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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1989-02-28 to 1989-03-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report equivalent or similar to OECD guideline 474: GLP.

Data source

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

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
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Oronite XS 101 (KWA 88-01217)
- Physical state: colorless liquid
- Storage condition of test material: room temperature in the dark

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 49 days
- Weight at study initiation: 19.5-30.1g (females) and 25.8-35g (males)
- Assigned to test groups randomly: [no/yes, under following basis: ] yes so weights did not exceed ± 20% group mean
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 21
- Humidity (%): 35-53
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: peanut oil
- Concentration of test material in vehicle: 0.470, 0.332, 0.210 g/ml
- Amount of vehicle (if gavage or dermal): 4.0 g/kg (4.40 ml/kg)
Duration of treatment / exposure:
The test material solution/suspension was administered intraperitoneally at dose levels of 5 g/kg (6.25 ml/kg), 2.5 g/kg (3.13 ml/kg), and 1.25 g/kg (1.56 ml/kg). All treated animals received 4.0 g/kg (4.40 ml/kg) peanut oil in addition to the test material. The vehicle groups were dosed with 4.0 g/kg of peanut oil by the same route. The positive control animals received triethylenemelamine (TEM) administered intraperitoneally in isotonic saline at a dose of 0.25 mg/kg delivered at a rate of 5ml/kg.
Frequency of treatment:
one treatment
Post exposure period:
Five males and five females from each treatment group and the vehicle control were sacrificed for bone marrow sampling at 24, 48 and 72 hours after the start of treatment. Positive controls were sampled only at 24 hours.
Doses / concentrations
Remarks:
Doses / Concentrations:
1.25, 2.5, 5.0 g/kg
Basis:
actual ingested
No. of animals per sex per dose:
Negative control (peanut oil): 36 mice (18/sex)
Positive control: 10 mice (5/sex)
low dose test material (1.25g/kg): 36 mice (18/sex)
mid dose test material (2.50 g/kg): 36 mice (18/sex)
high dose test material (5.0 g/kg): 42 mice (21/sex)
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Route of administration: intraperitoneally
- Doses / concentrations: 0.25mg/kg in isotonic saline

Examinations

Tissues and cell types examined:
polychromatic erythrocytes
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The test dose was administered intraperitoneally and animals sacrificed 24, 48, and 72 hours post administration


DETAILS OF SLIDE PREPARATION: Bone marrow from five males and five females from each treatment group were sacrificed for a given time point (10 samples/treatment group). Mice were sacrificed by cervical dislocation and a femur was removed from each animal. The marrow was aspirated from the bone and placed into a conical bottom beaker with fetal bovine serum and centrifuged. The supernatant was decanted and the pellet was resuspended in residual serum. Two bone marrow smears were made from each animal according to the method of Schmid. The slides were fixed in methanol and stained with 4% Giemsa for 20 minutes.


METHOD OF ANALYSIS: The polychromatic erythrocytes were recognized as bluish-gray non-nucleated cells of the same approximate size as the mature orange-pink normochromatic erythrocytes. Micronuclei were recognized as round, darkly stained, blue-purple, sharply contoured particles which were 1/20-1/5 the size of the cell, and which were in the same focal plane as the cell. One slide per animal was selected for scoring. The selected slides were coded and analyzed blindly using a Zeiss Photoscope III with 63x Planapo oil immersion lens. The ratio of NCE to PCE and the %PCE was calculated by counting a total of 1000 erythrocytes. On each slide, 1000 PCE were evaluated for the presence of micronuclei. The number of micronuclei in NCE was also tallied.

Evaluation criteria:
The dispersion test was used to test for excess animal-to-animal variability in the spontaneous frequency of micronucleated polychromatic erythrocytes (MN-PCE) among vehicle controls within a day and to test for excess day-to-day variability in vehicle control groups. If excess intra-day variability was detected, the data was inspected to determine if the outlier(s) was outside the historical range (0-6 micronucleated cells/1000 cells). If it was not, the data was unacceptable. If it was, but the group mean was less than or equal to 5 micronucleated cells/1000 cells, the data was acceptable. If no excess inter-day variability was detected, vehicle control data from the three sampling points were combined for each sex. If there was excess day-to-day variability, the vehicle control data were not combined and the treated groups were compared to the concurrent controls.
Statistics:
A one-tailed Fisher exact test and a binomial approximation test were performed to assess significance compared to vehicle control groups. Since multiple comparisons were made, Bonferroni corrections were made to adjust the probability value required for significance. If no difference was detected at the 5% levels of significance, the results were negative. If a differences was detected, the dose response was analyzed using the one-tailed Cochran-Armitage test for trend in binomial proportions. If this test detected a trend at the 5% level, the results were considered to be positive. If a trend was not detected, the Study Director evaluated the variability observed in the vehicle control and the nature of the statistically significant responses.

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

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The in vivo micronucleus assay of the test material in mice was negative. This finding does not warrant the classification of the test material as a genotoxin under EU GHS guidelines and does not warrant classification under the EU requirements for dangerous substances and preparations.
Executive summary:

The test material was examined for its potential to induce chromosomal damage in mouse bone marrow erythrocytes in mice dosed interperitoneally with the test material at 1250, 2500 and 5000 mg/kg. Vehicle and positive control animals received peanut oil and triethylenemelamine, respectively. Bone marrow was collected from 10 animals (5/sex) from each treatment and vehicle control group at 24, 48, and 72 hours after administration.  Positive controls were sampled at 24 hours.  No clinical signs of toxicity were observed during the study.  Cytotoxicity was detected in males in the positive control and at the 1250 mg/kg dose level sampled at 24 hours and in females at the 2500 and 5000 mg/kg dose levels sampled at 48 hours. No significant increase in the number of micronucleated polychromatic erythrocytes was observed in any treated group when compared to control animals.  The positive control induced micronuclei as expected.  The test material was considered to be non-genotoxic and non-clastogenic under the conditions of this test. This finding does not warrant the classification of the test material as a genotoxin under EU GHS guidelines and does not warrant classification under the EU requirements for dangerous substances and preparations.