2,2-bis(methylthio)propane

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

other: Body responsible for the test

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

478-900-1

EC Name:

-

Cas Number:

6156-18-9

Molecular formula:

C5 H12 S2

IUPAC Name:

2,2-bis(methylthio)propane

Details on test material:

- Name of test material (as cited in study report): BMTP
- Synonym: BISMETHYLTHIOPROPANE
- Chemical name: 2,2-bis-methylthiopropane
- Batch number: 2KS149
- Description: colorless liquid
- Storage conditions: at room temperature and protected from light
- Purity: 97.22%
- Date of analysis: 19 November 2007
- Expiry date: November 2008

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: approximately 7 weeks old
- Weight at study initiation: 218 g for males (ranging from 174 to 261 g) and 184 g for females (ranging from 147 to 221 g)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: by groups in Individually Ventilated Cages (IVC) (polycarbonate 900 cm2, Tecniplast) containing sawdust
- Diet : free access to SsniffR/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 22
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 28 January 2008 to 21 March 2008

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The Sponsor supplied the dosing solution ready to use. All the preparations were carried out under nitrogen atmosphere. The preparations were sent to the laboratory CIT for dosing of animals purposes. An aliquot from each preparation was kept by the sposonr to determine the concentration and stability.

Duration of treatment / exposure:

2 administrations

Frequency of treatment:

24-hour interval

Post exposure period:

24 h

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg/day (males)
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
375, 750 and 1500 mg/kg/day (females)
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide, once by oral route at the dose-level of 15 mg/kg

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose-level of 2000 mg/kg/day for males and 1500 mg/kg/day for females were considered to be the maximum tolerated dose and were retained as the highest dose-levels for the main test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
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.

DETAILS OF SLIDE PREPARATION:
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).

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.
If 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 (¿ three groups) followed by a Dunnett test (if necessary).
If normality or homogeneity of variances was not demonstrated, a Mann/Whitney test (two groups) or a Kruskall Wallis test (¿ three groups) was performed followed by a Dunn test (if necessary).

All these analyses were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS Institute Inc), with a level of significance of 0.05 for all tests.

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:

RESULTS OF RANGE-FINDING STUDY
At 2000 mg/kg/day, no mortality was observed in the two treated males. Sedation, hypoactivivty, half-closed eyes, unsteady gait and rhinorrea were noted.
One of the two females treated at 2000 mg/kg/day was found dead 24 hours following the first treatment. The other female showed clinical signs following the first treatment and was sacrificed without any second administration of the test item.

At 1500 or 1000 mg/kg/day, no mortality was induced in the treated females. Hypoactivity, unsteady gait, half closed eyes, and in addition ocular secretion at 1500 mg/kg/day, were observed in treated animals.

The dose-level of 2000 mg/kg/day for males and 1500 mg/kg/day for females were considered to be the maximum tolerated dose and were retained as the highest dose-levels for the main test.
The two other selected dose-levels were 1000 and 500 mg/kg/day for males and 750 and 375 mg/kg/day for females.


RESULTS OF DEFINITIVE STUDY
In males, no clinical signs were noted at 500 mg/kg/day, hypoactivity and unsteady gait were noted following the first treatment at 1000 mg/kg/day. At 2000 mg/kg/day, one male out of 8 was found dead 24 hours following the second treatment. Clinical signs were noted in all treated animals (hypoactivity, unsteady gait, sedation, lateral recumbency and sometimes half-closed eyes).

In females, no clinical signs were noted at 375 mg/kg/day, unsteady gait was observed following the first treatment at 750 mg/kg/day. At 1500 mg/kg/day, one female out of 8 was found dead 24 hours following the first treatment. Clinical signs were noted in all treated animals (hypoactivity, unsteady gait, sedation, lateral recumbency, half-closed eyes and ocular secretions).

In either males or females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test item BMTP, were equivalent to those of the vehicle control group.

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data.
Cyclophosphamide induced a significant increase 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:

BMTP did not induce any damage to chromosomes or mitotic apparatus of rat bone marrow cells after two oral administrations separated by a 24-hour interval at dose-levels of 500, 1000 and 2000 mg/kg/day for males and 375, 750 and 1500 mg/kg/day for females.

Executive summary:

The potential of BISMETHYLTHIOPROPANE (BMTP) to induce structural or numerical damage was evaluated in bone marrow cells of rat. The study was performed according to the OECD guideline 474 and in compliance with the Principles of Good Laboratory Practice Regulations. Several preliminary toxicity tests were 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 Sprague-Dawley rats received two oral treatments of BMTP at dose-levels of 500, 1000 and 2000 mg/kg/day for males and 375, 750 and 1500 mg/kg/day for females, 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 15 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 dose-level of 2000 mg/kg/day for males and 1500 mg/kg/day for females were considered to be the maximum tolerated dose and were retained as the highest dose-levels for the main test. The two other selected dose-levels were 1000 and 500 mg/kg/day for males and 750 and 375 mg/kg/day for females. In either males or females, the mean values of MPE as well as the PE/NE ratio in the groups treated with BMTP, were equivalent to those of the vehicle control group. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data. Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

BISMETHYLTHIOPROPANE did not induce any damage to chromosomes or mitotic apparatus of rat bone marrow cells after two oral administrations separated by a 24-hour interval at dose-levels of 500, 1000 and 2000 mg/kg/day for males and 375, 750 and 1500 mg/kg/day for females.