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:
From September 27, 1999 to November 26, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
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
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Reaktiv-Orange F97-0318

Test animals

Species:
mouse
Strain:
other: HsdWin:NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchen
- Age at study initiation: Male/female animals approximately 8 weeks
- Body weight range at treatment: Males: 32-40 g; Females: 27-32 g
- Housing: In fully air-conditioned rooms in makrolon cages type 3 (five animals/cage) on soft wood granulate
- Diet: Rat/mice diet ssnif R/M-H (V 1534), ad libitum
- Water: Tap water in plastic bottles, ad libitum
- Acclimation period: 5 d under study conditions
- Animal identification: Fur marking with KMn04 and cage numbering
- Randomization procedure: Randomization schemes 1999.0432 and 1999.0433

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 50±20%
- Photoperiod: 12 h light/dark cycle

IN-LIFE DATES: From: To: September 27, 1999 to November 26, 1999

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Deionized water
- Concentration of test material in vehicle: 20% (w/v)
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SUSPENSION: On the days of administration the test substance was dissolved in deionized water at the appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.
Duration of treatment / exposure:
2 d
Frequency of treatment:
twice at an interval of 24 h
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5/sex/group
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control: Endoxan® containing cyclophosphamide
Dissolved in: Distilled water
Dose: 50 mg/kg bw
Concentration: 0.5% (w/v)
Route and frequency of administration: Oral (gavage), once
Volume Administered: 10 mL/kg bw

Examinations

Tissues and cell types examined:
- 2,000 polychromatic erythrocytes were counted for each animal.  
- The number of cells with micronuclei was recorded, not the number of individual micronuclei.  
- The ratio of polychromatic erythrocytes to 200 total erythrocytes was determined.
- Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2,000 counted erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on the results of preliminary dose range finding study the dose of 2,000 mg/kg bw which did not cause any toxic effects in male and female mice over an observation period of 7 d, was selected as the highest dose, for the main study. This selected dose level also represents the limit dose according to the regulations.

-Preparation and Staining: Animals were killed by carbon dioxide asphyxiation 24 h after dosing. For each animal, about 3 mr fetal 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 approximately 1,200 rpm, after which almost all the supernatant 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 h.

-Staining procedure: The staining was performed as follows:
-5 minutes in methanol
-5 minutes in May-Gronwald's solution
-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:
Both biological and statistical significances were considered together for evaluation purposes. A test substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
After confirming the validity of the study the following sequential test procedure for the examination of the mutagenicity was applied: Based on a monotone-dose-relationship, one-sided Wilcoxon tests were performed starting with the highest dose group. These tests were performed with a multiple level of significance of 5%.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
orange discolored urine and red discolored feces were observed as clinical signs after administration of test substance
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- All animals survived after treatment. Orange discolored urine and red discolored feces were observed as clinical signs. 24 h after the applications all animals were free of clinical signs.
- The dissection of the animals revealed no test substance related macroscopic findings.
- The bone marrow smears were examined for the occurrence of micronuclei in red blood cells. The incidence of micronucleated polychromatic erythrocytes in the dose group of test substance was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test substance and was not less than 20% of the control values.
- Cyclophosphamide (i.e., endoxan) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of mice. Therefore, the test substance was not considered to be mutagenic in this micronucleus assay.
Executive summary:

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of HsdWin:NMRI mice according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP.

 

The test substance was dissolved in deionized water and given twice at an interval of 24 h as an oral dose of 2,000 mg/kg bw/day to male and female mice. The dose was selected based on the results of a previous dose range finding study. The animals were sacrificed 24 h after the last administration and bone marrow cells were collected for micronuclei analysis.

The positive control (endoxan) induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. After treatment with the test substance, the number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected and was not less than 20% of the control value.

 

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of mice. Therefore, the test substance was not considered to be mutagenic in this micronucleus assay.