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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Version / remarks:
2016
Deviations:
yes
Remarks:
50 metaphases instead of 200 metaphases per animal were scored, mitotic index was not determined, evaluation criteria are not reported
GLP compliance:
not specified
Type of assay:
other: chromosome aberration

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Source: Sigma-Aldrich
Batch no.: 13975

Test animals

Species:
rat
Strain:
other: CD (a remote Sprague-Dawley-derived strain)
Sex:
male/female
Details on test animals and environmental conditions:
All the animals were located in a room which was separate from but adjacent to the area where the exposures were conducted. They were housed individually in cages in a room with a light intensity of approximately 200 lux, a 12 h light-dark cycle, approximately 10 air changes per hour, temperature maintained at ca 22°C with extreme limits of 17°C and 24°C, and relative humidity ca 50%, with extreme limits of 32% and 52%. Food and water were freely available to the rats at all times. The animals to be dosed were individually identified using brass ear tags bearing the aninal number and the suffix letter showing the compound designation. Each rat was ascribed a cage card which identified that animal by project number, animal niunber, sex and treatment group.

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
none
Details on exposure:
Animals were sacrificed 6, 24, and 48 hours following inhalation exposure to vapors of 50 or 400 ppm for 1 or 5 days.
Each rat was injected i.p. with 3 mg/kg colchicine dissolved in Hank's Balanced Salt Solution (HBSS) 4 h after the last dose was given. Two hours later the rats were killed by neck dislocation. One femur from each animal was dissected out, cleaned of adherent tissue and the marrow aspirated into a 10 ml plastic blood sample tube containing 4 ml HBSS at ambient temperature and lithium hepari. Each tube was labelled with the appropriate random number from a slide coding sheet. Hence, from this time until the completed result sheets were de-coded, the rat number and group were unknown to the scientists and technicians. The cell suspension was centrifuged at 1,500 r.p.m. for 5 min, the supernatant fluid discarded and replaced with 4 ml fresh HBSS. The cells were suspended, then centrifuged again and the supernatant fluid discarded. 4-5 ml 0 .075 M-KC1 pre-heated to 37°C was added to the cells while they were agitated on a vortex mixer. Following incubation for 20 min in a 37°C water bath, the cells were centrifuged, the supernatant fluid decanted and the cells fixed in 4 ml freshly prepared fixative (methanol : glacial acetic acid; 3:1). The fixative was removed after centrifugation and replaced with 2 ml fresh fixgtive. Tubes containing fixed cells were stored in a 4°C refrigerator overnight. The following morning (or later, up to 3 days) the fixative was changed and cell suspensions dropped onto clean slides labelled with the same number as the tube and allowed to dry thoroughly. Slides were stained in a bath of Giemsa R66 (Gurr) diluted with 10 parts distilled water for 30 min, rinsed briefly in distilled water, dehydrated in alcohol, cleared in xylene and mounted in DePeX.
Slide Reading: Leitz binocular microscopes were used for this purpose. Magnification was nominaily x 1,000 using x 10 magnification eye pieces and x 100 objectives. Wherever possible, for each animal 50 cells with a minimum of 41 well spread chromosomes were examined and scored. The location of all spreads examined were recorded using the microscope stage vernier. The slide number was always located on the right hand side. Abnormalities looked for were: gaps, breaks, fragments, dicentrics, translocations (within the limitations of the staining methods) and pulverisation.
Duration of treatment / exposure:
single dose 1 x 7 h or 1 x 7 h repeated dosing 5 days
Frequency of treatment:
7 h daily for repeated dosing
Post exposure period:
no data
Doses / concentrationsopen allclose all
Dose / conc.:
50 ppm
Dose / conc.:
400 ppm
No. of animals per sex per dose:
30 for single dosing
10 for repeated dosing
Control animals:
yes, concurrent no treatment
Positive control(s):
ethyl methanesulphonate

Examinations

Tissues and cell types examined:
All animals were inspected for clinical signs/mortality twice daily except at weekends when they were observed once only. During the dosing period, all animals subjected to the exposure manipulations were weighed upon removal from the exposure chambers.
Details of tissue and slide preparation:
Dosing solutions were prepared daily 5 min before administration to the animals was started. The desired amount of ethyl methanesulphonate was weighed into a volumetric flask and diluted with distilled water to obtain the correct concentration.
Positive control animals were not allowed access to food or water whilst the remaining test groups were being exposed. Ethyl methanesulphonate was administered orally by gavage to the rodents at a constant dose volume of 10 mL/kg at around 16.00 h on each day that dosing was required.
Evaluation criteria:
Not specified
Statistics:
A one-sided Student's t test was used on the transformed values.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the multiple exposure cytogenetic test, there were no indications of induction of chromosomal damage in either the male or female rats exposed to 50 ppm or 400 ppm cyclohexanone atmospheres. Responses to the positive control substance, ethyl methanesulphonate, were significant in the females, but not in the males. The single exposure test male rats showed a significant increase in gap frequency at the 6 h sample time in the 400 ppm cyclohexanone atmosphere exposed group (p < 0.005). No similar increase in aberration frequency was observed in the female rats at this or any other sampling time. Also, aberrations excluding gaps were not increased in any of the male rat groups eNposed to cyclohexanone. Responses to ethyl methanesulphonate were not uniformly significant. Frequencies of all aberrations were increased in male rats at the 6 h and 24 h sampling times and in female rats at the 24 h and 48 h sampling times. Aberrations excluding gaps were increased in male rats at the 24 h and 48 h sampling times and in female rats at the 24 h and 48 h sampling times.

Applicant's summary and conclusion

Conclusions:
In a chromosomal aberration assay doses of 50 and 400 ppm were applied to male and female rats via the inhalation route (single dose 7 h/day or 7 h/d for 5 days). The assay followed in general the current OECD guideline 474, showing minor deviations, but the studies are well documented and considered scientifically valid. Single and repeated dosing of cyclohexanone did not lead to relevant increases in chromosomal aberrations in the bone marrow of the animals. The study is considered to sufficiently cover the endpoint investigated.
The bioavailability of cyclohexanone following inhalation exposure is very good. This was demonstrated by a significant increase in cyclohexanone plasma levels in rats exposed to 400 and 1600 ppm for 6h (Industrial Health Foundation Inc., 1987; see toxicokinetics) and a significant increase in urinary cyclohexanone metabolites following exposure of human volunteers to 200 mg/m3 for 8h (Mraz et al. 1994; see toxicokinetics).