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:
27 November to 30 November 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU test guidance in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Reaktiv-Rot F-66813 FW
- Physical state: solid
- Appearence: red powder

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 : x= 30.4 g (27 - 37 g) females: x= 23.7 g (21 - 28 g)
Acclimatization: at least 5 days
Food / water: rat/mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe), ad libitum, tap water in plastic bottles, ad libitum
Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
Room temperature: 22 ± 2 °C
Relative humidity: 55 ± 10 %
Lighting time: 12 hours daily
Animal identification: fur-marking with KMn04 and cage numbering

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Deionised water
Details on exposure:
The test compound dilutions were prepared fresh each day. 6250 mg Reaktiv-Rot F-66 813 FW was weighed in a beaker, mixed with deionisized water, washed out in a 25 ml flask and topped up to the calibration mark. A solution was formed.

For the Endoxan® stock solution, 5 ml distilled water were added to 100 mg Endoxan® in an injection phial and shaken to form a clear solution. The solutions for administration were prepared from this stock solution. For this purpose, 2 ml of the 2 % stock solution were mixed with 6 ml distilled water.

The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 5000 mg Reaktiv-Rot F-66 813 FW 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.
Duration of treatment / exposure:
0 and 5000 mg/kg divided into 2 administrations of 10 ml/kg
Killing of animals (5 males + 5 females per group) and evaluation of micronuclei 24, 48, and 72 hours after administration
Frequency of treatment:
2 administrations within 2 hours on Day 1
Post exposure period:
24, 48, or 72 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5000 mg/kg
Basis:
nominal in water
test article
Remarks:
Doses / Concentrations:
50 mg/kg
Basis:
nominal in water
Endoxan
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
other: positive control: Endoxan
Positive control(s):
Endoxan

Examinations

Tissues and cell types examined:
Bone marrow smears:
- Polychromatic erythrocytes
- Normochromatic erythrocytes
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-Griinwalds solution
2 minutes in May-Griinwalds 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 micro-nuclei 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), (4).

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) (4).
Statistics:
The statistical evaluations were performed using the "Diamant" 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:
no effects
Remarks:
tested up to highest applicable dose
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Animals were treated with 0 and 5000 mg Reaktiv-Rot F-66 813 FW per kg body-weight to study the induction of micronuclei in bone marrow cells of mice.
All animals survived after application of 5000 mg Reaktiv-Rot F-66 813 FW per kg bodyweight. No signs of toxicity were observed.
The bone marrow smears were examined for the occurance of micronuclei in red blood cells.
The incidence of micronucleated polychromatic erythrocytes in the dose groups of Reaktiv-Rot F-66 813 FW 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. A small reduction in the ratio of polycromatic to normochromatic erythrocytes was within the normal range of the historical controls and was considered as of no toxicological significance.
Cyclophosphamid (EndoxanR) induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females indicating the sensitivity to the test system.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Summarizing it can be stated that, under the conditions described, administration of Reaktiv-Rot F-66 813 FW did not lead to a substantial increase of micro-nucleated polychromatic erythrocytes. It is concluded that Reaktiv-Rot F-66 813 FW is not mutagenic in the micronucleus test.
Executive summary:

This test was performed according to OECD guideline for testing of chemicals 474, 1983; Genetic Toxicology, Micronucleus test. No unforeseen cir­cumstances were observed, which may have affected the quality and integrity of this study. The study was conducted in compliance with the principles of Good Laboratory Practice.

 

Reaktiv-Rot F-66 813 FW 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 Reaktiv-Rot F-66 813 FW per kg bodyweight.

 

The 5000 mg per kg bodyweight dose level was chosen since a preliminary studyhad 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.

Endoxan®was used as positivecontrol 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 Reaktiv-Rot F-66 813 FW 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 remained essentially unaffected by the test compound. A small reduction in the ratio of polychromatic to normochromatic erythrocytes was noted for male animals at 24 h killing time only. However this value was within the normal range of the historical controls and was considered as of no toxicologocal significance.

 

Endoxan®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 showed a significant difference to the negative control values.

 

The results indicate that, under the conditions of the present study, Reaktiv-Rot F-66 813 FW is not mutagenic in the micronucleus test.