Registration Dossier

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:
1985-Sep-16 to 1985-Oct-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
No major deviations
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Reactive Blue FC 15353

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: F. Winkelmann, Borchen
- Age at study initiation: 8-12 weeks
- Weight at study initiation:25-37 g
- Assigned to test groups randomly: yes, under following basis: according to a plan from the Institute for Biometry, Bayer AG, Wuppertal
- Housing: 3 or 5 animals (males/females separated) per macrolon cage (type I or III) on wood granulate
- Diet (e.g. ad libitum): Altromin 1324, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22 °C
- Humidity (%): 60-90 %
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
test substance: 0.5 % aqueous Cremophor-emulsion
positive control: deionized water
negative control: 0.5 % aqueous Cremophor-emulsion
Details on exposure:
volume of oral application:
test substance and negative control: 20 ml/kg body weight
positive control: 10 ml/kg body weight
Duration of treatment / exposure:
test substance: 24, 48 and 72 hours
positive and negative control: 24 hours
Frequency of treatment:
single dose
Doses / concentrations
Remarks:
Doses / Concentrations:
7500 mg/kg body weight
Basis:
no data
No. of animals per sex per dose:
5/sex
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): well-known cytostatic drug and clastogene with bifunctional alcylating effect
- Route of administration: oral, gavage
- Doses / concentrations: 20 mg/kg body weight

Examinations

Tissues and cell types examined:
bone marrow of femur
1000 polychromatic erythrocytes counted per animal plus number of mature eryhtrocytes per 1000 polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based of a pretest with 5000 mg/kg body weight and 7500 mg/kg body weight (5 animals each); test substance application: gavage; single dose.
Both doses caused clinical symptoms within 72 hours, i.e. somnolescence, blue discoloration of skin and feces, pale eyes and bristled fur. No death occured.

DETAILS OF SLIDE PREPARATION:
erythrocytes were prepared according to the method reported by W. Schmid, Mutation Res. 31, 9-15, 1975
samples were stained using a slide stainer Typ Haematek, Fa. Miles and than wahsed with methanol and water, and subsequently dried
prior to analysis, samples were covered by glass after 10 min incubation in xylene

METHOD OF ANALYSIS:
1000 polychromatic erythrocytes and mature eryhtrocytes per 1000 polycrhomatic erythrocates were counted using a light microscop with 1000x magnification.
Evaluation criteria:
The test substance is considered positive if the mean number of micronucleated polychromatic erythocytes of the test substance group is higher than in the negativ control group; and, if so, the significance level calculated by the Wilcoxon signed-rank test is < 5 %.
Statistics:
Numbers of micronucleated polychromated erythrocytes per 1000 polychromated erythrocytes were counted from 5 animals/sex for each exposure time plus negative and positive control and means were calculated. A Wilcoxon signed-rank test was performed when the mean of the test substance was higer than the negative control.
Additional the 1s-intervals were calculated.
The usage of the Wilcoxon signed-rank test is appropiate; the significance level of < 5 % is appropiate.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
somnolescence, blue discoloration of skin and feces, pale eyes and blistered fur
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
the test item had no effect on erythopesis.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Reactive Blue FC 15353 did not cause an increase of micronucleated polychromatic erythrocytes and is therefore considered not-clastogenic in the micronucleus in vivo test.
Executive summary:

In a NMRI mouse bone marrow micronucleus assay, (5/sex) were treated once by gavage with Reactive Blue FC 15353 at a dose of 7500 mg/kg bw. Bone marrow cells were harvested at 24, 48, and 72 hours post-treatment. The vehicle was 0.5 % aqueous cremophor-emulsion.

There were signs of toxicity (somnolescence, blue discoloration of skin and feces, pale eyes and blistered fur) during the study. Reactive Blue FC 15353 was tested at an adequate dose based on pretests with 5000 mg/kg bw and 7500 mg/kg bw. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

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