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 the 29th Aaugust to the 7th November, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to the European Guidelines based on the OECD Test Guideline, in GLP

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
EEC Directive 84/449, L 251, B 12, p. 137 - 139
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Environmental fate/Ecotoxicological/Toxicological studies:
Storage: Room temperature, 20-24 °C, in the dark
Stability: pure, for 5 years; in solvent > 24 hours in water, DMSO, DMF, stable for at least 2 hours in aqueous medium.
Evidence of chemical instability at pH 6: none
Safety precaution: routine hygienic procedures were sufficient to assure personnel health and safety.
Toxicological studies:
Preparation: On the day of the experiment, the test article was suspended in Carboxymethylcellulose (1 %).
The vehicle was chosen to its nontoxicity for the animals. All animals received a single standard dose volume of 10 mL/kg body weight orally.

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Strain: NNRI
Source: BRL Tierfarm Füllinsdorf CH- 4414 Füllinsdorf/Basel, Switzerland
Number of Animals: 84 (42 males/42 females)
Initial Age at Start of Acclimatization: minimum 10 weeks
Acclimatization: minimum 5 days
Initial Body Weight at Start of Treatment: approximately 30 g
According to the suppliers assurance the animals were in healthy condition.
The animals were under quarantine in the animal house of CCR for one week after their arrival. During this period the animals did not show any signs of illness or altered behaviour.
The animals were distributed into the test groups at random and identified by cage number.

HUSBANDRY
Housing: single
Cage Type: . Makrolon Type I, with wire mesh top (EHRET GmbH, D-7830 Einmendingen)
Bedding: granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
Feed: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
Water: tap water, ad libitum (Gemeindewerke, D61O1 Rossdorf, F .R.G.)
Enviromnent: temperature 21 ± 3 °C
relative humidity 30-70 %
artificial light 6.00 a.m.- 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The vehicle of the test article was used as negative control.
Carlioxymethylcellulose
Source: SERVA, D-6900 Heidelberg
Batch: 17681
Somministration: orally, singly
Volume: 10 mL/kg b.w.
Details on exposure:
DOSE SELECTION
It is generally recommended to use the maximuiri tolerated dose or the highest dose that can be formulated and administered reproducibility. The volmne to be administered should be compatible with physiological space available.
The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on sur vival within 72 hours.
Duration of treatment / exposure:
24, 48 and 72 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5000 mg/kg b.w.
Basis:
actual ingested
pre-experiment 1°
Remarks:
Doses / Concentrations:
1500 mg/kg bw
Basis:
actual ingested
pre-experiment 2°
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested
pre-experiment 2°
No. of animals per sex per dose:
6 males
6 females
Positive control(s):
CPA; Cyclophosphamide
Source: SERVA, D-6900 Heidelberg, F.R.G.
Batch: 17681
Dissolved in: physiological saline
Somministration: 40 mg/kg li.w., orally, singly
Volume: 10 mL/kg b.w.
Solution prepared on day of administration.
The stability of CPA at room temperature is good. At 20 °C only 1 % of CPA is hydrolised per day in aqueous solution.

Examinations

Tissues and cell types examined:
To describe a cytotoxic effect the ratio between polychromatic and normochrcmatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.
Details of tissue and slide preparation:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1.000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polycliromatic erythrocytes nor a
statistically significant and reproducible positive response at anyone of the test points is considered nonmutagenic in this system.
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
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
pre-experiment 1°(4 males/4 females 5000 mg/Kg bw)
All surviving animals expressed toxic reactions: reduction of spontaneous activity, eyelid closure and apathy. One male and one female died within the first hour after treatment.
pre-experiment 2° (2 males/2 females 1500 mg/Kg bw)
All treated animals expressed toxic reactions: reduction of spontaneous activity. One female expressed abdominal position.
Additionally, one male and one female expressed eyelid closure and apathy.
2000 mg/Kg bw
All treated animals died within 6 hours after treatment.

After treatment with 1500 mg/kg b.w.of the substance one male and one female died at preparation interval 24 hours, one female died at preparation interval 48 hours, and one female died at prepa ration interval 72 hours.
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 no cytotoxic properties.

In comparison to 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 with were in the same range as comparedto the nega tive control groups.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
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.
Therefore, the substance is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

This study was performed to investigate the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was suspended in Carboxymethylcellulose (1 %). This suspending agent was used as negative control. The volume administered orally was 10 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.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 1500 mg/kg b.w.

In pre-experiments this dose level was estimated to be the maximum tolerated dose. The animals expressed toxic reactions.

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 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.

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

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.

Therefore, the substance is considered to be non-mutagenic in this micronucleus assay.