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:
disregarded due to major methodological deficiencies
Study period:
1994
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Purity is out of range, Insufficient analytic data

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
not specified

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Moellegaard Breeding Centre Deutschland GmbH, HauptstraBe 62, 16352 Schönwalde
- Age at study initiation: 8 weeks
- Weight at study initiation: m: 31-48 g; f: 26 - 34 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: groups of 5 per sex
- Diet (e.g. ad libitum): ad lib.
- Water (e.g. ad libitum):ad lib.
- Acclimation period: >6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%):50-90
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Aqueous starch mucilage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: single administration
Duration of treatment / exposure:
12, 24 or 48 hours
Frequency of treatment:
single
Post exposure period:
12, 24 or 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg (Limit dose)
Basis:
actual ingested
(gavage)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide;
- Justification for choice of positive control(s):
- Route of administration: oral
- Doses / concentrations: 0,5 % (w/v)

Examinations

Tissues and cell types examined:
Bone marrow from femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Limit dose based on "no toxicity"

DETAILS OF SLIDE PREPARATION:
Extraction of the bone marrow
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 12, 24 or 48 hours after application. For each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube.
Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tUbe. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supematant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.
Staining procedure
- 5 minutes in methanol
- 5 minutes in May-GrOnwalds solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution 10 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan
METHOD OF ANALYSIS: Manual countig of cells with micronuclei in 1000 polychromatic erythrocytes per slide. A Wilcoxon-Test (one-sided) was evaluated to check the validity of the study. This is done by comparing the number of polychromatic erythrocytes with micronuclei in the positive control with the negative control.
A Wilcoxon-Test (one-sided) was performed for each measurement group (12h, 24h, 48h) and for polychromatic and normochromatic erythrocytes. These tests are performed sequentially with a munlple level of significance of 5%
Evaluation criteria:
Both biQlogical and statistical significances were considered together fQr evaluatiQn purposes.
A substance is considered as positive if there is a significant increase in the number Qf micronucleated polychromatic erythrocytes fQr at least Qne Qf the time points. A test substance producing nQ significant increase in the number Qf micronucleated polychromatic erythrocytes is considered nQn-mutagenic in this system.
Statistics:
comparing
the number of polychromatic erythrocytes with micronuclei in the positive control with the negative
control.
A Wilcoxon-Test (one-sided) [6,7) was performed for each measurement group (12h, 24h, 48h) and for
polychromatic and normochromatic erythrocytes. These tests are performed sequentially with a munlple
level of significance of 5% (5).
The following comparisons are performed only if there is a difference between the positive conlrol and the
negative control (24h). This Is tested with a Wilcoxon-Test (two-sided) [6,7) with a 5 %-Ievel of significance.
Wilcoxon-Tests (two-sided) are performed sequenlially for the ratio of polychromatic erythrocytes for each
measurement group (12h, 24h, 48h) al a muniple level of significance of 5 % (5). Actual data were also
compared with historical controls.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
positive
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): positive
Fluorescent Red YJGG lead tQ a substantial increase of micronucleated polychromatic erythrocytes and is mutagenic in the micronucleus test.
Executive summary:

Fluorescent Red YJGG was tested in the micronucleus test. The test compound was suspended in starch mucilage and dosed once orally at 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay (see preiiminary study). According to the test procedure the animals were killed 12, 24 or 48 hours after administration.

Endoxan* was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was statistically significant increased at the 24 hours killing time in male and female mice. The ratio of polychromaticlnormochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Fluorescent Red YJGG and was statistically not different from the control values.

Endoxan* induced in both males and females a , .arked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that Fluorescent Red YJGG is mutagenic in the micronucleus test.