MIRAN No. 05
- Concentration to be read (ppm): 650, 1400
- Pathlength, dial setting: 0.65
- Sensitivity: high
- Typical reading at target conc.: 0.40, 0.70

MIRAN CALIBRATION
- A heating jacket was used around the MIRAN cell. The heating jacket was set to 50 °C and maintained for both the calibration and chamber assay. The MIRAN was allowed to warm up for approximately 20 minutes after being turned on. Heating tape was wrapped around the injection tee on the in-line pump and around the entire length of the 1/4" Teflon tubing which connected the exhaust side of the pump and the inlet of the MIRAN. The Variamp was turned to a setting of 45. The cleanness of the cell was checked by flushing it with room air for approximately one minute. The loop was closed, the unit was zeroed and the appropriate injection series performed.

DAILY MIRAN CALIBRATION CHECK
- A three-point calibration check of the MIRAN was conducted each exposure day prior to sampling the chambers:MIRAN No. 02- Concentration to be sampled (ppm): 0, 300- Total vol. of test material injection (µL): 2.4, 7.3, 12.2 - Experimental absorbance reading (0 - 1.0 scale): 0.28, 0.70, 1.0- Calculated concentration (ppm): 110, 335, 559 MIRAN No. 04 and 05- Concentration to be sampled (ppm): 650, 1400- Total vol. of test material injection (µL): 9.8, 29.4, 49.0 - Experimental absorbance reading (0 - 1.0 scale): No.4: 0.34, 0.78, 1.02; No. 5: 0.29, 0.69, 0.97- Calculated concentration (ppm): 450, 1350, 2240Injections were made with a 10 µL Hamilton syringe (Model 7001 SN or 701 N). The absorbance was recorded after each injection; it must be within 15 % of the original calibration series.The calculated concentration may be determined by the following equation:mg/L = [(µL injected) (941 g/L) / 5.641] (mL/1000 µL) (1 1/1000 mL) (1000 mg/g)ppm = [(mg/L) (328 °K) (µ mole/98.14 µg) / 273 °K] (1000 µg/mg) (22.4 µL/µ mole)The temperature of the heated cell was 328 °K

INHALATION EXPOSURE CHAMBER SAMPLING
- A 1/4" Teflon line was connected to the front of the chamber. Atmospheric measurements of the cyclohexanone were taken using a MIRAN equipped with an air pump. The pump was turned off to let the cell come to equilibrium before taking the reading. The actual absorbance was read from the MIRAN absorbance scale.

