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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was performed according to accepted but older guidelines and under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Cited as Directive 84/449/EEC, B.12
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
CAS 1761-71-3 (cyclohexylamine,4,4'-methylenebis-), purity not indicated ("rein")

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
- Age: 7 to 8 weeks
- Weight at study initiation: 33-40 g (males) & 30-35 g (females)
- Housing: Makrolon cages
- Food and water: free access to feed and water

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
DMSO
Details on exposure:
- The animals received a single intraperitoneal injection of the test substance dissolved in DMSO
- The dose level used as the test dose was selcted to produce mortality of about 10% (based on preliminary LD50 study)
- Volume injected: 0.1 mL/10 g bw
Duration of treatment / exposure:
single application
Frequency of treatment:
once
Post exposure period:
24, 48 and 72 h after administration, 6 animals of each group were sacrificed
Doses / concentrations
Remarks:
Doses / Concentrations:
50 mg/kg
Basis:

No. of animals per sex per dose:
No. of animals per dose: at least 20/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (20 mg/kg)

Examinations

Tissues and cell types examined:
femoral bone marrow smears
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION
- The bone marrow preparations were prepared, stained and evaluated based on the method of Schmid
- The two femora were excised from the test animals, the attached tissue was removed and the femora were dissected at the condyles
- The bone marrow was rinsed with fetal calf serum by means of a syringe and cannula and transferred to a centrifuge tube with 4 mL calf serum
- The cell suspension was centrifuged at 100 g for 5 minutes, and the serum was sucked off except for a small amount
- The cells were resuspended by careful pipetting, dropped onto clean microscopic slides and smeared

METHOD OF ANALYSIS
- After one day, the microscopic slide preparations were stained with May-Gruenwald and Giemsa solution, rinsed in distilled water, dried and mounted in DePeX via xylene
- The coded preparations were evaluated under the microscope at a 500 fold enlargement
- 1000 erythrocytes were counted and differentiated according to polychromatic (young) and normochromatic (old) erythrocytes
- The fraction of polychromatic cells indicates the function of erythropoiesis and allows detecting possible cytostatic or cytotoxic effects of the test substance
- The proportion of cells carrying miconuclei was determined in 2000 polychromatic erythrocytes
- The mean and standard deviation were calculated from the individual values of the 6 animals of each group
Evaluation criteria:
CRITERIA FOR EVALUATING RESULTS:
- Significant increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of male and female mice
- Statistical method: U test
Statistics:
- The test group and control group were investigated for differences by means of the U-test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
- Mortality : Male: 2 out of 22, Female: 3 out of 22
- Clinical signs: slight to moderate convulsion and piloerection

STATISTICAL RESULTS:
% PCE and %PCE with micronucleus were not increased in males or females at 24, 48, and 72 hours after treatment.

EFFECT ON PCE/NCE RATIO (%PCE):
Males
- Control group: 52.9, 53.3, 51.8 at 24, 48 and 72 hours, respectively
- Treatment group: 50.4, 48.4, 53.6 at 24, 48 and 72 hours, respectively
- Positive control: 52.2 at 24 hours

Females
- Control group: 53.4, 51.9, 52.2 at 24, 48 and 72 hours, respectively
- Treatment group: 53.0, 53.2, 54.1 at 24, 48 and 72 hours, respectively
- Positive control: 51.7 at 24 hours

GENOTOXIC EFFECTS:
Mean number of micronucleated PCE:
Males
- Control group: 0.16, 0.27, 0.21 at 24, 48 and 72 hours, respectively
- Treatment group: 0.26, 0.24, 0.20 at 24, 48 and 72 hours, respectively
- Positive control: 1.40 at 24 hours

Females
- Control group: 0.13, 0.25, 0.19 at 24, 48 and 72 hours, respectively
- Treatment group: 0.17, 0.22, 0.21 at 24, 48 and 72 hours, respectively
- Positive control: 1.39 at 24 hours

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
According to the test results available, there are thus no significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the dose group (50 mg Dicykan/kg body weight) or between the different sacrifice intervals (24, 48 and 72 hours). Thus, under the experimental conditions chosen here, the test substance Dicykan has no chromosome damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.