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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Apparently well conducted GLP study.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
The relative humidity recorded in the animal room was sometimes outside the target range specified in the protocol. This minor deviation was not considered to have compromised the validity or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
additive
Test material form:
other: colorless liquid
Details on test material:
The test substance was identified as follows:
· name:
- protocol and labelling: t-AMYL PEROXYPIV ALATE
· batch number:
- protocol and labelling: 11623-9801-708
· description:
- at receipt: colorless liquid
- on the test article description: colorless liquid
container: one plastic flask
· date of receipt 13 April 2000
· storage conditions: at -20°C and protected from light
· purity: 75.9% in Isododecane
· expiry date: March 2001.

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals or test system and environmental conditions:
Number: . 12 male and 12 female mice for the preliminary toxicity test
56 mice: 28 males and 28 females for the cytogenetic study.
Strain: Swiss leo: OFI (lOPS Caw).
Reason for this choice: rodent species generally accepted by regulatory authorities for this type
of study.
Breeder: Iffa Credo, I'Arbresle, France.
Age: on the day of treatment, the animals were approximately 6 weeks old.
Veterinary care at CIT: upon their arrival at CIT, the animals were given a complete examination
to ensure that they were in good clinical conditions.
Acclimatisation: at least 5 days before the day of treatment.
Constitution of groups: upon arrival, the animals were randomly allocated to the groups by sex'
Subsequently, each group was assigned to a different treatment group.
Identification: individual tail marking upon treatment.

Environmental conditions
Upon their arrival at CIT, the animals were housed in an animal room, with the following
. environmental conditions:
· temperature: 21 ± 2°C,
· relative humidity: 30 to 70%,
· light/dark cycle: 12 hl12 h (07:00 - 19:00),
· ventilation: about 12 cycles/hour of filtered non-recycled fresh air.
The housing conditions (temperature, relative humidity, light/dark cycle and ventilation) were
checked regularly. .
The animals were housed by groups in polycarbonate cages. Each cage contained autoclaved
sawdust (SICSA, 94142 Alfortville, France).
Bacteriological and chemical analysis of the sawdust, including the detection of possible
contaminants (pesticides, heavy metals) of the sawdust are performed by the suppliers.

Food and water
All animals had free access to A04 C pelleted maintenance diet (UAR, 91360 ViIlemoisson-surOrge,
France) and tap water (filtered using a 0.22 micron filter) ..
Each batch of food was analysed (composition and contaminants) by the supplier.
Bacteriological and chemical analysis of water, including the detection of possible contaminants
(pesticides, heavy metals and nitrosarnines) are performed regularly by extemallaboratories. The
results of these analyses are archived at CIT.
No contaminants are known to be present in the diet, drinking water or bedding material at
levels which may be expected to interfere with or prejudice the outcome of the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Administration
· Route for the vehicle and the test substance: oral, since it is a possible route of exposure in man
· Frequency: two treatments separated by 24 hours,
· Volume: 10 m/kg,
· CPA: oral route, one treatment.
The quantity of each substance administered to each animal was adjusted according to the most
recently recorded body weight.
Frequency of treatment:
Two treatments separated by 24 hours
Post exposure period:
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.
Doses / concentrations
Remarks:
Doses / Concentrations:
62.5, 125 or 250 mg/kg/day (expressed as active material), at a 24-hour interval
Basis:
other: percent active
No. of animals per sex per dose:
In the main study, three groups of five male and five female mice.
Control animals:
yes, concurrent vehicle
Positive control(s):
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.

Examinations

Tissues and cell types examined:
Bone marrow smears were then prepared. For each animal, the number of the micronuc1eated 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).
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 normochrornatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
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 and colI., 1989), 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 x2 value (Lovell and colI., 1989). 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).
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
Positive controls validity:
valid
Additional information on results:
In order to select the top dose-level for the cytogenetic study, 2000, 1000, 500 and 250 mg/kg/day were administered, to three males and three females. The interval between each administration was 24 hours.
At 2000 and 1000 mg/kg/day, all animals were found dead 24 hours after the first treatment. At 500 mglkg/day, hypoactivity and/or piloerection were noted in 113 males and 1/3 females, 2 and 6 hours after the second treatment. These two animals were found dead 24 hours after the second treatment.
At 250 mglkg/day, piloerection was observed in all animals 6 hours after the second treatment and persisted 18 hours later. The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since observable toxic effects were noted, the top dose-level was based on the toxicity level, such that a higher dose-level was expected to induce lethality. . .
Consequently, 250 mg/kg/day was selected as the top dose-level. The two other dose-levels were 125 and 62.5 mg/kg/day.

Any other information on results incl. tables

No clinical signs and no mortality were observed in the animals of both sexes given 125 and 62.5 mg/kg/day. At 250 mg/kg/day, piloerection was noted in 3/5 males as well as in 1/5 females from the main group. For both males and females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test substance, were equivalent to those of the vehicle group. The mean values of MPE as wen as the PE/NE ratio for the vehicle control groups were consistent with our historical data. For one female from the positive control group (corresponding to slide 26) no significant increase in the frequency of MPE was noted. However since for the remaining nine animals cyclophosphamide induced a clear increase in the frequency of MPE and since a very significant increase (p < 0.001) in the mean frequency was noted, this was considered as indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Under our 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 62.5, 125 or 250 mg/kg/day (expressed as active material).

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
Under our 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-hourinterval, at the dose-levels of 62.5, 125 or 250 mg/kg/day (expressed as active material).
Executive summary:

The objective of this study was to evaluate the potential of the test substance to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of mice.

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 leo: OFI (lOPS Caw) mice received two oral treatments of the substance at dose-levels of 62.5, 125 or 250 mg/kg/day (expressed as active material), at a 24-hour interval. One group of five males and five females received the vehicle (com 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. 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 observable toxic effects were noted in the preliminary test, the top dose-level was based on the toxicity level, such that a higher dose-level was expected to induce lethality. Consequently, 250 mg/kg/day was selected as the top dose-level. The two other dose-levels were 125 and 62.5 mg/kg/day. For both males and females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test substance, were equivalent to those of the vehicle group. The mean values of MPE as well as the PE/NE ratio for the vehicle control groups were consistent with our historical data. For one female from the positive control group, no significant increase in the frequency of MPE was noted. However since for the remaining nine animals cyclophosphamide induced a clear increase in the frequency of MPE and since a very significant increase (p <0.001) in the mean frequency was noted, this was· considered as indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Under our 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 62.5, 125 or 250 mg/kg/day (expressed as active material).