RESULTS
- The nominal results were slightly higher than analytical results throughout the study: The 300, 650 and 1400 ppm dose groups had average analytical values of 95, 96 and 99% of nominal. In addition, the overall analytical values were within 101% of the target concentrations for the dose groups. The concentration in the control chamber was determined to be 0 at all sampling intervals. 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. No particulate was found to be present in any test chamber.
Details on mating procedure:
- Impregnation procedure: Cohabitation; females selected for mating were placed with male rats nightly.
- M/F ratio per cage: 2:1 ratio (females:males)
- Proof of pregnancy: Vaginal smears were taken early in the morning following intervals of cohabitation and females were considered to have mated if sperm was observed at microscopic evaluation of the vaginal smear. Day on which evidence of mating was observed was defined as Day 0 of gestation.
Duration of treatment / exposure:
Days 6 - 19 of gestation
Frequency of treatment:
Daily; 6 hours/day
Duration of test:
Until day 20 of gestation
Dose / conc.:
0 ppm
Remarks:
Control animals were exposed to pre-conditioned air (using filters and humidifiers) drawn from the room.
Dose / conc.:
300 ppm
Dose / conc.:
650 ppm
Dose / conc.:
1 400 ppm
No. of animals per sex per dose:
26 females/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: Females which mated were assigned to groups daily in such a way as to most nearly equalise the Day 0 mean group body weights. A manual sorting procedure was used.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined for mortality and gross signs of toxicologic or pharmacologic effects twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were subjected to detailed physical examinations on days 0, 6, 10, 15 and 20 of gestation. In-chamber observations for a "startle-type" response of the animals were initiated approximately one week after exposures had begun. Animals, as a group, were observed prior to exposure, at approximately 1 and 3 hours during the exposure period and at the end of the exposure period. These examinations were performed three times/week. Physical observations scheduled on Day 6, 10 and 15 (treatment days) were performed following removal from the inhalation chamber at the end of the exposure day.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 15 and 20 of gestation. On gestation day 20 the actual body weight was recorded and the weight corrected to exclude the uterine weight was determined.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20; animals were sacrificed by lethal exposure to ether.
- Post-mortem: A complete gross post-mortem examination was performed on all animals. The following were examined for all animals: External surface, all orifices, the cranial cavity, carcass, the external surface of the spinal cord and the external and sectioned surfaces of the brain, nasal cavity and paranasal sinuses, the thoracic, abdominal and pelvic cavities and their viscera and the cervical tissues and organs.
- Examination of reproductive system: The intact uterus (ovaries attached) was removed from the abdominal cavity and weighed. The number and location of the following were recorded for each horn: Live foetuses, dead foetuses (dead foetus with no visible degeneration), late resorptions (recognisable dead foetus undergoing degeneration regardless of size), early resorptions (evidence of implantation but no recognizable foetus), and implantation sites. The number of corpora lutea was recorded for each ovary.Late resorptions were weighed and evaluated grossly for external malformations; only those with obvious external malformations were saved (10 % neutral formalin).
Ovaries and uterine content:
- The ovaries and uterine content were examined after termination: Yes
- Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The numbers of live and dead foetuses were recorded.
Fetal examinations:
- Foetal weight data, foetal sex distribution data, foetal external examination data, foetal visceral evaluation data, foetal skeletal evaluation data, malformation data, ossification variation data
- External examinations: Yes; all per litter
- Soft tissue examinations: Yes; half per litter
- Skeletal examinations: Yes; half per litter
- Head examinations: Yes; half per litter

ALL FOETUSES
- Each foetus was given a gross external examination for malformations, weighed, sexed (external criteria: ano-genital distance) and tagged for identification.

FOETAL SOFT TISSUE EVALUATION
- Approximately one-half of the foetuses in each litter (alternating foetuses within the litter) were evaluated for soft tissue malformations using the microdissection procedure of Staples. The foetuses were decapitated and the heads were preserved in Bouins solution. The dissection and evaluation of each foetus was performed under a dissecting microscope.Following the foetal evaluation, the foetuses were eviscerated (internally sexed by inspection of the gonads) and stored in 70 % ethanol. Following a period of fixation, the heads were sectioned using a razor blade and tissues were evaluated under a dissecting microscope.

