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in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD)

Data source

Reference Type:
other company data
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): 2,5,6-Trimethyl-2-cyclohexen-1-on
- Test substance No.: 90/731
- Date of manufacturing: November 13, 1990
- Physical state: light yellow liquid
- Analytical purity: 94.3%
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance had been verified analytically over a period of 18 months (November 1990 - April 1992) . Therefore, it can be assumed that the test substance was also stable 6 months later at the time of the performance of this study (October 1992).

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River GmbH, WIGA, D-W8741 Sulzfeld, Germany
- Weight at study initiation: about 28 g
- Assigned to test groups randomly: yes, acc. randomization plan prepared with an appropriate computer program
- Housing:individually in Makrolon cages, type M I
- Diet (e.g. ad libitum): ad libitum, Standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum, drinking water
- Acclimation period: about one week in Makrolon cages, type M III, in groups of 5 separately according to sex

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

- The feed used in the study was assayed for chemical and microbiological contaminants.
- The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Technical Services of BASF Aktiengesellschaft as well as for the presence of germs by a contract laboratory.

Administration / exposure

Route of administration:
- Vehicle(s)/solvent(s) used: olive oil
- Concentration of test material in vehicle: 4.25, 8.5, 17 g/100 ml
- Amount of vehicle (if gavage or dermal): 10 ml
Details on exposure:
- substance formulations were prepared immediately before administration
- The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the experiment
Frequency of treatment:
Post exposure period:
16 - 48 h
Doses / concentrations
Doses / Concentrations:
425, 850, 1700 mg/kg bw (in 10 ml/kg)
nominal conc.
No. of animals per sex per dose:
5 for all test groups and negative controls;
2 male and 3 female for cyclophosphamide;
3 male and 2 female for vincristine
Control animals:
Positive control(s):
cyclophosphamide for clastogenicity
- Route of administration: oral, gavage
- Doses / concentrations: 20 mg/kg bw

vincristine for spindle poison
- Route of administration: oral, gavage
- Doses / concentrations: 0.15 mg/kg bw


Tissues and cell types examined:
polychromatic and normochromatic erythrocytes of bone marrow
Details of tissue and slide preparation:
In a pretest for the determination of the acute oral toxicity deaths were observed down to a dose of 2000 mg/kg body weight . The amount which was survived by all animals was 1780 mg/kg body weight. Therefore, a dose of 1700 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 850 mg/kg and 425 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

Test Group - Sampling time - Dose
1 - 24 h - negative control
2a - 16 h - 1700 mg/kg bw
2b - 24 h - 1700 mg/kg bw
2c - 48 h - 1700 mg/kg bw
3 - 24 h - 850 mg/kg bw
4 - 24 h - 425 mg/kg bw
5 - 24 h - 20 mg/kg bw cyclophosphamide
6 - 24 h - 0.15 mg/kg bw vincristine

The bone marrow was prepared according to the method described by SCHMID, W . (1976, 1977):
- The two femora were prepared from the animals, and all soft tissues were removed .
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended.
- 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette . Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
- The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or 3 minutes . They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Corbit-Balsam.

In general, 1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei . The normochromatic erythrocytes (= normocytes), which occur, are also scored.

Evaluation criteria:
- The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.
- The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and may serve as a control value .
- A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals .
- Ratio of polychromatic to normochromatic erythrocytes indicates an influence of the test substance specifically on the bone marrow .
The number of polychromatic micronucleated erythrocytes after test substance treatment was nearly the range of the actual control value and within the historical values .

Results and discussion

Test results
Vehicle controls validity:
Positive controls validity:
Additional information on results:
- Dose range: 1780, 2000 mg/kg bw
- Solubility: The homogeneity of the test substance in the solvent was guaranteed by constant stirring during the removal and administration of the test substance formulation and by analytical determination of 3 individual samples of each concentration .
- Clinical signs of toxicity in test animals: deaths occured at 2000 mg/kg bw; no deaths at 1780 mg/kg bw

Any other information on results incl. tables


Test group Interval: 16 h Interval: 24 h Interval: 48 h
Polychromatic erythrocytes Normocytes / total amount polychromatic erythrocytes Cells with micronuclei (%) Polychromatic erythrocytes Normocytes / total amount polychromatic erythrocytes Cells with micronuclei (%) Polychromatic erythrocytes Normocytes / total amount polychromatic erythrocytes Cells with micronuclei (%)
No. Dose (mg/kg bw) polychromatic normochromatic polychromatic normochromatic polychromatic normochromatic
1 solvent control 10000 3008 2.1 1
2 1700 10000 3497 2.1 0.3 10000 5681 1.8 1.4 9000 4823 1.9 0.8
3 850 10000 3690 2.2 1.6
4 425 10000 3574 2.1 1.1
5 20, cyclophosphamide  10000 1710 10.4 1.2
6 0.15, vincristine 10000 2912 72.4 2.1

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.

Thus, the test substance 2,5,6-Trimethyl-2-cyclohexen-1-on did not lead to any increase in the rate of micronuclei . The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d > D/4) did not deviate from the solvent control value at any of the sacrifice intervals. No inhibition of erythropoiesis induced by the treatment of mice with 2,5,6-Trimethyl-2-cyclohexen-1-on was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.


- The single oral administration of the solvent in a volume of 10 ml/kg body weight was tolerated by all animals without any signs or symptoms.

- The dose of 1700 mg/kg led to clear signs of toxicity such as irregular respiration, piloerection, staggering and, in a few cases, to abdominal position, squatting posture and closed eyelids ; the general state of some animals was poor. Some of these signs were still observed the day after treatment; two days after test substance administration one animal was found dead.

- Doses of 850 mg/kg and 425 mg/kg only led to irregular respiration and piloerection which lasted for about 3 hours.

- Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight- caused any evident signs of toxicity.

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
Under the experimental conditions chosen above, the test substance 2,5,6-Trimethyl-2-cyclohexen-1-on has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.

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