2-methylpropane-2-thiol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro gene mutation study in mammalian cells, conducted according ot OECD TG 476 and in compliance with GLP, reported a negative mutagenic response in both the presence and absence of metabolic activation (CPChem, 1982, Klimisch score = 2).

An in vitro sister chromatid exchange assay conducted according to OECD 479 and in compliance with GLP, reported that 2-methylpropane-2-thiol did not induce a statistically significant genotoxic effect (CPChem, 1982, Klimisch score = 2).

An in vitro gene mutation study in bacteria, conducted according to OECD TG 471 and in compliance with GLP, reported a negative mutagenic potential in a S. typhimurium reverse mutation assay as well as in an E. coli reverse mutation assay in both the presence and absence of metabolic activation (Verspeek-Rip, 2015, Klimisch score = 1).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

cytotoxicity data and individual culture SCE data were not presented in the report

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S-9 liver microsomes from male Sprague Dawley rats treated with a single intraperitoneal injection of Aroclor 1254 at a dose of 500 mg/kg 5 days before sacrifice.

Test concentrations with justification for top dose:

With and without metabolic activation:
Experiment 1: mg/ml
Experiment 2: mg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethanesulfonate (EMS, 400 µg/ml) without metabolic activation and CP (1.4 µg/ml) with metabolic activation

Details on test system and experimental conditions:

TOXICITY EVALUATION:
The test compound dose levels were determined by a preliminary multidose-range finding study with the highest concentration of the chemical tested depending upon its solubility. t-Butyl mercaptan solubilized at approximately 270 mg/ml in dimethylsulfoxide. The toxicity was determined by testing a wide range of concentrations for relative inhibition of the growth of the cells following a 4-hr exposure. Twenty-four hrs after exposure, cell counts were determined. Based on the cell counts, the relative toxicity of the chemical doses compared to the solvent control was determined. The maximum dose selected for the mutagenicity test was approximately 1350 ug/ml because it exhibited growth inhibition.

SCE ASSAY:
Cells were treated in an exponential stage of growth by setting up cultures with 2 to 5E05 cells per 25 cm2 flask, 24 hours prior to treatment. Cells were exposed to the chemical for 2 hours, washed twice and then 5-bromodeoxyuridine (BrdU) was added to each culture. All cultures were sampled a minimum of 24 hours after addition of BrdU to ensure completion of two full cell cycles. Duplicate cultures were set up for each dose level and all controls. Twenty-four hours after the above initiation of the cultures, the cells were treated with the test chemical in the presence of an S-9 rat liver activation system for 2 hours and washed twice in a balanced salt solution. The cells were then sampled and treated as described above. Two hours after, colcemid (0.2 ug/ml) was added to each tube and metaphases were collected by mitotic shake-off. The cells were swollen in a 0.075M KCL hypotonic, and washed three times in an acetic alcohol fixative. Slides were prepared and stained. Fifty cells in the metaphase stage of mitosis were scored at each dose level for the number of sister chromatid exchanges (SCE).

Evaluation criteria:

Test substance considered positive if a statistically significant increase in SCEs and a concomitant two-fold increase in SCEs relative to solvent control occurs.

Statistics:

Statistical analysis conducted - details not provided.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1350 µg/ml exhibited growth inhibition

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Following exposure to five graded doses of t-Butyl Mercaptan, in the presence of metabolic activation, a statistically significant increase in the number of SCEs per cell (10.88 vs 7.58 in the DMSO control [1.4-fold] at 1350 µg/ml and 10.66 vs 7.58 [1.4-fold] at 450 µg/ml) and SCEs per chromosome (0.55 vs 0.37 [1.5-fold] at 1350 µg/ml and 0.53 vs 0.37 [1.4-fold] at 450 µg/ml); but no significant increase was seen in the remaining dose levels, and no dose level showed a two-fold increase in SCEs. The increases in SCEs ranged from 0.9 to 1.2-fold in the absence of metabolic activation and 1.0 to 1.5-fold in the presence of activation. Therefore t-butyl mercaptan is not considered to be positive in this test system.

