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Toxicological information

Genetic toxicity: in vivo

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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:
supporting study
Study period:
From 28 NOV 1988 to 1 DEC 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
o-anisidine
EC Number:
201-963-1
EC Name:
o-anisidine
Cas Number:
90-04-0
Molecular formula:
C7H9NO
IUPAC Name:
2-methoxyaniline
Details on test material:
- Name of test material (as cited in study report): o-Anisidin D

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF-breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: males mean: 32 g; females mean: 25.1 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:
for test compound: sesame oil
for positive control: water
- Concentration of test material in vehicle: 250 mg/ml
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test compound:
Preparation of compounds dilution was done freshly each day.
2500 mg o-Anisidin D were weight in a 10 ml flask, mixed with sesame oil and topped up to the calibration mark. A solution was formed.

Positive control:
For Endoxan(R) 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:
24, 48 or 72 h
Frequency of treatment:
once
Post exposure period:
24, 48 or 72 h
Doses / concentrations
Remarks:
Doses / Concentrations:
0 and 1000 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
- Dose: 50 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 to test the acute toxicity, a dose of 1000 mg o-Anisidin D per kg bodyweight was the maximum tolerated dose level.

DETAILS OF SLIDE PREPARATION:
At the indicated times a suspension of the bone marrow of both femora 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 an 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.
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 "Diamant" computer program Version 2.0. 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
Remarks:
clinical signs: increased spontaneous activity, ataxic galt, abdominal position, widened palpebral fissures, piloerection, urine brown-orange coloured, 72 hours after application all animals were free of clinical signs of toxicity
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Applicant's summary and conclusion

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

o-Anisidin D was tested in the micronucleus test conducted according to OECD guideline 474. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 1000 mg o-Anisidin D per kg bodyweight.

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 incidence of micronucleated polychromatic erythrocytes of the animals treated with o-Anisidin D 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 o-Anisidin D. The ratio of polychromatic to normochromatic erythrocytes in the treatment group was statistically different from control values but this effect was considered as of no toxicological significance.

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

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