Registration Dossier

Administrative data

Description of key information

Oral route

NOAEL = 143 mg/kg bw/day, 3 months, read-across from cyclohexanone, OECD 408, BASF (1994)

NOAEL = 462 mg/kg bw/day, 2 years, read-across from cyclohexanone, OECD 453, Lijinsky (1986)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
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:
143 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects
Key result
Critical effects observed:
no
Conclusions:
Read-across from cyclohexanone to cyclohexanol is considered valid for the repeated dose toxicity endpoints as well as classification and labelling for specific target organ toxicity after repeated exposure hazards. A NOAEL of 143 mg/kg body weight/day was derived from the study on cyclohexanone.
Executive summary:

In the 90-day drinking water study based on OECD Guideline 408 and according to GLP, signs of toxicity were observed at a cyclohexanone concentration of 7000 ppm. A reduction of water and food consumption at 2000 and 500 ppm was not considered to be an adverse health effect, but caused by the strong odor and taste of the test substance. Therefore, the NOAEL for this drinking water study is 2000 ppm cyclohexanone, corresponding to a dose of 143 mg/kg body weight/day. This result can be directly applied to cyclohexanol due to the close physico-chemical and toxicological similarity.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
143 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Based on a sound read-across position, data from two studies using cyclohexanone are considered to provide suitable data on which to base a risk assessment.
System:
other: lipid metabolism
Organ:
not specified

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
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. Read-across justified in analogue reporting format.
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
Parental animals were exposed by inhalation on 5 days per week for 6 hours to cyclohexanone vapours during 10 weeks prior to mating, then during the 15 days of the mating period and for additional 28 days until weaning (resulting in total in about 16 weeks of exposure). This exposure can be considered as sub-chronic.
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 Fema
les: 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:
whole body
Vehicle:
other: conditioned air
Details on inhalation 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

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
TEST ATMOSPHERE
- Samples taken from breathing zone: Yes
- Brief description of analytical method used:
TEST MATERIAL ANALYSIS
- The 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)
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.
Dose / conc.:
0 ppm
Remarks:
F0 and F1 generation
Dose / conc.:
250 ppm
Remarks:
F0 and F1 generation
Dose / conc.:
500 ppm
Remarks:
F0 and F1 generation
Dose / conc.:
1 000 ppm
Remarks:
F0 generation
Dose / conc.:
1 400 ppm
Remarks:
F1 generation (dose was increased after one week of exposure to 1000 ppm)
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.
Observations and examinations performed and frequency:
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.
Sacrifice and pathology:
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.
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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
First parental generation: 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.
Second parental generation: 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 postexposure 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):
First parental generation: 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.
Second parental generation: 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):
First parental generation: Body weight data for the F0 parent animals exposed to the test material were comparable to the untreated control animals.
Second parental generation: 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, treatment-related
Description (incidence and severity):
First parental generation: not examined
Second parental generation: 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:
no effects observed
Description (incidence and severity):
First parental generation: 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.
Second parental generation: not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
First parental generation: not examined
Second parental generation: 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):
First parental generation: Necropsy examination of sacrificed F0 parental animals revealed no treatment-related lesions.
Second parental generation: 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):
First parental generation: not examined
Second parental generation: 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):
First parental generation: No microscopic changes were seen in the reproductive organs from the 1000 ppm animals and the untreated control animals.
Second parental generation: 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:
no effects observed
Description (incidence and severity):
First parental generation: Specific investigations of the male and female reproductive organs did not reveal any histopathological changes.
Second parental generation: Specific investigations of the male and female reproductive organs did not reveal any histopathological changes.
Key result
Dose descriptor:
NOAEC
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive performance
Key result
Dose descriptor:
NOAEC
Effect level:
2 007.2 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive performance
Key result
Critical effects observed:
no
Organ:
other: no findings during microscopical histopathological examination
Conclusions:
Inhalation exposure of rats to cyclohexanone vapours (6 hours per day, 5 days per week) over a period of about 16 weeks resulted in a NOAEC of 500 ppm (corresponding to 2007 mg/m3), based on effects on the reproductive performance. No clinical signs of systemic toxicity were seen up to a dose of 1000 ppm. Gross pathology and microscopical histopathology did not reveal any target organ specific toxicity at higher doses of 1000 or 1400 ppm.
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. The parental generations in this study were exposed for 6 hours per day, five days per week during a period of about 16 weeks. Thus, this study provides useful information on the potential adverse health effects resulting from sub-chronic inhalation exposure to cyclohexanone.

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 conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
2 007.2 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Based on a sound read-across position, data from two studies using cyclohexanone are considered to provide suitable data on which to base a risk assessment.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to Regulation (EC) No 1907/2006, Annex VIII, 8.6 Repeated dose toxicity, column 2: The short-term toxicity study (28 days) does not need to be conducted if a reliable sub-chronic (90 days) or chronic toxicity study is available, provided that an appropriate species, dosage, solvent and route of administrations were used. A reliable two-generation study is available, in which rats were exposed to cyclohexanone by inhalation for a period of about 16 weeks. Relevant hazard information for the inhalation route can be derived from this sub-chronic study. Furthermore, a reliable sub-chronic study is available for the oral route, which can be used to derive appropriate hazard information for the inhalation route by route-to-route extrapolation.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to Regulation (EC) No 1907/2006, Annex VIII, 8.6 Repeated dose toxicity, column 2: The short-term toxicity study (28 days) does not need to be conducted if a reliable sub-chronic (90 days) or chronic toxicity study is available, provided that an appropriate species, dosage, solvent and route of administrations were used. A reliable sub-chronic study is available for the oral route. The reliable sub-chronic study for the oral route is used to derive appropriate hazard information for the dermal route by route-to-route extrapolation.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to Regulation (EC) No 1907/2006, Annex VIII, 8.6 Repeated dose toxicity, column 2: The short-term toxicity study (28 days) does not need to be conducted if a reliable sub-chronic (90 days) or chronic toxicity study is available, provided that an appropriate species, dosage, solvent and route of administrations were used. A reliable sub-chronic study is available for the oral route. The reliable sub-chronic study for the oral route is used to derive appropriate hazard information for the dermal route by route-to-route extrapolation.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral

