3,3,5-trimethylcyclohexan-1-one

Genetic toxicity: in vivo

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:

weight of evidence

Study period:

2004-05-25 to 2004-07-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

3,3,5-trimethylcyclohexan-1-one

EC Number:

212-855-9

EC Name:

3,3,5-trimethylcyclohexan-1-one

Cas Number:

873-94-9

Molecular formula:

C9H16O

IUPAC Name:

3,3,5-trimethylcyclohexan-1-one

Details on test material:

3,3,5-Trimethylcyclohexanone of Atofina. Batch No. 1R010101. Purity 99.6 %; 0.110 % 3,3,5-trimethylcyclohexanol, 0.066 % isophorone,
0.070 % water, 0.024 % 1,1,3-trimethylcyclohexane

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Source: Charles River Laboratories France, l'Arbresle (France)
- Age: approx. 6 weeks
- Weight at study initiation: not reported
- No. of animals per dose: 5 males + 5 females; additional 3 animals per  sex at the high dose level were not evaluated due to the absence of  
mortalities
- Housing: in groups
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): at least 12 cycles per hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

corn oil

Details on exposure:

ADMINISTRATION: 
- Vehicle: Corn oil
- Control groups and treatment:    
negative: vehicle (2 i.p. administrations separated by 24 hours)   
positive: 50 mg cyclophosphamide (CPA)/kg bw (1 oral administration in  distilled water)
- Total volume applied: 10 ml/kg bw/administration
- Duration of test: 48 hours
- Sampling times and number of samples: 24 hours after last treatment

Duration of treatment / exposure:

2 Doses separated by 24 hours

Frequency of treatment:

2 times for vehicle and test item
1 time for positive control

Post exposure period:

24 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 175; 350; 700 mg/kg bw/day
Basis:

No. of animals per sex per dose:

5 animals, except high dose level: 8 animals

Control animals:

yes, concurrent vehicle

Positive control(s):

50 mg cyclophosphamide (CPA)/kg bw (1 oral administration in  distilled water)

Examinations

Tissues and cell types examined:

femora, bone marrow

Details of tissue and slide preparation:

EXAMINATIONS: 
- Criteria for selection of M.T.D.: Initial study with 2 applications  each of 500; 700; 1000; 1500; 2000 mg/kg bw separated by 24 hours; 3  males + 
3 females / dose level. The top dose for the cytogenetic test was  selected such that a higher dose-level was expected to induce lethality.
- Clinical observations: First 4-6 hours after administration; once daily  on days 2 and 3
- Organs examined at necropsy: femur bone marrow   2000 PCE (polychromatic erythrocytes) per animal were analysed for  micronuclei   
1000 erythrocytes were scored for PCE/NCE ratio

Evaluation criteria:

- Criteria for evaluating results: Statistically significant (p<=0.05)  and biologically relevant increase in frequency of micronucleated  polychromatic 
erythrocytes of at least one test group as compared to the  negative control group

Statistics:

Mann-Whitney test

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Main test: No deaths occurred.
EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO:    
No reduction in PCE/NCE ratio was present in the positive control group  or in any test group when compared with the negative control animals.
GENOTOXIC EFFECTS:    
For the positive control a significant and clear increase in the  frequency of micronucleated polychromatic erythrocytes was observed.   In general, 
the mean frequencies of micronucleated polychromatic  erythrocytes in the groups treated with the test item were equivalent to  those of the vehicle 
control group. A statistically significant increase  (p<0.05) was observed only in the low dose female group. This  significance was considered to be 
artefactual and mainly due do a very  low control value. The absolute result was well within the usual vehicle  control range and thus not biologically 
relevant, and there was no  dose-response relationship. 

Any other information on results incl. tables

MORTALITY: 
- Dose finding
  2000 mg/kg bw/day: All animals were dead within one hour after the  first administration.
  1500 mg/kg bw/day: 1/3 males and 1/3 females were dead within 2 and 24 hours following the first administration, respectively. 

