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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:
December 14, 2010 - February 11, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in compliance with GLP and in accordance with OECD Guideline 474 and EU Method B12.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-methylenebis(N-sec-butylcyclohexamine)
EC Number:
679-514-8
Cas Number:
154279-60-4
Molecular formula:
C21H42N2
IUPAC Name:
4,4'-methylenebis(N-sec-butylcyclohexamine)
Details on test material:
MTDID 25575 Batch No. 30103 260308 031 Expiry date: 06 December 2011
Purity 98,69 Wt%
Clear colourless liquid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
Young NMRI BR mice (SPF) were supplied by Charles River, Sulzfeld, Germany, 5 weeks old upon arrival.
The animals were group housed (5 animals per sex per cage) in labelled polycarbonate cages, containing sterilised sawdust as bedding material. The housing conditions were under daily monitoring (T = 21 ±3 °C, relative humidity 40-70%). 12 hours light/dark cycle was employed. Conventional laboratory diet was provided ad libitum. Drinking water was provided ad libitum.

The body weights of the mice at the start of the treatment were within 20% of the sex mean. The mean body weights were for males 35.2 ±1.8 g and for females 26.9 ± 2.0 g and the range for males 33-39 g and for females 23-33 g.

The acclimatisation period was at least 5 days before the start of treatment. The animals were 6 weeks old at the start of treatment.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: MTDID 25575 was dissolved in corn oil (Sigma, Zwijndrecht, The Netherlands)
THE DOSING VOLUME: 10 ml/kg bw

Frequency of treatment:
Single dose.
Post exposure period:
24 and 48 hours, second sampling time at 48 hours for high dose group only.
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 50 and 100 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 male and 5 female per dose
Control animals:
yes
Positive control(s):
The positive control used was cyclophosphamide (CAS: 50-18-0, from Baxter B.V.,Utrecht, The Netherlands) dissolved in physiological saline (B. Braun, Melsungen AG, Germany) dosed as a single intraperitoneal injection of 40 mg/kg bw.

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Selection was based on a dose range findng study. The highest concentration selected caused signs of toxicity, but not mortality.

TREATMENT AND SAMPLING TIMES: The mice received an intraperitoneal injection of a maximum tolerated (high), an intermediate and a low dose of MTDID 25575. The route of administration was chosen to maximize the chance of the test substance reaching the target tissue. The first sampling time was 24 h after treatment for all groups and the second sampling time was 48h after treatment for the high dose group only
Evaluation criteria:
A test substance is considered positive if:
- It induces a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p< 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocyte in the animals are above the historical control data range.

A test substance is considered negative if:
- none of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p< 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocyte in the animals are within the historical control data range.

Statistics:
Wilcoxon Rank Sum Test, one-sided

Results and discussion

Test resultsopen allclose all
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Sex:
female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Doses: 100 and 200 mg/kg bw
- Clinical signs of toxicity in test animals: ataxia, lethargy, hunched posture, rough coat and closed eyes.

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): There was no decrease in the ratio of polychromatic to normochromatic erythrocytes.
- Appropriateness of dose levels and route: The highest concentration selected caused signs of toxicity, but not mortality. The route of administration was chosen to maximize the chance of the test substance reaching the target tissue.
- Statistical evaluation: Wilcoxon Rank Sum Test, one-sided.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance (trade name Clearlink 1000) was evaluated for its genotoxical potency with miclronuclus test in bone marrow cells of mice. Male and female mice (5/sex/treatment group, beside 10/sex in 100 mg/kg bw dose group) were dosed via intraperitoneal injection with vehicle or with 100, 50 and 25 mg/kg/bw of test substance. A positive control group was dosed via intraperitoneal injection with 40 mg/kg bw with cyclophosphamide. The test was conducted according to OECD Guideline 474 and in compliance with GLP.

All the animals treated with the test substance showed the following signs after dosing: hunched posture and rough coat. All the animals in 100 mg/kg bw dose group were lethargic. 7/10 males and 5/10 females in dose group 100 mg/kg bw showed ataxia. No treatment related clinical signs or mortality were noted in control animals. The animals treated with the test substance did not show decrease in the ratio of polychromatic too normochromatic erythrocytes compared to the vehicle control group. Positive control group showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to vehicle controls.

Conclusion: Based on the results of this test the test substance is not clastogenic or aneugenic in mice.

This study is classified as acceptable and fulfils the requirement of the in vivo micronucleus test.