FOETAL SKELETAL EVALUATION
- The remaining foetuses in each litter were eviscerated (internally sexed by inspection of the gonads) and processed for staining of the skeletal structures with Alizarin Red S. Stained foetal skeletal specimens were evaluated for malformations and ossification variations using a dissecting microscope.
Statistics:
First, Bartlett's test was performed to determine if groups had equal variance. If 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 means were indicated, Dunnett's test was used to determine which means were significantly different from the control. The Kruskal-Wallis test was used as nonparametric procedure for testing equality of means; 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, 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 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. 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. If any one cell had an expected value less than 5, the chi-square and Armitage's tests were not reported; only the Fisher Exact test (corrected via Bonferroni inequality) was performed and reported.All tests were reported at the 5 and 1% level of significance.
Historical control data:
The testing laboratory has historical control data for the reproductive performance of the strain of rat used, along with data that show 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. Likewise, many of the high-dose animals were noted as lethargic when evaluated post-exposure on Day 6 but this condition was noted with decreased frequency at Day 10 and was not noted thereafter. Nasal discharge was also noted with increased frequency in the high-dose animals, particularly at Days 10 and 15. A brown/red vaginal discharge was seen in several high-dose females on Day 15 of gestation; these same females had a normal distribution of uterine implantations at the Day 20 sacrifice and the significance of this observation is unclear.Animals were also evaluated as a group in the chambers during the exposure intervals for the startle-response. In the control group, animals exhibited a normal reflex response during the exposure interval throughout the recording period. Likewise, low-dose animals were noted to respond throughout the exposure interval. In a few instances the responses of some low-dose animals were noted as sluggish. The mid-dose animals were noted to respond to stimulus; however, a greater number of animals were noted as lethargic or sluggish in their response. This was noted as early as one hour after exposure had initiated. In the high-dose group, animals exhibited a sluggish response after one hour of exposures and no response at the three and six hour intervals.
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):
Mean body weight data during gestation (Days 0, 6, 15 and 20 including actual and corrected Day 20 weights) were comparable between the control, low- and mid-dose groups. In the high-dose group, mean body weights were comparable to control at Days 0 and 6 of gestation and were significantly lower than control at Day 15. At Day 20, mean actual and corrected body weight data for the high-dose group were also significantly lower than control data.Mean body weight gain during the Day 0-6 (pre-treatment) interval was comparable between the control and treated groups. Mean body weight gain data during the Day 6-20 interval, using actual Day 20 weight data, were comparable between the control, low- and mid-dose groups. In the high-dose group, mean body weight gain during the Day 6-20 gestation interval using the actual Day 20 weight data was significantly lower than control data.Mean weight gain during the Day 6-20 gestation interval, using corrected Day 20 weight data, was comparable between the control and low-dose group and slightly lower than control in the mid-dose group; however, this latter difference from control data was not statistically significant. In the high-dose group, mean weight gain during the Day 6-20 gestation interval using corrected Day 20 weight data, was significantly lower than control.Mean weight data during gestation were not considered to be adversely affected by treatment at the low- or mid-dose exposure levels. At the highest exposure level, mean weight gain data were depressed during the treatment period.
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 specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
Postmortem findings observed grossly occurred in the treated and control animals or they occurred sporadically; they were not considered to be related to the administration of the test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean number of uterine implantation sites was comparable between the control, mid- and high-dose groups, and was slightly higher than control in the low-dose group (not considered indicative of an adverse effect of treatment).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The percentage of litters with at least one resorption site was comparable between the control and low-dose group, but was slightly higher than control in the mid- and high-dose group. These latter differences from control data were not statistically significant. None of the control or treated females had in utero litters comprised entirely of resorption sites.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy rates for the control, low-, mid- and high-dose groups were 92.3, 88.5, 92.3 and 88.5%, respectively. No treatment-related effects were noted.
Other effects:
not specified
Key result
Dose descriptor:
NOAEC
Effect level:
