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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:
14 January 2003 - 23 February 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed according to OECD guidelines and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
• name: 2,5-DIMETHYL-2,5-DI(2-ETHYLHEXANOYLE PEROXY)-HEXANE
• CAS number: 13052-09-0

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, l'Arbresle, France
- Age at study initiation: on the day of treatment, the animals were approximately 6 weeks old.
- Weight at study initiation: no data
- Assigned to test groups randomly: yes, under following basis: by sex
- Fasting period before study: no
- Housing: by groups in polycarbonate cages
- Diet (e.g. ad libitum): ad libitum, A04 C pelleted maintenance diet (SAFE, Villemoisson-sur-Orge, France).
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before the day of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30-70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 14 January 2003 - 23 February 2003

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
The vehicle was corn oil, batch Nos. 81K2204 and 062k0006 (Sigma, Saint-Quentin-Fallavier, France).
Details on exposure:
Route for the vehicle and the test substance: at the request of the Sponsor, the intraperitoneal route was used,
• Volume: 10 mL/kg,
• CPA: oral route, one treatment
Frequency of treatment:
Two treatments separated by 24 hours
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
5 except for the high dose group where 8 animals were used
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPA)
- Justification for choice of positive control(s): guideline recommendation
- Route of administration: Oral
- Doses / concentrations: Batch No. OD203A (Laboratoire Asta Médica, Mérignac, France) dissolved in distilled water at a concentration of 5 mg/mL. The preparation was stored at -20°C and thawed immediately before use.

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In order to select the top dose-level for the cytogenetic study, 2000 mg/kg/day were administered twice, to three males and three females. The interval between each administration was 24 hours. Except for piloerection noted in both males and females, no clinical signs were noted. The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no severe toxic effects were observed, the top dose-level was 2000 mg/kg/day. The two other selected dose-levels for the main test were 500 and 1000 mg/kg/day.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 after the last treatment bone marrow samples were taken

DETAILS OF 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 flushed out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).

METHOD OF ANALYSIS: 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). The analysis of the slides was performed at Microptic, cytogenetic services (2 Langland Close Mumbles, Swansea SA3 4LY, UK), in compliance with GLP, and the Principal Investigator was Natalie Danford.
Evaluation criteria:
For a result to be considered positive, a statistically significant increase in the frequency of MPE 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 (Lovell et al., 1989) (d), 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 χ2 value (Lovell et al., 1989) (d). 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 (Schwartz, 1969) (e). 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
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No clinical signs and no mortality were observed in the animals of both sexes given 500 and 1000 mg/kg/day. Piloerection was noted in both males and females at 2000 mg/kg/day.
For both males and females, the mean values of MPE in the groups treated with the test item, were equivalent to those of the vehicle control group.
The PE/NE ratio was significantly (p < 0.001) lower when compared to that of the vehicle control group, in the males treated at 2000 mg/kg/day, showing that the bone marrow cells were effectively exposed to the test item.
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.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under our experimental conditions, the test item did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mg/kg/day.
Executive summary:

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 mice received two intraperitoneal treatments at dose-levels of 500, 1000 or 2000 mg/kg/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 item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.

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).

The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no severe toxic effects were observed, the top dose-level was 2000 mg/kg/day.

The two other selected dose-levels for the main test were 500 and 1000 mg/kg/day. For both males and females, the mean values of MPE in the groups treated with the test item, were equivalent to those of the vehicle control group. The PE/NE ratio was significantly lower when compared to that of the vehicle control group, in the males treated at 2000 mg/kg/day, showing that the bone marrow cells were effectively exposed to the test item.

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

Under our experimental conditions, the test item did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells

after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mg/kg/day.