3-cyano-3,5,5-trimethylcyclohexanone

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:

key study

Study period:

1991-02-04 to 1992-02-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

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-cyano-3,5,5-trimethylcyclohexanone

EC Number:

411-490-4

EC Name:

3-cyano-3,5,5-trimethylcyclohexanone

Cas Number:

7027-11-4

Molecular formula:

C10H15NO

IUPAC Name:

3-cyano-3,5,5-trimethylcyclohexanone

Details on test material:

Isophorone nitrile of Degussa AG. Batch No. 64-II/78/90. Purity 99.6 % (GC).

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Source: Winkelmann, Borchen (Germany)
- Age: 6 weeks
- Weight at study initiation: 25-33 g (males); 23-29 g (females)
- No. of animals per dose: 18 males + 18 females (control groups); 21  males + 21 females (test group)
- Housing: single in conventional Makrolon Typ II cages
- Diet: standard diet ssniff M, ad libitum
- Water: tab water ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 2 °C
- Humidity (%): approximately 55 +/- 15 %
- Photoperiod (hrs dark / hrs light): 12 h/ 12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle: Traganth (1 % suspension in water - aqua ad iniectabilia)

Details on exposure:

- Control groups and treatment:    
negative: vehicle    
positive: 51.1 mg cyclophosphamide (CPA)/kg bw in physiological NaCl  solution (0.9 %)
- Total volume applied: 10 ml/kg bw
- Duration of test: 24 hours; 48 hours; 72 hours
- Clinical observations: First 4-6 hours after administration; once daily  on days 2 and 3
- Sampling times and number of samples: 24 hours; 48 hours; 72 hours: 6  mice per sex and control group, 
7 mice per sex of test group; normally  first 5 per sex and group evaluated
- Organs examined at necropsy: femur bone marrow; gross necropsy on  animals deceased intercurrently (no further details in report)   
1000 PCE (polychromatic erythrocytes) per animal were analysed for  micronuclei

Duration of treatment / exposure:

single oral application

Frequency of treatment:

1 time

Post exposure period:

24h, 48 h, 72 h

Doses / concentrations

Remarks:

Doses / Concentrations:
82.5 mg/kg bw (males and females)
Basis:
nominal conc.

No. of animals per sex per dose:

18 control groups; 21  test group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamid (Endoxan) 51.4 mg/kg body weight in physiological saline solution by oral gavage

Examinations

Tissues and cell types examined:

polychromatic erythrocytes of the bone marrow from femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: maximum dose without mortalities
DETAILS OF SLIDE PREPARATION: Bone marrow from both femurs were flushed with 1.5 ml fetal calf serum, centrifuged, suspended upon a thin layer of serum and smeared on a slide. The smears were stained using the panoptic stain method developed by PAPPENHEIM.

Evaluation criteria:

- Criteria for evaluating results: statistically significant (p<0.05) and  reproducible increase in frequency of micronucleated polychromatic 
 erythrocytes of at least one test group sampling time as compared to the  same sampling time of the negative control group

Statistics:

BIOMETRY:
- Normally 1000 polychromatic erythrocytes were counted per animal.
-The frequencies of micronucleated polychromatic erythrocytes of the test material group and the positive control group were compared with those of the the negative control group at each sampling time.
-A POISSON test was applied, the data from each treatment group for each sex as well as for both sexes combines were compared with the respective negative conrol group data using a VAX 8200 computer. Values of 5 animals of each sex and sampling time were used for statistical evaluation.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

other: negative control=vehicle control

Positive controls validity:

valid

Additional information on results:

MORTALITY: 
- Dose finding   
100 mg/kg bw: Severe symptoms of toxicity and death.   
82.5; 75.0; 68.1 mg/kg bw: Clear symptoms of toxicity, no death.
- Main test: 1 male from the test group died 9 minutes after  administration.
CLINICAL SIGNS (test group): Slight to severe clonic convulsions in the  test group (9/21 males, 6/21 females). No abnormal clinical signs in the  
control groups.
NECROPSY FINDINGS:    Main test: The male that died was necropsied: No abnormalities were  detected.
EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO:    No reduction in PCE/NCE ratio was present in test material group  animals when compared with 
the corresponding negative control animals,  with the exception of one male at the 24 hours sampling time.   
The average PCE/NCE ratio of the positive control animals was  significantly lower than that of the corresponding vehicle controls at 48  and 
72 hours. Due to technical error in the preparation of the slides,  realistic PCE/NCE ratios for 72 hour control males cannot be given  
(accidental damage of NCE cell membranes, PCE unaffected). 
GENOTOXIC EFFECTS:    For the positive control a significant and clear increase in the  frequency of micronucleated polychromatic erythrocytes
 was observed  (except females at 72 hours). Upon administration of the test substance,  no significant increase over the corresponding 
negative controls was  observed in either male or female animals, as well as for both sexes  combined, at any of the sampling times.

Any other information on results incl. tables

Summary of results

Treatment  Sex   Time   Micron./1000 PCE     PCE/NCE
--------------------------------------------------------
Test subst. m    24 h    2.2 +- 0.84      0.48 - 2.09
Vehicle     m    24 h    1.8 +- 0.84      1.08 - 1.33
CPA         m    24 h   35.6 +- 3.91 *    0.52 - 1.22
Test subst. f    24 h    1.2 +- 1.10      0.75 - 1.20
Vehicle     f    24 h    1.4 +- 0.55      0.69 - 0.98
CPA         f    24 h   23.6 +- 6.73 *    0.53 - 1.28
Test subst. m    48 h    2.0 +- 1.41      1.04 - 1.75
Vehicle     m    48 h    0.8 +- 1.30      1.24 - 1.60
CPA         m    48 h    9.6 +- 3.13 *    0.26 - 1.11 *
Test subst. f    48 h    2.0 +- 1.00      0.92 - 1.70
Vehicle     f    48 h    2.0 +- 1.00      0.92 - 1.51
CPA         f    48 h    5.0 +- 2.82 *    0.47 - 0.64 *
Test subst. m    72 h    1.4 +- 2.07      1.36 - 1.95
Vehicle     m    72 h    1.0 +- 1.22        no data
CPA         m    72 h    4.4 +- 2.88 *    0.13 - 0.57
Test subst. f    72 h    2.6 +- 1.82      1.07 - 1.86
Vehicle     f    72 h    2.0 +- 1.73      0.73 - 1.65
CPA         f    72 h    1.6 +- 1.14      0.20 - 0.43 *
--------------------------------------------------------
CPA = cyclophosphamide;   * = statistical significance

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Isophorone nitrile is not a mutagenic substance under the in vivo conditions in this micronucleus assay using male and female NMRI mice.

Executive summary:

The results of this study indicate that under the test conditions, isophorone nitrile did not induce micronucleated polychromatic erythrocytes in male and female mice. 82.5 mg/kg bw isophorone nitrile was administered oral to 21 male and 21 female NMRI mice per group. This doses were selected as the maximum tolerated dose (MTD) based upon a preliminary toxicity study. Bone marrow polychromatic erythrocytes, collected from 5 male and female mice 24, 48 and 72 hours after single treatment, were examined microscopically for micronucleated polychromatic erythrocytes (PCE). No significant increase in the frequency of PCE over the control  was found with any group treated  with the test substance. For the positive control (cyclophosphamid, CPA) a significant increase in the frequency of PCE was observed. Therefore, isophorone nitrile is not a mutagenic substance under the in vivo conditions in this micronucleus assay using male and female NMRI mice.