650 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean foetal weight data, distinguished by sex, were comparable between the control, low- and mid-dose groups and significantly lower than the control in the high-dose group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Mean foetal weight data, distinguished by sex, were comparable between the control, low- and mid-dose groups and significantly lower than the control in the high-dose group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean number of live foetuses was comparable between the control, mid- and high-dose groups, and was slightly higher than control in the low-dose group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The percentage of male foetuses per group was comparable between the control and treated groups. No adverse effect of treatment was evident from the foetal sex distribution data.
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external malformations were seen in the 331 control foetuses (24 litters), 326 mid-dose foetuses (24 litters) or 306 high-dose foetuses (23 litters) evaluated. One high-dose foetus was noted to have a shiny (glass-like) appearance, which was not considered indicative of a malformation. In the low-dose group, one foetus had an umbilical hernia; this same foetus did not have prominent eye bulges. A second low-dose foetus was pale in colour, which was considered a minor malformation. No other external malformations were observed in the low-dose group (346 foetuses in 23 litters). The incidence of external malformations in the low-dose group was 0.6%. No adverse effect of treatment was evident from foetal external examination data.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No skeletal malformations were seen in the 168 low-dose foetuses (23 litters), 158 mid-dose foetuses (24 litters) and 145 high-dose foetuses (23 litters) evaluated. In the control group, three foetuses (an incidence of 1.9%) had skeletal malformations. One foetus had wavy ribs (unilateral), one foetus had fused ribs (unilateral) and thoracic vertebral malformations (misaligned and/or unossified vertebral elements) and one foetus had a misaligned thoracic vertebral centrum. No adverse effect of treatment was evident from foetal skeletal malformation data.The incidence of foetuses with at least one ossification variation for the control, low-, mid- and high-dose groups was 84.2% (133/158), 89.3% (150/168), 87.3% (138/158) and 97. % (142/145), respectively. These incidences were considered comparable between the control, low- and mid-dose groups and higher than control at the high-dose level.The incidence of foetuses with the various types of ossification variations was considered similar between the control, low- and mid-dose groups. At the high-dose level, the incidence of foetuses with incomplete ossification of the cranial bones (e.g., nasals, frontals, parietals, interparietals, and supraoccipitals) and/or ossification irregularities of the hyoid (incomplete or unossified) was notably increased from control data. Likewise, at the high-dose level, the incidence of incomplete or unossified sternebrae and unossified metatarsals and phalanges (fore-limbs) was also increased. Thus, at the high-dose level, there was a generalised retardation in ossification.In summary, evaluation of foetuses recovered from females treated at the low- or mid-dose levels for external, visceral or skeletal malformations did not reveal an increase in malformations. At the high-dose level, no increase in malformation rate was evident during the foetal external, visceral or skeletal evaluations; however, the incidence of foetuses with at least one ossification variation was increased at this dose level and some retardation in ossification was indicated among foetuses at this dose level, particularly in cranial ossifications, sternebrae and phalanges.
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral malformations were seen in 11/173 control foetuses (6.4%), 8/180 low-dose foetuses (4.4%), 1/168 mid-dose foetuses (0.6%) and 1/161 high-dose foetuses (0.6%). The incidence of litters containing foetuses with soft tissue malformations for the control, low-, mid- and high-dose groups was 29.2% (7/24), 26.1% (6/23), 4.2% (1/24) and 4.3% (1/23), respectively.The most common malformation seen during the visceral evaluations was distended renal pelvis. This malformation noted alone or in association with a tortuous or distended ureter, was seen in nine control foetuses (5.2%), seven low-dose foetuses (3.9%) and one mid-dose foetus (0.6%). This observation was not seen in the 161 high-dose foetuses evaluated. The distention noted was graded as slight to moderate; none of the foetuses were noted with extreme distention of the renal pelvis. This observation is noted at low frequency historically in the testing laboratory.Major ocular malformations (anophthalmia, microphthalmia) were seen in one control, one low-dose and one high-dose foetus. Unilateral anophthalmia was seen in one control foetus.In the low-dose group, bilateral anophthalmia was seen in the foetus noted with an umbilical hernia at external evaluation. This foetus also had moderate distention of the lateral ventricles of the cerebral hemispheres. In the high-dose group, bilateral microphthalmia was seen in one foetus. No adverse effect of treatment was evident from visceral malformation data.Tortuous ureter(s), a soft tissue variation, was noted in ten control foetuses (an incidence of 5.8%) and eight low-dose foetuses (an incidence of 4.4%). This observation was not noted in the 168 mid-dose or 161 high-dose foetuses evaluated. Absence of the innominate segment between the right subclavian and right carotid artery, a soft tissue variation, was seen in one mid-dose foetus (an incidence of 0.6%). No adverse effect of treatment was indicated from the foetal visceral evaluations.
Other effects:
not specified
Key result
Dose descriptor:
NOAEC
Effect level:
650 ppm
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: Mean Foetal Weight Data

