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:
1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

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
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: Remazol-Brillantblau R FWTRG

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
Species: NMRI mouse
Strain: Hoe: NMRKf (SPF71)
Origin: HOECHST AG, Kastengrund, SPF breeding colony
Initial age at test: 7 weeks
Number of animals: 70 (35 males / 35 females)
Bodyweight at start of study: males mean= 30.5 g (26 - 35 g); females: mean= 25.6 g (23 - 31 g)
Acclimatization: at least 5 days
Food / water: mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe), ad libitum; tap water in plastic bottles, ad libitum
Housing: 5/cage
Room temperature: 22 +/- 2°C
Relative humidity: 55 +/- 10%
Lighting time: 12 h/12 h light/dark cyclus

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
deionised water
Details on exposure:
The dose levels for micronucleus testing were selected on the basis of a preliminary study to determine the acute toxicity and the maximal applicable dose. Oral administration of 5000 mg Remazol-Brillantblau R FWTRG per kg bodyweight did not lead to a partial lethality in male and female mice. It is considered the maximal applicable dose and was selected as dose level for the main study.

The 5000 mg/kg bw dose was prepared as a 25% (w/v) solution and administerd at a volume of 20 mL/kg bw devided into two equal parts of 10 mL/kg bw administered within 2 hours

The test compound dilutions were prepared fresh each day. 6250 mg Remazol-Brillantblau R FWTRG were weight in a beaker, mixed with deionisized water, washed out in a 25 ml flask and topped up to the calibration mark. A suspension was formed.
Duration of treatment / exposure:
animals were killed after 24, 48 or 72 hours after administration of the test compound
Frequency of treatment:
The test compound was given in two equal parts within two hours
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg
Basis:
nominal in water
No. of animals per sex per dose:
5 for each killing time
Control animals:
yes, concurrent vehicle
Positive control(s):
endoxane, 50 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
Extraction of the bone marrow
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 24, 48 or 72 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 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.
Staining procedure
- 5 minutes in methanol
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan
Evaluation criteria:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group to which they belonged remains unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically; comparison
of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).
The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided).
Statistics:
The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).
The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided). The statistical evaluations were performed using the I1Diamant l1 computer program Version 2.0, supplied by the Department of Information and Communication Hoechst AG. All statistical results are based on a 95 %level of significance. Actual data were also compared with historical controls.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
All animals survived after application of 5000 mg Remazol-Brillantblau R FWTRG per kg bodyweight. The following signs of toxicity were observed: faeces dark blue coloured and diarrhea. 24 hours p.a. in the cages no. 3, 4, 9 and 14 the soft-wood granulate was red coloured.
48 hours after application all animals were free of clinical signs of toxicity.
The dissection of the animals revealed the following macroscopic findings: At the killing time 24 hours p.a. at the dosage 5000 mg Remazol-Brillantblau R FWTRG per kg bw in some male and females the content of appendix and colon was blue coloured.

The incidence of micronucleated polychromatic erythrocytes in the dose groups of Remazol-Brillantblau R FWTRG was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes has been observed. The number of normochromatic erythrocytes with micronuclei did not differ significantly from the values of the simultaneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
not mutagenic
Executive summary:

Remazol-Brillantblau R FWTRG was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 5000 mg Remazol-Brlllantblau R FWTRG per kg bodyweight.

The 5000 mg per kg bodyweight dose level was chosen since a preliminary study had shown it to be the maximum applicable dose.

The animals were treated once with the test compound and according to the test procedure the animals were killed 24, 48 or 72 hours after administration of the test compound.

The test compound was given in two equal parts within two hours and according to the test procedure the animals were killed 24, 48 or 72 hours after administration of the test compound.

EndoxanR was used as positiv control substance and was administered orally at a dose of 50 mg per kg bodyweight.

The incidence of micronucleated polychromatic erythrocytes of the animals treated with Remazol-Brillantblau R FWTRG was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Remazol-Brillantblau R FWTRG and was statistically not different from the control values.

EndoxanR induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a signifi cant extend.

The results indicate that, under the conditions of the present study, Remazol-Brillantblau R FWTRG is not mutagenic in the micronucleus test.