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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:
key study
Study period:
May 2001 - November 2001
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
none

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males: 32.3 g +/- 1.6 g; females: 26.4 +/-1.5 g
- Assigned to test groups randomly: yes, under following basis: 25 male and female animals each were assigned to 5 experimental groups to 5 animals per group and sex using random-charts. The cages were labelIed with coloured cage cards showing the study number, dose group, time
of sacrifice, animals' sex and numbers and date of dosing.41
- Fasting period before study: Overnight fasting before oral administration and until 3 hours after administration food was available ad libitum.
- Housing: Altromin Type S8/15, granulated soft wooed bedding, batch no 060801
- Water (e.g. ad libitum): tap water (municipal supply), Makrolon bottles, changed daily
- Acclimation period: 7 days before start of dosing (pre-experiment for toxicity)
13 days before start of dosing (main experiment)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-24°C
- Humidity (%): 45-65%
- Air changes (per hr): air conditioned
- Photoperiod (hrs dark / hrs light): artificial light was set to give a cycle of 12 hours light and 12 hours dark; Light from 6.30 a.m. until 6.30 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: The test item was solved in deionised water shortly before administration
- Concentration of test material in vehicle: 2000 mg/kg body weight
- Amount of vehicle (if gavage or dermal): 10 ml/kg body weight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
test item: limit dose of 2000 mg/kg body weight
vehicle control : Deionised water (10 mI/kg body weight)
positive control : 40 mg/kg body weight CPA (Cyclophosphamide)
Duration of treatment / exposure:
Bone marrow smears were prepared at 12 hours (dose group), at 24 hours (vehicle contral, positive control, dose graup) and at 48 hours (dose group) after dosing.
Frequency of treatment:
single treatment with limit dose of 2000 mg/kg body weight
Post exposure period:
After administration the animals were monitored at approximately 1, 6, 24 and 48 hours for any symptoms of acute toxicity.
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal in water
No. of animals per sex per dose:
25 male and female animals each were assigned to 5 experimental groups to 5 animals per group and sex.
2 animals per sex were treated.
Control animals:
yes
Positive control(s):
Cyclophosphamide (CPA), dissolved in deionised water
- Route of administration: oral administration using a metal catheter
- frequency of administration: single dose
- dose: 40 mg/kg body weight
- volume administered: 10 ml/kg body weight

Examinations

Tissues and cell types examined:
bone marrow removed from the femurs.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the results of the pre-experiment the limit dose of 2000 mg/kg body weight could be chosen

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
sampling intervals of 12, 24 and 48 hours after treatment. Only the sampling interval of 24 hours was chosen for the vehicle and the positive
contral group.

DETAILS OF SLIDE PREPARATION:
Bone marrow was removed from the femurs and smears prepared on microscope slides. The smears were aged for approximately 24 hours before staining with May-Grünwald/Giemsa solution.

METHOD OF ANALYSIS:
All slides were coded before scoring and scored blind. A minimum of 2000 polychromatic erythrocytes (PCE) was scored for the presence of micronuclei (= MPCE = micronucleated polychromatic erythrocytes) for each animal. The proportion o fPCEs among total erythrocytes
(PCEs + normochromatic erythrocytes [NCE]) was determined for each animal on the basis of 200 erythrocytes.
Evaluation criteria:
The test item is classified mutagenic, if it induces a statistically significant increase at the sampling times of 12, 24 or 48 hours with biological relevance. A statistically significant increase might require further confirmation by the demonstration of a dose response relationship at the respective sampling time.
Statistics:
Micronucleus scores (MCPE) and the proportion of PCEs among total erythrocytes are presented as individual values. In addition group means and standard deviations are calculated for each sex and experimental group. The statistical significance compared to the vehicle control were proved by means the Welch t-test (Rasch et al., Verfahrensbibliothek Versuchsplanung und -auswertung, Berlin 1981).
Statistical significance is declared at the 5 % level (one-sided).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
2000 mg/kg bw
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg body weight
A preliminary study on acute toxicity was performed with a small group of animals under identical conditions to these in the mutagenicity study with respect to animals, vehicle, route, frequency and volume of administration to assess the approximate MTD (maximum tolerated dose).
Two animals per sex were treated with a single oral dose of 2000 mg/kg body weight. This dose corresponds to the limit dose in accordance with the OECD Guideline. After administration the animals were monitored at approximately 1, 6, 24 and 48 hours far any symptoms of acute toxicity.
None ofthe animals died.

RESULTS OF DEFINITIVE STUDY
Mean values of micronuc1eated polychromatic erythrocytes (MPCEs) of 0.20 % (males) and 0.22 % (females) were found for the vehic1e control group. The range ofthe historical negative controls in the testing facility (since 1997) was 0.11 to 0.24 % in males and 0.12 to 0.27 % in females.
In BRÜGGOLIT FF6 treated groups mean MPCE values were in the range from 0.19 to 0.26 % in males and from 0.18 to 0.27 % in females.
The statistical analysis of the MPCE counts did not show any statistically significant difference in comparison to the vehicle control.
Treatment with the positive control chemical (CycJophospharnide, 40 mglkg body weight) induced statistically significant increases in the incidence of MPCEs with a group mean value of 2.79 % in males and 2.49 % in females.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
not to be classified
Executive summary:

Groups of 5 male and 5 female NMRI-mice were exposed to the test item at the limit dose of 2000 mglkg body weight. The test item was administered orally using a metal catheter. Deionised water (10 mI/kgbw) served as vehicle control and Cyclophosphamide (CPA) at a dose of 40 mglkg body weight - also administered orally - was used as positive control.

A dose of 2000 mg/kgbw was chosen as the maximum dose in accardance with the guideline because no of the animals died at this dose in the pre-experiment.

Bone marrow smears were prepared at 12 hours (dose group), at 24 hours (vehicle contral, positive control, dose graup) and at 48 hours (dose group) after dosing.

Two thousand polychromatic erythrocytes per animal were analysed far the presence of mieranuclei. To investigate bone marrow toxieity the proportion of polyehromatie erythrocytes among total erythrocytes was evaluated on the basis of 200 erythrocytes.

The frequency of mieronucleated polychromatie erythroeytes (MPCEs) in the vehicle control group was within the physiologieal range. Treatment with CPA induced statistieally significant inereases in the ineidenee of MPCEs.

In none ofthe experimental groups treated with the test item an increase in MPCEs was observed.

The treatments induced statistically significant decreases of the proportion of polychromatic erythrocytes among total erythrocytes in all cases in female animals and for the last sacrifice time in the male animals. But these values are in the range of the historical control data in the testing facility. Therefore it is deemed these changes are ineidental and not eaused by the administration ofthe test item.

Based on the results of the study reported here it is concluded that the test item does not induce micronuclei in polychromatic erythrocytes of NMRI-mice under the described experimental conditions. The test item is therefore considered to be non-mutagenic in the mouse bone marrow micronucleus test.