Dose Group (ppm)

 

Average Foetal Body Weight (g)

Male

Female

Both

0

Mean

SD

N

3.8

0.3

24

3.6

0.3

24

3.7

0.3

24

300

Mean

SD

N

3.8

0.3

23

3.5

0.2

23

3.7

0.2

23

650

Mean

SD

N

3.8

0.3

24

3.6

0.2

24

3.7

0.2

24

1400

Mean

SD

N

2.8

0.4

23

2.6

0.4

23

2.7

0.4

23

 Where N is the number of litters

Conclusions:
Under the conditions of this study, the NOAEC for both maternal toxicity and embryotoxicity was 650 ppm (equivalent to 2.65 mg/L). The NOAEC for teratogenicity was 1400 ppm (equivalent to 5.71 mg/L), the highest dose tested.
Executive summary:

A study was conducted to evaluate the embryotoxic and/or teratogenic effects of cyclohexanone in the rat. The study was conducted in accordance with the U.S. EPA "Health Effects Test Guidelines" (Teratogenicity Study).

Mated CD® rats (26 females/group) were treated via the inhalation route by administering the test substance in the breathing zone for 6 hours/day during the Day 6-19 gestation interval. Target exposure levels in the inhalation chambers were 0, 300, 650 and 1400 ppm. Animals were weighed and given detailed physical evaluations at regular intervals during gestation; additionally, animals in each group were evaluated in the chambers during the exposure interval for responsiveness to a startle-type stimulus.

On Day 20 of gestation, females were sacrificed. Each female was given a gross post-mortem examination, uterine weight recorded, corpora lutea counted and uterine implantation data recorded. Foetuses recovered at this time were weighed, sexed and given a gross external examination. Subsequently, one-half of the foetuses in each litter were evaluated for visceral malformations and the remainder evaluated for skeletal malformations or ossification variations.

No mortality occurred in the control or treated groups. Pregnancy rates for the control, low-, mid- and high-dose groups were 92.3, 88.5, 92.3 and 88.5%, respectively.

No adverse effect of treatment was evident in body weight data during gestation in the low-dose and mid-dose groups. 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.

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.

No adverse effect of treatment was evident in uterine implantation data. 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.

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

No increase in type or incidence of malformation was evident in foetuses recovered from treated females during either the external, visceral or skeletal evaluations. In the high-dose group, the incidence of foetuses with at least one ossification variation was increased and foetuses 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).

Thus, cyclohexanone administered at exposure levels of 300 and 650 ppm 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 an increased incidence of lacrimation, lethargy and nasal discharge) and embryotoxicity occurred (reduced foetal weight data; increase in ossification variation data); however, no teratogenicity was evident at this dose level.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
READ-ACROSS CONSIDERATIONS
Cyclohexanol is structurally very similar to cyclohexanone, differing only in the oxidation level of the functional group (secondary alcohol vs. ketone). As expected the similarity in chemical structure is reflected in the similarity of the physico-chemical properties. The low melting points show that both substances could be toxicologically tested as liquids at temperatures slightly above ambient conditions. As expected due to the higher polarity of the hydroxyl group, the boiling point of cyclohexanol is a little higher than that of cyclohexanone and the vapour pressure a little lower. Both substances are slightly soluble in water, which in turn leads to relatively low partition coefficients. The similarity in chemical structure and physico-chemical properties would suggest that the substances should have a similar toxicity profile, and that is supported by studies reported in the literature common to both Cyclohexanone and Cyclohexanol. Both Cyclohexanone and Cyclohexanol are readily absorbed and are reversibly interconvertible in vitro and in vivo. In animals, Cyclohexanol is the major metabolite of Cyclohexanone, which itself clears rapidly from circulation. Both the administration of Cyclohexanone and of Cyclohexanol lead to the excretion of the glucuronic acid conjugate of cyclohexanol. In humans, cyclohexanol is the primary metabolite of cyclohexanone (which is short-lived in circulation), but it is further metabolized to 1,2- and 1,4-cyclohexanediols. The excretion of the latter metabolites is qualitatively and quantitatively independent of the parent compound administered, be it ketone or alcohol. Although it is highly likely that all toxic effects seen upon treatment with Cyclohexanone are in fact caused by its rapidly formed metabolite Cyclohexanol, it cannot be fully excluded that either substance has some additional intrinsic mode of action. If this were the case for Cyclohexanone, read-across from the ketone to the alcohol would lead to an overestimation of the toxicity of the latter. However, it was shown that the ketone is actively formed from Cyclohexanol administered to rabbits, so that it could exert its intrinsic effect even in Cyclohexanol studies. The opposite possibility, specific effects of Cyclohexanol, is fully covered by toxicity studies on Cyclohexanone, since it was shown that peak plasma concentrations of Cyclohexanol are similar, irrespective whether the ketone or the alcohol were administered. Given the reversible conversion of Cyclohexanone into Cyclohexanol, their consistent pattern of toxicokinetics and their identical metabolic fate, it is concluded that the endpoint data on the source substance, Cyclohexanone, are relevant to the human risk assessment on Cyclohexanol, and that the proposed read-across for this endpoint is justified (see read across document attached to Section 13 of the dossier).

Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Dose descriptor:
NOAEC
Effect level:
650 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEC
Effect level:
650 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Read-across from cyclohexanone to cyclohexanol is considered valid for the developmental toxicity endpoints as well as classification and labelling. Under the conditions of this study, the NOAEC for both maternal toxicity and embryotoxicity was 650 ppm (equivalent to 2.65 mg/L). The NOAEC for teratogenicity was 1400 ppm (equivalent to 5.71 mg/L), the highest dose tested.

Executive summary:

A study was conducted to evaluate the embryotoxic and/or teratogenic effects of cyclohexanone in the rat. The study was conducted in accordance with the U.S. EPA "Health Effects Test Guidelines" (Teratogenicity Study).

No mortality occurred in the control or treated groups. Pregnancy rates for the control, low-, mid- and high-dose groups were 92.3, 88.5, 92.3 and 88.5%, respectively.

No adverse effect of treatment was evident in body weight data during gestation in the low-dose and mid-dose groups. 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.

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.

No adverse effect of treatment was evident in uterine implantation data. 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.

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

No increase in type or incidence of malformation was evident in foetuses recovered from treated females during either the external, visceral or skeletal evaluations. In the high-dose group, the incidence of foetuses with at least one ossification variation was increased and foetuses 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).

Thus, cyclohexanone administered at exposure levels of 300 and 650 ppm 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 an increased incidence of lacrimation, lethargy and nasal discharge) and embryotoxicity occurred (reduced foetal weight data; increase in ossification variation data); however, no teratogenicity was evident at this dose level.

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
Quality of whole database:
Reliable information on the developmental toxicity via the oral route is available for cyclohexanone that is appropriate for hazard and risk characterisation and the purpose of classification and labelling.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
5 620 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable information on the developmental toxicity via the inhalation route is available for cyclohexanone that is appropriate for hazard and risk characterisation and the purpose of classification and labelling.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
No reliable information on the developmental toxicity via the dermal route is available for cyclohexanol or cyclohexanone.
Additional information

Description of key information

The developmental toxicity endpoint is covered with reliable and relevant information on cyclohexanone for the oral and inhalation routes of exposure, based on a robust read-across argumentation.

Inhalation route of exposure

Potential embryotoxic and teratogenic effects of cyclohexanone in the rat were studied via the inhalation route (Schroeder 1984). Mated CD rats were exposed to 0, 300, 650 and 1400 ppm. No adverse effects in dams or foetuses related to treatment were observed in the low and medium dose groups. At the highest dose, maternal toxicity was evident as reduced body weights and increased incidence of lacrimation, lethargy and nasal discharge. Furthermore, embryotoxicity was observed at the highest dose, which resulted in reduced foetal body weights and an increase in ossification variation. However, no teratogenic effects were observed at all dose levels, including the highest exposure concentration.

