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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
(16 h for highest dose is performed 2 hours earlier than guideline recommendation, only 1000 MN PCE evaluated /animal)
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclohexanol
EC Number:
203-630-6
EC Name:
Cyclohexanol
Cas Number:
108-93-0
Molecular formula:
C6H12O
IUPAC Name:
cyclohexanol
Test material form:
not specified
Details on test material:
Supplier: BASF.
State: crystalline, white solid.

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, WIGA, D-W8741 Sulzfeld, FRG
- Weight at study initiation: 26.4 g
- Assigned to test groups randomly?: yes
- Housing: individua
l- Diet: ad libitum; (Kliba Haltungsdiaet, Klingentalmuehle AG, CH-4303 Kaiseraugst, Switzerland)
- Water: ad libitum, feed and water analysis indicated that the contaminants occuring in these medium would not alter result outcome
- Acclimation period: 1 wk

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s): aqueous 0.5% CMC (carboxymethyl cellulose)
- Justification for choice: no data
- Concentration of test material in vehicle: 5, 10 and 15%
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Details on exposure:
- Justification for choice of concentrations: based on preliminary toxicity test
Duration of treatment / exposure:
- Negative and positive control: 24h
- 500 mg/kg bw: 24h
- 1000 mg/kg bw: 24h
- 1500 mg/kg bw: 16, 24, 48h
Frequency of treatment:
single dose application
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 020 mg/kg bw/day (actual dose received)
Dose / conc.:
1 550 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
- Negative control: 5
- 500 mg/kg bw: 5
- 1000 mg/kg bw: 5
- 1500 mg/kg bw: 15 (5 animals/sex for 16, 24, 48h sacrifice time point)
Control animals:
yes, concurrent vehicle
Positive control(s):
For clastogenicity: Cyclophosphamide
- No. of animals: 2M/3F
- Dose: 20 mg/kg bw
- Vehicle: aqua dest
- Route of exposure: orally
- Frequency: single dose application
- Volume of application: 10 mL/kg bw

For spindle poison effect: Vincristine
- No. of animals: 3M/3M
- Dose: 0.15 mg/kg bw
- Vehicle: aqua dest
- Route of exposure: ip
- Frequency: single dose application
- Volume of application: 10 mL/kg bw

Examinations

Tissues and cell types examined:
Bone marrow
- Number of polychromatic erythrocytes (PCE) examined: 1000/animal. Following parameters were measured: Number of polychromatic erythrocytes, number of polychromatic erythrocytes containing micronuclei, number of normochromatic erythrocytes, number of normochromatic erythrocytes containing micronuclei, ratio of polychromatic to normochromatic erythrocytes, number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A pretest was performed to determine the oral acute toxicity of cyclohexanol after single oral administration of 2000 mg/kg bw. This dose caused no mortality, however the severe toxical signs were seen. The mice exhibited; irregular respiration, piloerection, staggering and squatting posture directly after test substance treatment. After about 15 minutes, abdominal position and a narcotic-like state was additionally observed. The general state of the animals was poor. In the main study however, 11 mortalities out of 30 animals occured at this dose. Hence the study protocol was amended. The highest dose was reduced to 1500 mg/kg bw

SACRIFICE AND SAMPLING TIMES:
- Hours after application: 16h & 48h (1500 mg/kg bw), 24h (all dose groups)

DETAILS OF SLIDE PREPARATION:
- Preparation of the bone marrow were based on the method of SCHMID, W. 1976 & 1977- Schmid W. (1976). The micronucleus test for cytogenetic analysis. In : Hollaender, A . (ed), Chemical Mutagens, Principles and Methods for their Detection, Volume 4, Plenum Press, New YorK- Schmid W., (1977) In: Kilbey et al . (eds), Handbook of Mutagenicity Test Procedures, Elsevier Scientific Publishing Company, Amsterdam - New York - Oxford.
Statistics:
According to the authors, perfoming a statistical evaluation was not necessary since the number of polychromatic micronucleated erythrocytes from the dosed groups was within/nearly the range of the actual control value and within the historical values.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CLINICAL SIGNS
- Control: Animals of the negative control group as well as both positive control groups did not show any clinical signs of toxicity.
- 500 mg/kg bw: about 30 minutes - 2 hours after test substance administration; irregular respiration, piloerection and squatting posture. After about 4 hours all symptoms had disappeared
- 1000 mg/kg bw: about 30 minutes - 4 hours after treatment; irregular respiration, piloerection, staggering, squatting posture and the general state of the animals was poor. The day after treatment no clinical signs were exhibited by the animals1500 mg/kg bw: about 30 - 60 minutes after treatment; irregular respiration, piloerection, abdominal position, narcotic-like state, staggering and the general state of the animals was poor. Some of these signs were still present two days after administration.

MISCROSPCOPIC EVALUATIONS (1000 PCE/dose were investigated)

TEST SUBSTANCE
- Percentage of PCE with micro nuclei after 24h: 1.2% (control), 1.5% (500 mg/kg bw), 1.2% (1000 mg/kg bw), 0.8% (1500 mg/kg bw)
- Percentage of PCE with micro nuclei after 16 and 48h in 1500 mg/kg bw dose group: 1.5% (16h), 1.1% (48h)
- The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals
- Percentage of NCE with micro nuclei after 24h: 1.59% (control), 0.97% (500 mg/kg bw), 0.88% (1000 mg/kg bw), 1.65% (1500 mg/kg bw), 1.54% (CP), 1.86% (Vincristine)- Percentage of NCE with micro nuclei after 16 and 48h in 1500 mg/kg bw dose group: 2.11% (16h), 0.24% (48h)
- Ratio normocytes/total amount of polychromatic erythrocytes after 24 hours: 6301 (control), 5134 (500 mg/kg bw), 4520 (1000 mg/kg bw), 5467 (1500 mg/kg bw), 3255 (CP), 4848 (Vincristine)
- Ratio normocytes/total amount of polychromatic erythrocytes aster 16 and 45h in the 1500 mg/kg bw dose group: 5203 (16h) and 4191 (24h)

POSITIVE CONTROLS
- Cyclophosphamide (20 mg/kg bw): With 8.2%, the positive control substance cyclophosphamide for clastogenicity, produced polychromatic erythrocytes containing exclusively small micronuclei
- Vincristine (0.15 mg/kg bw): With 84.0%, the positive control vincristine for spindle poisoning effects producd a clearly enhanced number of micronuclei containing polychromatic erythrocytes with the expected amount of large micronuclei, i.e. 13.2%.

Any other information on results incl. tables

The number of NCE/total amount of PCE did not show a clear dose-dependent effect decrease. However, the values of the treated groups are below that of control animals. In addition the time-dependent decrease of the value from 5467 to 4191 in the high dose indicates that the test substance has reached the bone marrow.

Applicant's summary and conclusion

Conclusions:
The test substance did not show any clastogenic or aneugenic activity in this test system in vivo.
Executive summary:

In a standard in vivo micronucleus test with cyclohexanol in mice conducted to GLP and OECD Testing guideline 474, there were no biologically relevant, significant differences in the frequency of polychromatic erythrocytes containing micronuclei either between the solvent control and the three dose groups (1500, 1000 and 500 mg/kg bw) at any sacrifice intervals time (16, 24 and 48 hours). Thus, under the experimental conditions chosen, the test substance cyclohexanol did not show any chromosome-damaging (clastogenic) effects nor did it lead to any impairment of chromosome distribution in the course of mitosis.