No reliable repeated dose oral toxicity studies are available for cyclohexanol. Based on a sound read-across position, data from studies with cyclohexanone are considered to provide suitable data on which to base a risk assessment.

A 90-day drinking water study was performed with cyclohexanone according to the OECD Guideline 408 (BASF 1994). Ten female and ten male Wistar rats per dose group were continuously exposed to 500, 2000 or 7000 ppm cyclohexanone through the drinking water (corresponding to 40, 143 and 407 mg/kg bw/day) for 13 weeks. Weighing of all animals was done daily, food and water consumption was determined weekly, and detailed observations (ophthalmological examination, haematology, clinical biochemistry and urinalysis), as well as gross necropsy and histopathology, were performed at the end of the study. At the concentration of 7000 ppm, signs of toxicity were observed. In this dosage group, an increase in total cholesterol, total protein and globulins in both sexes, and an increase in platelets in the females, were observed. The only substance related pathomorphological effect was a decreased terminal body weight. A reduction of water and food consumption was caused by the strong odour and taste of the test substance, and therefore the decreased body weight was not considered to be an adverse health effect. Haematological changes were also observed, and are probably a result of slight changes in lipid metabolism or of reduced water consumption. The NOAEL for this drinking water study was 2000 ppm, corresponding to a dose of 143 mg/kg bw/day. This study is considered to be relevant, adequate, and reliable for the purposes of classification and risk assessment.

A 2-year chronic toxicity assay of cyclohexanone was conducted according to OECD Guideline 453 in F344 rats and (C57BL/6 X C3H)F1 mice by administering a solution of cyclohexanone in drinking water acidified with HCl to pH 2.5 (Lijinsky 1986). Two concentrations were given to rats, 6500 and 3300 ppm (wt/vol). Male mice received 13000 and 6500 ppm, while female mice were given three concentrations, 25000, 13000, and 6500 ppm. Each treatment group consisted of 50 or 52 male and 50 or 52 female rats or mice, except 47 male mice treated with the highest dose and 41 female mice treated with the highest dose. There was a group of untreated controls of each species. Survival and weight gain were similar to those of controls at the lowest cyclohexanone dose in both sexes of both species, but weight gain was depressed at all higher doses. Survival was good in all groups except in female mice at the 2 highest doses. Most of the neoplasms in the treated groups did not differ significantly in number from those in the controls. The evidence for carcinogenic activity of cyclohexanone was marginal and the effect, if any, was weak. A nominal NOAEL of 462 mg/kg bw/day, corresponding to 3300 ppm, was derived. This study reported comparable but less severe results than the previous study (BASF 1994), and it is considered to be supporting information.  

Inhalation

Two parental generations of Sprague-Dawley rats were exposed by inhalation to cyclohexanone vapours for 6 hours per day, 5 days per week over a period of about 16 weeks in a reliable two-generation study on growth, development and reproduction (Mayhew 1986). Due to the long exposure period, this inhalation study can be interpreted as a sub-chronic repeated dose inhalation toxicity test and used to cover this toxicological endpoint. The overall NOAEC in this study was 500 ppm (corresponding to 2007 mg/m3) based on a reduction in the reproductive performance over two generations. The NOAEC for clinical effects was 1000 ppm, and the NOAEC for specific target-organ toxicity was 1400 ppm.

Treon et al. (1943) investigated the sub-acute inhalation toxicity of cyclohexanol in the rabbit and monkey. Although these studies provide useful information for the metabolism of this compound, the details on the toxicology are not sufficient to reliably evaluate the sub-acute inhalation toxicity of cyclohexanol. The number of animals utilised in the studies was low and from the data presented it was not possible to fully describe the pathology of any organs and therefore to determine meaningful NOAEL/LOAEL values. According to Regulation (EC) No 1907/2006, Annex VIII, column 2: The short-term toxicity study (28 days) does not need to be conducted if a reliable sub-chronic (90 days) or chronic toxicity study is available, provided that an appropriate species, dosage, solvent and route of administration were used.  

Dermal

No studies on the repeated dose toxicity via the dermal route are available. According to Regulation (EC) No 1907/2006, Annex VIII, column 2: The short-term toxicity study (28 days) does not need to be conducted if a reliable sub-chronic (90 days) or chronic toxicity study is available, provided that an appropriate species, dosage, solvent and route of administration were used. A reliable sub-chronic study is available for the oral route (BASF 1994), which can be used to derive appropriate hazard information for the dermal route by route-to-route extrapolation.  

Other routes

No reliable studies are available for other routes.

Justification for classification or non-classification

Based on the results of the key study (BASF 1994), the substance is not classified for specific target organ toxicity after repeated exposure according to the criteria set out in Regulation (EC) No. 1272/2008, since the NOAEL of 143 mg/kg bw/day is above the threshold of 100 mg/kg bw/day for a 90-day study, which would trigger the classification as STOT RE category 2.