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:
17/05/2010 – 06/07/2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France origin, Saint-Germain-surl’Arbresle; FRANCE
- Age at study initiation: 5 to 10 weeks old
- Weight at study initiation: approximately 200 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: animals were housed in polypropylene cages measuring 42.5 x 26.6 x 15 cm, covered by a stainless steel netted lid, in which they will be placed in groups of 3 or 2
- Diet (ad libitum): 801175 RM1(P)DU IRR 9Kgy irradiated from Special Diets Services
(ENGLAND).
- Water (ad libitum): softened by reverse osmosis and filtered on 0.2 µm membrane
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 20 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC at 0.5% in distilled water
Details on exposure:
Dose volume : 10 mL/kg b.w.
Duration of treatment / exposure:
2 treatments
Frequency of treatment:
daily
Post exposure period:
Number of sampling times : for the negative control and the 3 treated groups: 24 hours after the second treatment for the positive control group: 24 hours after the single treatment
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5 (excepted 7 for the high dose level)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (25 mg/kg i.p.)

Examinations

Tissues and cell types examined:
Bone Marrow
Details of tissue and slide preparation:
At the sampling time, the 5 animals per group were sacrificed by CO2 asphyxia; the femurs were removed, and the bone marrow was extracted with foetal calf serum (1 mL per animal). The cell suspensions were centrifuged for 5 minutes at 1000 rpm. The supernatant was removed. The centrifugate was spread on slides. The smears were stained using a technique, derived from the May Grunwald Giemsa technique (Schmid, 1975), which makes it possible to distinguish between polychromatic (PCE) and normochromatic erythrocytes (NCE): PCE are purple whereas NCE are red.
After coding the slides by a person not involved in the study, two slides per animal were read by two independent operators; for each animal, the number of polychromatic erythrocytes having one or more Howell-Jolly bodies (micronuclei) was determined for at least 2000 polychromatic erythrocytes. (In case of divergence, 2000 new polychromatic erythrocytes were examined; the retained value was the mean of all readings).
The polychromatic/normochromatic erythrocyte ratio will be determined by analyzing at least 1000 erythrocytes per animal.
Evaluation criteria:
For a test item to be considered negative in the micronucleus test, there must be no statistically significant increase in the number of MNPCE observed compared with negative control animals.
For a test item to be considered positive in the micronucleus test, there must be seen a statistically significant increase in MNPCE for at least one dose group and/or a statistically significant dose-related increase in MNPCE, compared to the negative control animals. Statistical significance will not be the only determinant of a positive response, and the Study Director will consider the biological relevance of the results in the final evaluation.
Statistics:
Statistical analysis was performed for micronucleus number using a non-parametric test, the Mann Whitney U rank test. An analysis of a large number of control results has shown that the distribution of the numbers of micronuclei does not correspond to a Gaussian distribution, but to a Poisson-type distribution. This makes it necessary to use a non-parametric statistical test, and the Mann Whitney U rank test is recommended by UKEMS (LOVELL et al., 1989).

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
but tested up to the limit dose of 2000 mg/kg
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Clinical signs of toxicity in test animals: No clinical signs were observed after up to 24 hours after the second treatment in male and female rats. As no difference between both sexes were noted, the main assay was performed in male rats only.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The results obtained on negative control animals and those treated with the positive reference
substance were similar to those generally obtained in the laboratory. A statistically significant increase in the frequency of micronuclei was noted in the group treated with Cyclophosphamide, demonstrating the sensitivity of the animal strain used to a clastogenic agent. The validity criteria for the test were fulfilled and the test was validated. Regarding the frequency of micronuclei, no statistically significant increase in the frequencies of micronucleated polychromatic erythrocytes was found in the animals treated with di-tert-dodecyl polysulfides (TPS 32) at any dose.
- Ratio of PCE/NCE (for Micronucleus assay): No statistically significant decrease in the ratio PCE to NCE was noted in the 3 di-tertdodecyl polysulfides (TPS 32) treatment groups when compared to the negative control group. In consequence, no proof of systemic exposure was evidenced.

Applicant's summary and conclusion

Conclusions:
The validity criteria for the results were fulfilled. The study was thus considered as valid. The test item di-tert-dodecyl polysulfides (TPS 32) was investigated by means of the in vivo micronucleus test, in male OFA Sprague Dawley rats treated orally twice with 2000 – 1000 and 500 mg/kg/day, followed by one sampling time 24 hours after the last treatment. As a conclusion, di-tert-dodecyl polysulfides (TPS 32) induced no genotoxic activity under these experimental conditions.
Executive summary:

The potential clastogenic activity of di-tert-dodecyl polysulfides (TPS 32) was tested using the in vivo micronucleus test in male OFA Sprague Dawley rats, in compliance with the Commission Regulation (EC) No. 440/2008 and the OECD Guideline 474, by oral route, using 2 successive daily treatments at the maximum dose recommended by OECD guidelines, i.e. 2000 mg/kg, followed by one sampling time 24 hours after the last treatment. The two lower doses of 1000 and 500 mg/kg/day (x2) were also analysed. The validity criteria for the results were fulfilled. The study was thus considered as valid. Di-tert-dodecyl polysulfides (TPS 32) induced no genotoxic activity under these experimental conditions.