No second  treatment was performed.
  1000 mg/kg bw/day: 1/3 males was dead within 24 hours following the  second treatment.
  700 and 500 mg/kg bw/day: No deaths occurred within the 48 hour period.
- Main test: No deaths occurred.
CLINICAL SIGNS: 
- Dose finding
  1000 mg/kg bw/day: The surviving animals showed hypoactivity, sedation,  tremors, staggering gait, dyspnea and/or piloerection.
  700 mg/kg bw/day: Lateral recumbency and dyspnea (2/3 males) or  sedation (3/3 females) were noted 30 min following the first 

treatment.  These signs diappeared the following day and only piloerection was noted.
  500 mg/kg bw/day: Lateral recumbency, dyspnea, tremors, hypoactivity  and/or staggering gait were noted one hour following 

treatment in 2/3  males and 2/3 females. Only piloerection was observed 17 hours later and  sometimes until sacrifice.
- Main test
  700 mg/kg bw/day: In all treated animals, sedation was noted 15 minutes  following the first treatment and piloerection was 

observed two hours  following the second treatment.

  350 or 175 mg/kg bw/day: No clinical signs were observed. 
EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO: 
  No reduction in PCE/NCE ratio was present in the positive control group  or in any test group when compared with the negative 

control animals.
GENOTOXIC EFFECTS: 
  For the positive control a significant and clear increase in the  frequency of micronucleated polychromatic erythrocytes was observed.
  In general, the mean frequencies of micronucleated polychromatic  erythrocytes in the groups treated with the test item were 

equivalent to  those of the vehicle control group. A statistically significant increase  (p<0.05) was observed only in the low dose  female group. This  significance was considered to be artefactual and mainly due do a very  low control value. The absolute result 

was well within the usual vehicle  control range and thus not biologically relevant, and there was no  dose-response relationship. 
--------------------------------------------------------
Treatment          Sex    Micron./1000 PCE     PCE/NCE
--------------------------------------------------------
Vehicle             m        0.5 +- 0.5      0.3 +- 0.1
175 mg/kg bw/day    m        0.0 +- 0.0      0.3 +- 0.2
350 mg/kg bw/day    m        0.2 +- 0.3      0.3 +- 0.1
700 mg/kg bw/day    m        0.5 +- 0.4      0.4 +- 0.1
Cyclophosphamide    m       17.0 +- 6.8 ***  0.7 +- 0.1
Vehicle             f        0.0 +- 0.0      0.4 +- 0.2
175 mg/kg bw/day    f        0.6 +- 0.4 *    0.4 +- 0.2
350 mg/kg bw/day    f        0.4 +- 0.2      0.5 +- 0.2
700 mg/kg bw/day    f        0.3 +- 0.3      0.4 +- 0.2
Cyclophosphamide    f       17.0 +- 8.3 ***  0.6 +- 0.1
--------------------------------------------------------
* p<0.05; *** p<0.001

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
3,3,5-Trimethylcyclohexanone is not a mutagenic substance under conditions of this in vivo micronucleus assay using male and female mice.

Executive summary:

In a Swiss mouse Erythrocyte Micronucleus assay, 5 mice/sex/dose were treated with two I.p. injections at a 24 hour interval, with 3,3,5-Trimethylcyclohexanone (99.6 %) at doses of 0, 175, 350, and 700 mg/kg bw. The maximum tolerated dose (MTD) was determined in a preliminary toxicity study. Bone marrow polychromatic erythrocytes, collected 24 hours after last treatment, were examined microscopically for micronucleated polychromatic erythrocytes (PCE).

There were no signs of toxicity in the 175 and 350 mg/kg group. At 700 mg/kg sedation was noted in all treated animals after 15 minutes following the first treatment. 

3,3,5-Trimethylcyclohexanone was tested at an adequate dose. The positive control (cyclophosphamid, CPA) induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.