Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was classified as reliable with restriction because the results are obtained by valid read-across. The read-across study was conducted according to OECD TG 474 guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

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
Details on test material:
TS: t-butyl mercaptan (2-methylpropane-2-thiol)
Source: Elf Atochem NA
Batch 1700TBM95
Purity 99.7%

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD, USA
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation:
Pilot study: Males, 29.0 - 38.1 grams, females, 26.4 - 30.1 grams at randomization
Micronucleus assay: Males, 27.4 - 35.8 grams , females, 24.5 - 30.6 grams at randomization
- Assigned to test groups randomly: yes, using a computer-generated program which is based on distribution according to body weight.
- Fasting period before study: no data
- Housing: up to five of the same sex per cage in plastic autoclavable cages with filter tops
- Diet (e.g. ad libitum): Purina Certified Rodent Chow 5002
- Water (e.g. ad libitum): tap water
- Acclimation period: no less than 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 74 ± 6
- Humidity (%): 50 ± 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solutility of the TS
- Amount of vehicle (if gavage or dermal): 20 ml/kg
Details on exposure:
For the pilot study, one group of mice (5/sex) was dosed with 5000 mg/kg and four additional groups (2 male mice/group) were dosed with 1, 10, 100 or 1000 mg/kg via oral gavage at a dose volume of 20 ml/kg. Corn oil was used as the vehicle. The animals were weighed immediately prior to dose administration and 1 and 3 days after dose administration. Mice were observed after dose administration and daily thereafter until sacrifice for clinical signs of chemical effect. In the absence of mortality in the high dose group, the following dose levels were used for the definitive assay: 1250, 2500 or 5000 mg/kg.
Fifteen mice/sex were dosed at 0, 1250 or 2500 mg/kg. Twenty mice/sex were dosed at 5000 mg/kg (five additional animals as replacement animals). Five mice/sex were dosed with cyclophosphamide at 60 mg/kg. Five animals/sex from each test group and the vehicle control group were sacrificed at 24, 48 and 72 hours post-dose. The five animals in the positive control group were sacrificed 24 hours post-dose. Bone marrow cells were collected at sacrifice and were examined microscopically for micronucleated polychromatic erythrocytes. The mice for the definitive assay were weighed immediately prior to dose administration and observed after dose administration for clinical signs of chemical effect.
Duration of treatment / exposure:
Single exposure
Post exposure period:
24, 48 or 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
1250, 2500, 5000 mg/kg
Basis:

No. of animals per sex per dose:
Fifteen mice/sex were dosed at 0, 1250 or 2500 mg/kg. Twenty mice/sex were dosed at 5000 mg/kg.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 60 mg/kg

Examinations

Tissues and cell types examined:
At the scheduled sacrifice times, up to five mice per sex per dose were sacrificed by carbon dioxide asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes, and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May Gruenwald Giemsa and permanently mounted.
Details of tissue and slide preparation:
To control for bias, slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification (10 x 40), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (10 x 100), 1000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. Micronuclei are round, darkly-staining nuclear fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 1000 polychromatic erythrocytes was enumerated for each animal. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
Evaluation criteria:
Evaluation of Test Results
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each mouse and treatment group.
In order to quantify the test article effect on erythropoiesis, as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
The test article was considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p_< 0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.

Criteria for a Valid Test
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p<= 0.05, Kastenbaum-Bowman Tables).
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970; Mackey and MacGregor, 1979). All analyses were performed separately for each sex and sampling time.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Slight reductions (up to 20%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls.
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
No mortality occurred in male or female mice in the micronucleus study. Clinical signs following dose administration included lethargy in male and female mice at 1250, 2500 and 5000 mg/kg. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Slight reductions (up to 20%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls.

No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control group was observed in male or female mice at 24, 48 or 72 hours after dose administration (p > 0.05, Kastenbaum-Bowman).

Any other information on results incl. tables

SUMMARY OF BONE MARROW MICRONUCLEUS STUDY USING t-BUTYL MERCAPTAN

TREATMENT

SEX

TIME

(HR)

NUMBER OF

MICE

PCE/TOTAL

ERYTHROCYTES

MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES

NUMBER PER 1000 PCE'S

NUMBER PER

PCE'S SCORED'

(MEAN ± S.D.)

Corn Oil

20 ml/kg

M

24

5

0.53

0.8 ± 0.84

4/5000

48

5

0.58

1.4 ± 1.14

7/5000

72

5

0.57

0.8 ± 0.84

4/5000

F

24

5

0.70

2.0 ± 1.00

10/5000

48

5

0.59

1.0 ± 0.71

5/5000

72

5

0.61

1.2 ± 0.84

6/5000

t-Butyl mercaptan

1250 mg/kg

M

24

5

0.58

1.0 ± 1.00

5/5000

48

5

0.60

1.6 ± 1.34

8/5000

72

5

0.68

0.4 ± 0.55

2/5000

F

24

5

0.73

1.6 t 2.07

8/5000

48

5

0.60

2.2 ± 0.45

11/5000

72

5

0.61

0.6 ± 0.89

3/5000

2500 mg/kg

M

24

5

0.59

1.4 ± 1.14

7/5000

48

5

0.55

2.0 ± 2.24

10/5000

72

5

0.63

1.4 ± 1.52

7/5000

F

24

5

0.66

1.0 ± 1.00

5/5000

48

5

0.54

2.2 ± 1.30

11/5000

72

5

0.67

2.4 ± 1.14

12/5000

5000 mg/kg

M

24

5

0.57

1.4 ± 1.34

7/5000

48

5

0.49

1.2 ± 0.84

6/5000

72

5

0.61

1.2 ± 0.84

6/5000

F

24

5

0.60

0.4 ± 0.55

2/5000

48

5

0.47

1.0 ± 0.71

5/5000

72

5

0.68

1.6 ± 1.34

8/5000

CP,

60 mg/kg

M

24

5

0.54

17.4 ± 3.21

87/5000*

F

24

5

0.46

34.4 ±2.41

172/5000*

*, p <= 0.05 (Kastenbaum-Bowman Tables)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
2-methylpropane-2-thiol is negative in the mouse micronucleus assay (Putman, 1995).
Executive summary:

A mouse micronucleus assay (Putman, 1995) was conducted with 2 -methylpropane-2-thiol via oral exposure to male and female ICR mice according to the OECD 474 Guideline (Microbiological Associates, 1995). The mice received a single dose of 2 -methylpropane-2 -thiol by gavage at 0, 1250, 2500 (15/sex) or 5000 mg/kg bw (20/sex). A positive control group of mice was included. Five mice/sex were sacrificed at 24, 48 and 72 hours after the single dose and bone marrow was evaluated for cytotoxicity and micronucleus formation. No mortality occurred at any dose and lethargy was noted following all test substance treatments. Slight reductions in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the treated animals indicating the test material reached the bone marrow. No significant increase in micronucleated polychromatic erythrocytes was noted in test material treated groups when compared to vehicle control animals. It was concluded that 2 -methylpropane-2-thiol did not induce chromosome mutations under the conditions of this study.