The developmental toxicity of cyclohexanol was investigated in an unpublished screening study in the rat conducted to the OECD TG 422 (Newton 2005). A summary of the report has been published in the US HPV Programme, but the registrant has no access to the full study report. No robust study summary has been written and included in the registration dossier for this reason. However, a short description of the study derived from the published HPV summary has been included here for completeness and to provide all relevant data, in accordance with Annex I requirements for Chemical Safety Report. The study is not used to conclude on the endpoint of developmental toxicity of cyclohexanol. Cyclohexanol administered by inhalation did not produce maternal toxicity or teratogenicity at the highest dose of 450 ppm (1843 mg/m3). A slightly increased incidence of pregnancies with no viable foetuses and lower pup weights was observed at the highest exposure level of 450 ppm, and the NOEL in this study was therefore considered to be 150 ppm (614 mg/m3).

Conclusion for the inhalation route: No teratogenic effects of cyclohexanone were seen up to a dose of 1400 ppm (5620 mg/m3) in the inhalation developmental toxicity study in the rat.

Oral route of exposure

Cyclohexanone dissolved in water was administered to pregnant Himalayan rabbits by stomach tube at doses of 50, 250 and 500 mg/kg body weight from day 7 to 19 of pregnancy (i.e. after artificial insemination) (Hellwig 1994). Dams in the high-dose group had a significantly reduced food consumption and body weight gain related to dosing, and unsteady gait about 2 to 4 hours after dosing. No treatment-related effects were observed in the dams receiving oral doses of 50 or 250 mg/kg bw/day. There were no substance-related adverse effects on the gestational parameters and on the foetuses in all dose groups, and no substance-related teratogenic effects were observed.

Conclusion for the oral route: No teratogenic effects of cyclohexanone were seen up to a dose of 500 mg/kg bw/day in the oral developmental toxicity study in the rabbit.

Dermal route of exposure

No information available

Justification for classification or non-classification

REPRODUCTIVE TOXICITY

Human data on the potential reproductive toxicity of cyclohexanol are lacking. Relevant and reliable information from the 2-generation inhalation reproductive toxicity study conducted with cyclohexanone (Mayhew 1986) is taken to cover this endpoint. No adverse effects on the male and female reproductive organs were observed in animals exposed to a concentration of 5620 mg/m3 over several months. The reduction in the male reproductive performance occurring in the high-dose group may be explained by the likely narcotic effect of cyclohexanone at the high dose, which was expressed as lethargy of animals as the predominant post-exposure reaction after adaption to the treatment, and thus is considered as an effect of parental toxicity that does not warrant classification for effects on fertility.

The information on potential effects on fertility resulting from oral exposure to cyclohexanol available from a published non-guideline 2-generation study (Gondry 1972) and two published non-standard repeated dose toxicity studies (Lake et al. 1982, Dixit et al. 1980) is inconsistent and unreliable and not sufficient to warrant a classification of cyclohexanol.

A classification of cyclohexanol for effects on fertility is not warranted, based on a robust read-across from the relevant and reliable information on cyclohexanone for the inhalation route of exposure demonstrating no adverse effects on male and female reproductive organs and only slight effects on male reproductive performance in the presence of parental toxicity.

DEVELOPMENTAL TOXICITY

Human data on the potential developmental toxicity of cyclohexanol are lacking. Relevant and reliable information on the developmental toxicity of cyclohexanone from oral and inhalation developmental toxicity studies with cyclohexanone (Hellwig 1994, Schroeder 1984) is taken to cover this endpoint. No teratogenic effects were observed in these studies.

A classification of cyclohexanol for developmental toxicity is not warranted, based on a robust read-across from the relevant and reliable information on cyclohexanone for the oral and inhalation route of exposure demonstrating no teratogenic effects.