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:
7 September 1998 - 11 November 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test was conducted according to OECD Test Guideline No. 474, 1997, under GLP Standards, and QA
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
92/69/EEC
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
Statement of Compliance
Type of assay:
micronucleus assay

Test material

Constituent 1
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): Aluminiumhydroxichlorid
- Physical state: Solid
- Analytical purity: Confidential information
- Purity test date: Confidential information
- Lot/batch No.: Confidential information
- Expiration date of the lot/batch: Confidential information
- Stability under test conditions: Guaranteed for 4 hours
- Storage condition of test material: Darkness at room temperature in a fume cupboard

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH
- Age at study initiation: male/female animals approx. 7 weeks
- Weight at study initiation: Males: 35 – 43 g; Females: 29 – 32 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: in fully air-conditioned rooms in makrolon cages type 3 (five animals) on soft wood granulate
- Diet: rat/mice diet ssniff R/M-H (V 1534), ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: 5 days under study conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: soluble in deionized water
- Concentration of test material in vehicle: (volume) 20% (w/v)
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the days of administration the test substance was dissolved in deionized water at the appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

DIET PREPARATION: no data
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Twice at an interval of 24 hours
Post exposure period:
Animals were killed by carbon dioxide asphyxiation 24 hours after dosing.
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg kg bw
Basis:
analytical conc.
No. of animals per sex per dose:
Five male and five female mice were used per sampling time in each treatment group (negative and positive control, and test substance)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (Endoxan)
- Route of administration: oral gavage
- Doses / concentrations: 50 mg kg bw / 0.5 % (w/v)

Examinations

Tissues and cell types examined:
bone-marrow smears; micronucleated polychromatic erythrocytes, and ratio polychromatic erythrocytes (PCE)/total erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In a preliminary dose range finding study, oral administration of 2000 mg Locron P per kg body weight did cause only slight toxic effects in male and female mice over an observation period of 7 days. This dose level was therefore the regularly limit dose, selected as the highest dose for the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): two oral administrations at an interval of 24 hours, and one sampling time (24 hours).

DETAILS OF SLIDE PREPARATION: One drop of the thoroughly mixed sediment of bone marrow was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours. Staining was performed as follows: 5 min. in methanol, 5 min. in May-Grunwald's solution; brief rinsing twice in distilled water, 10 min. staining in Giemsa solution, rinsing in distilled water, drying, and coating with Entellan.

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.
Evaluation criteria:
Both biological and statistical significances were considered together for evaluation purposes. A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
Averages and standard deviations were calculated. One-sided Wilcoxon tests were performed.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg kg bw
- Clinical signs of toxicity in test animals: Spontaneous activity decreased up to 6 hours after application, but no deaths were recorded (total 3 males and 3 females tested)
- Evidence of cytotoxicity in tissue analyzed: No macroscopic findings were observed.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Locron P did not statistically significantly raise the frequency of micronucleated polychromatic erythrocytes.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the groups which were treated with Locron P showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis.
The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained essentialy unaffected by the treatment with Locron P and was not less than 20% of the control value.
- Appropriateness of dose levels and route: In the preliminary dose range finding study, oral administration of 2000 mg kg bw did cause only slight toxic effects in male and female mice over an observation period of 7 days. This dose level was therefore the regularly limit dose, selected as the highest dose for the main study.

- Statistical evaluation: 2000 mg Locron P/kg bw did not show a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The results lead to the conclusion that Locron P did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described in this report.
Executive summary:

The micronucleus test was carried out with Locron P. The test compound was dissolved in deionized water and was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay. According to the test procedure the animals were killed 24 hours after administration. Endoxan (Cyclophosphamide) was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight. The number of polychromatic erythrocytes containing micronuclei was not increased following treatment with Locron P. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with Locron P and was not less than 20% of the control value. The positive control, Endoxan, induced a marked and statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent. Under the conditions of the study the results indicate that Locron P is not mutagenic and not aneugenic in the micronucleus test.