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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:
2000-05-03 until 2000-06-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
additive
Test material form:
liquid
Specific details on test material used for the study:
Stabilizer: 25 % Isododecane

Test animals

Species:
mouse
Strain:
other: Swiss Ico: OF1 (IOPS Caw)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Iffa Crédo, l'Arbresle, France.
- Age at study initiation: approximately 6 weeks old
- Assigned to test groups randomly: yes, under following basis: upon arrival, the animals were randomly allocated to the groups by sex.
- Housing: in polycarbonate cages
- Diet: A04 C pelleted maintenance diet (UAR, 91360 Villemoisson-sur-Orge, France)
- Water: tap water (filtered using a 0.22 micron filter)
- Acclimation period: at least 5 days before the day of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ±2°C
- Humidity: 30 to 70 %
- Air changes: about 12 cycles/hour of filtered non-recycled fresh air
- Photoperiod: 12 h/12 h (07:00 - 19:00)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Amount of vehicle: 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in the vehicle (corn oil) in order to achieve the concentrations, expressed as active material, of 50, 100 or 200 mg/mL (corresponding to 66.31, 132.63 or 265.25 mg/mL of the test substance as supplied respectively) and then homogenized using a magnetic stirrer. The preparations were made immediately before use.
Duration of treatment / exposure:
Not applicable
Frequency of treatment:
two treatments separated by 24 hours
Post exposure period:
None
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CPA), dissolved in distilled water at a concentration of 5 mg/mL.

Examinations

Tissues and cell types examined:
erythrocytes (evaluation of micronucleated polychromatic erythrocytes) in bone marrow of male and female mice
Details of tissue and slide preparation:
- At the time of sacrifice, all the animals were killed by CO2 inhalation in excess. The femurs of the animals were removed and the bone marrow was eluted out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. All the slides were coded for scoring

- For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocytes ratio was established by scoring a total of 1000 erythrocytes (PE + NE)
Evaluation criteria:
must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.
Statistics:
When there was no significant within-group heterogeneity, using the heterogeneity chi-square test value, the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the %2 value.
When there was significant within-group heterogeneity, then that group was compared with the control group using a non-parametric analysis, the Mann-Whitney test. The student "t" test was used for the PE/NE ratio comparison. Probability values of p < 0.05 was considered as significant.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
At 2000 mg/kg bw/day piloerection and/or hypoactivity were noted in almost all animals 2 and 24 hours after the last treatment. One female from the supplementary group was found dead 24 hours after the last treatment.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In order to select the top dose-level for the cytogenetic study, 2000 mg/kg bw/day were administered, to three males and three females. The interval between each administration was 24 hours. Hypoactivity was noted in all animals 2 and 6 hours following the second treatment. Piloerection was also observed in 2/3 males 2 hours after the second treatment and persisted until sacrifice. In addition, half-closed eyes were noted in all animals 6 hours following the second treatment and persisted in 2/3 males and 1/3 females 18 hours later.
Since no mortality was noted in the preliminary test performed at 2000 mg/kg bw/day therefore this dose-level was selected as the highest dose-level for treatment.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: the mean values of MPE in the groups treated with the test substance, were equivalent to those of the vehicle group
- Ratio of PCE/NCE: the PE/NE ratio was significantly (p < 0.05) lower when compared to that of the vehicle group, in the groups given 2000 mg/kg bw/day (for both males and females) or 1000 mg/kg bw/day (for females only), showing that the bone marrow cells were effectively exposed to the test substance

- Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Any other information on results incl. tables

- The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data.

Applicant's summary and conclusion

Conclusions:
Under the study´s experimental conditions, the test substance does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mg/kg bw/day.
Executive summary:

In this study the test substance was examined for its potential to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of mice according to OECD guideline No. 474 and EU Method B.12.

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study.

In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice received two oral treatments of the test substance at dose-levels of 500,1000 or 2000 mg/kg bw/day, at a 24-hour interval. One group of five males and five females received the vehicle (corn oil) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test substance (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg bw.

The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared.

For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

For both males and females, the mean values of MPE in the groups treated with the test substance, were equivalent to those of the vehicle group. The PE/NE ratio was significantly (p < 0.05) lower when compared to that of the vehicle group, in the groups given 2000 mg/kg bw/day (for both males and females) or 1000 mg/kg bw/day (for females only), showing that the bone marrow cells were effectively exposed to the test substance. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data.

Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Under the experimental conditions, the test substance does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, with a 24-hour interval, at the dose-levels of 500,1000 or 2000 mg/kg bw/day.