Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 25 MAR 1981 to 2 APR 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given: comparable to guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar 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 animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, company breeding colony
- Age at study initiation: 7 to 12 weeks
- Weight at study initiation: males mean: 33 g; females mean: 26 g
- Housing: grouped (5 animals per cage) in macrolon cages (type 3) in fully airconditioned rooms
- Diet: rats/mice diet Altromin1324 (Altromin GmbH, Lage/Lippe, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2
- Humidity (%): 55+/-10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 2% starch mucilage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test compound:
Preparation of compounds dilution was done freshly each day.
6250 mg of test item were weight in a beaker, mixed with 2% starch mucilage, transferred into a 25 mL flask and topped up to the calibration mark. A solution was formed by stirring for 5 minutes on a magnetic stirrer.

Positive control:
For Endoxan(R) (= Cyclophosphamide) stock solution, 5 ml of distilled water were added to 100 mg Endoxan(R) 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.
Duration of treatment / exposure:
30 hours in total (1st application, lack phase of 24 hours, 2nd application, after 6 hours termination of test)
Frequency of treatment:
twice
Post exposure period:
6 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 500, 5000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: 100 mg/kg bw

Examinations

Tissues and cell types examined:
polychromatic erythrocytes derived from the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
According to a preliminary study, the maximal applicable dose of the test item is 5000 mg per kg bodyweight, which was administered twice.

DETAILS OF SLIDE PREPARATION:
At the indicated time a suspension of the bone marrow of both femora was formed. The mixture was then centrifuged for 5 minutes at about 1000 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared an a cleaned slide and air-dried for about 24 hours.

Staining procedure
- 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 distilled water
- rinsing in distilled water
- drying with filter paper
- cleaning the backside of the slide with methanol
- 5 minutes in xylene
- coating with Entellan
Evaluation criteria:
2000 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.
Statistics:
The number of polychromatic erythrocytes with micronuclei occurring in the 2000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically. Increases in dose groups compared to the simultaneous control group were determined using binominal distribution. Differences in dose groups compared to the simultaneous control group were checked using the method of Nemenyi (separately for both sexes).
The statistical evaluations were performed using a in-house computer program. All statistical results are based on a 95 % level of significance.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Faeces was stained orange in all animals of dose group 500 and 5000 mg/kg bw.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The results indicate that, under the conditions of the present study, the test item is not mutagenic in the micronucleus test.
Executive summary:

The test item was tested in a micronucleus test conducted similar to OECD guideline 474. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0, 50, 500 and 5000 mg per kg bodyweight.

The animals were treated twice within 24 h with the test compound and according to the test procedure the animals were killed 6 hours after the second administration of the test compound.

The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test item was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased compared to the control animals.The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment.

The positive control (Cyclophosphamide =EndoxanR) yielded positive results and therefore indicating the sensitivity of the system.