Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 September 2010 - ...........................................
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate coherence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Di-tertio-butyl polysulfides (TPS 44)
- Physical state: light yellow liquid
- Analytical purity: 97.29%
- Lot/batch No.: 27-janv-2009 on the certificate of analysis (27/01/2009 on the vial label)
- Expiration date of the lot/batch: January 2012
- Storage conditions of test material: at room temperature in a well-ventilated place

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: on the dfay of treatment, the animals were approximately 6 weeks old
- Weight at study initiation: 33.6 g (ranging from 31.8 to 37.5 g)
- Housing: the animals were housed by 2 or 3, in Individually Ventilated Cages (IVC) (polysulfone 900 cm2, Tecniplast) containing autoclaved sawdust
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered with a 0.22 µm filter) contained in bottles
- Acclimation period: at least 5 days before the day of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%,
- Air changes (per hr): at least 12 cycles/hour of filtered non-recycled fresh air
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: 0.5% aqueous carboxymethylcellulose
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Lot/batch no.: 060M0017
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was mixed with the required quantity of vehicle, under magnetic stirring
and until the obtention of a satisfactory homogenization.
For the preliminary test then for the main experiment, the test item was prepared in the vehicle at the concentration of 200 mg/mL.
The dosage forms were prepared daily by the CIT Pharmacy, within the 4 hours before use, and delivered to the study room in brown flasks, at room
temperature.
Duration of treatment / exposure:
A period of 2 days (2 treatments).
Frequency of treatment:
Two treatments separated by 24 hours.
Post exposure period:
24 hours
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
8 males.
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control was Cyclophosphamide.
- Route of administration: dissolved in distilled water
- Concentration: 5 mg/mL.

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In order to select the top dose-level for the cytogenetic study, the dose-level of 2000 mg/kg/day was administered twice 24 hours apart, to three males and three females.
At this dose-level, neither mortality, nor clinical signs were observed in the animals throughout the observation period.
Despite that the PE/NE ratio of the females given the test item at 2000 mg/kg/day was slightly lower than the corresponding vehicle control, all ratios remained consistent with the historical data.
The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no signs of toxicity were observed at 2000 mg/kg/day and in the absence of any clear differences between sexes, the main experiment was undertaken as a limit test at 2000 mg/kg/day using only male mice.

DETAILS OF SLIDE PREPARATION:
At the time of sacrifice, all the animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital, then killed by cervical dislocation. 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 CIT and the scoring was performed "blind".
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:
Normality and homogeneity of variances will be tested using a Kolmogorov Smirnov test and a Bartlett test.
When normality and homogeneity of variances were demonstrated, the statistical comparisons was performed using a Student t-test (two groups) or a one-way analysis of variance (¿ 3 groups) followed by a Dunnett test (when necessary).
When normality or homogeneity of variances was not demonstrated, a Mann/Whitney test (two groups) or a Kruskall Wallis test (¿ 3 groups) was performed followed by a Dunn test (when necessary).

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
PE/NE ratio decreased
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Neither mortality, nor clinical signs were observed in any animals given 2000 mg/kg/day throughout the observation period.

The mean values of MPE of the vehicle control and positive control groups were within the corresponding historical control ranges. The cyclophosphamide induced a significant increase (p < 0.05) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

The PE/NE ratio of the vehicle control was found above the historical data range. This value is mainly due one out of five animals and it is considered not to impact on the validity of the test.
When compared to the vehicle control, the PE/NE ratio of the test item treated group decreased significantly (p < 0.05), showing that the bone marrow cells were effectively exposed to the test item.

The mean value of MPE in the group treated with the test item was found not different from that of the vehicle group.

Applicant's summary and conclusion

Conclusions:
The test item Di-tertio-butyl polysulfides (TPS 44) did not induce damage to the chromosomes or the mitotic apparatus of male mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-level of 2000 mg/kg/day.
Executive summary:

The potential of the test item Di-tertio-butyl polysulfides (TPS 44) to induce structural or numerical damage was evaluated in bone marrow cells of mice. The study was performed according to the international guidelines (OECD 474 and Commission Directive No. B12) and in compliance with the Principles of Good Laboratory Practice Regulations.

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study. The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no signs of toxicity were observed at 2000 mg/kg/day and in the absence of any clear differences between sexes, the main experiment was undertaken as a limit test at 2000 mg/kg/day using only male mice. In the main study, one group of five maleSwiss Ico: OF1 (IOPS Caw) mice received oral administrations of Di-tertio-butyl polysulfides (TPS 44) at the dose-level of 2000 mg/kg/day, over a 2-day period. One group of five males received the vehicle (0.5% aqueous carboxymethylcellulose) under the same experimental conditions, and acted as control group. One group of five males received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg/day. 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).

Neither mortality, nor clinical signs were observed in any animals given 2000 mg/kg/day throughout the observation period. The mean values of MPE of the vehicle control and positive control groups were within the corresponding historical control ranges. The cyclophosphamide induced a significant increase (p < 0.05) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid. When compared to the vehicle control, the PE/NE ratio of the test item treated group decreased significantly (p < 0.05), showing that the bone marrow cells were effectively exposed to the test item. The mean value of MPE in the group treated with the test item was found not different from that of the vehicle group.

Di-tertio-butyl polysulfides (TPS 44) did not induce damage to the chromosomes or the mitotic apparatus of male mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-level of 2000 mg/kg/day.