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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 based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From November 26 to December 27, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted according to internationally accepted testing guidelines and performed according to GLP. Justification for Read Across is reported in the endpoint summary and in the Category Justification Report attached to the Section 13 of this dossier.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted May 26, 1983
Deviations:
no
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: BRL Tierfarm Fullinsdorf.
- Age at study initiation: minimum 10 weeks.
- Weight at study initiation: approximately 30 g.
- Assigned to test groups randomly: yes
- Fasting period before study: approximately 18 hours before treatment with the test article the animals received no food but water ad libitum.
- Housing: singly housed in Makrolon Type I, cages with wire mesh top.
- Bedding: granulated soft wood bedding.
- Diet: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe).
- Water: tap water, ad libitum.
- Acclimation period: minimum 5 days.
- Helth and quarantine: the animals were in healthy condition. The animals were under quarantine in the animal house of laboratory for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 25 to 70 %
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: water dest.
- Justification for choice of vehicle: the vehicle was chosen to its nontoxicity for the animals.
- Volume: 20 ml/kg bw
Details on exposure:
At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal’s body weight.
Frequency of treatment:
Single administration.
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
6 males, 6 females per test group; nevertheless only 5 animals per sex per group were treated. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide:
- Route of administration: single oral (gavage) dose.
- Solvent: physiological saline.
- Doses: 30 mg/kg bw.
- Volume administered: 10 ml/kg bw.
Solution prepared on day of administration.
The stability of CPA at room temperature is good. At 20 °C only 1 % of CPA is hydrolysed per day in aqueous solution.

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes from femurs of all animals
Details of tissue and slide preparation:
SAMPLING
Sampling of the bone marrow was done at 24, 48 and 72h after treatment.

DETAILS OF SLIDE PREPARATION
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum using a 5 mL syringe. The cell suspension was centrifuged at 1500 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Gruenwald (MERCK, D-6100 Darmstadt, F.R.G.)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G.). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.
Evaluation criteria:
A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points.

A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any one of the test points is considered non-mutagenic in this system. This can be confirmed by means of the nonparametric Mann-Whitney test.

However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In a pre.experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w. of the test item dissolved in aqua dest. The volume administered was 20 ml/kg b.w.
One of the treated animals expressed a reduction of spontaneous activity from 1 to 48 hours.
Higher dosing was not attainable:
- appropriate suspensions (homogeneity, viscosity) could be obtained only up to 250 mg/ml.
- application volumes higher than 20 ml/kg b.w. were not justifiable for the rodents used.

RESULTS OF DEFINITIVE STUDY
The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that the test item had no cytotoxic properties.
In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment were in the same range as compared to the negative control groups.

30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus frequency.

Any other information on results incl. tables

Experiment results

Sampling time Group/dose PCE's with micronuclei (%) Range PCE/NCE
24 h vehicle control 0.06 0 -2 1000/829
5000 mg/kg bw 0.05 0-2 1000/978
CPA 30 mg/kg bw 0.63 4 -11 1000/838
25 h vehicle control 0.07 0-2 1000/888
5000 mg/kg bw 0.06 0-3 1000/894
72 h vehicle control 0.06 0-2 1000/769
5000 mg/kg bw 0.13 0-4 1000/834

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Tthe test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
Executive summary:

Method

The study was performed to investigate the potential of the test compound to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was dissolved in aqua dest. This solvent was used as negative control. The volume administered orally was 20 ml/kg b.w.. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 5000 mg/kg b.w..

In a pre-experiment this dose level was estimated to be the maximum attainable dose.

An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency.

Results

After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.