Conclusions:

An in vitro sister chromatid exchange assay in a similar way to OECD TG 476 and cin compliance with GLP, concluded 2-methylpropane-2-thiol to be negative in both the presence and the absence of metabolic activation.

Executive summary:

An in vitro Sister Chromatid Exchange (CHO cells) assay was performed with 2 -methylpropane-2-thiol in a study similar to OECD Guideline 476. CHO cells were exposed to five concentrations of 2 -methylpropane-2-thiol (up to 1350 µg/ml) in the presence and absence of a metabolic activation system. In the presence of metabolic activation, a statistically significant increase in the number of SCEs per cell (10.88 [1.4-fold] at 1350 µg/ml and 10.66 [1.4-fold] at 450 µg/ml vs 7.58 in the DMSO control) and SCEs per chromosome (0.55 [1.5-fold] at 1350 µg/ml and 0.53 [1.4-fold] at 450 µg/ml vs 0.37 in control) was observed; but no significant increase was seen in the remaining dose levels, and no dose level showed a two-fold increase in SCEs. The increases in SCEs ranged from 0.9 to 1.2-fold in the absence of metabolic activation and 1.0 to 1.5-fold in the presence of activation. Therefore 2 -methylpropane-2-thiol is not considered to be positive in this test system. 

The study received a Klimisch Score of 2 and is classified as reliable with restrictions because it is comparable to a guideline study.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

only 0.5E06 cells were plated for selection of mutants, individual culture data were not provided, and large vs small colonies were not determined

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y mouse lymphoma cells subline 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

S-9 liver microsomes from male Sprague Dawley rats treated with a single intraperitoneal injection of Aroclor 1254 at a dose of 500 mg/kg 5 days before sacrifice.

Test concentrations with justification for top dose:

61, 90, 135.0, 202, 300, 449, 670, or 1000 mg/ml. 1000 mg/ml limit of solubility.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethanesulfonate (EMS, 620 µg/ml) without metabolic activation and 3-methylcholanthrene (3-MCA, 3 µg/ml) with metabolic activation

Details on test system and experimental conditions:

TOXICITY EVALUATION:
The test compound dose levels were determined by a preliminary multidose range-finding study with the highest dose tested being selected to give approximately 50 - 90% inhibition of suspension cell growth depending on the solubility of the compound. T-Butyl mercaptan solubilized at approximately 100 mg/ml in dimethylsulfoxide. One-tenth ml of each test substance concentration and the solvent were added to 50 ml tubes containing 6.0 x 10(6) TK+/- cells in 10 ml F10P. Cells were exposed to the chemical for 4 hrs, washed, resuspended in 20 ml F10P and incubated for 2 days. After the first and second day of incubation, the cell counts and viability were determined by counting a sample of the cells using a hemocytometer in the presence of trypan blue. Following the first day of incubation, the viable cell count was adjusted to 3.0 x 10(5) cells/ml. Based on the cell counts, the relative toxicity of the chemical compared to the solvent control was determined. The maximum dose selected for the mutagenicity test was 1000 ug/ml because it represented the limits of solubility of the test material.

MUTAGENICITY ASSAY:
Each test concentration was prepared to contain the test dose in 0.1 ml volumes. Six million precleansed TK+/- cells in 6 ml of F10P were added to centrifuge tubes. An additional 4 ml of the S-9 mix were added to half of the tubes. Immediately thereafter, 0.1 ml of the 100x concentrations of the test chemical dilutions or the positive controls, and 0.1 ml of the solvent were added to the appropriate tubes. Each tube was mixed, gassed with a mixture of C02 and air, and incubated at 37± 0.5°C on a revolving roller drum for 4 hours. Following incubation, the tubes were centrifuged and the treatment solutions decanted. The cells were washed twice with F10P and resuspended in 20 ml F10P after the second wash. The tube cultures were then gassed and reincubated for a 2 day expression time. The cell cultures were readjusted to 3.0 x 10(5) cells/ml as necessary. At the end of the expression period, a sample of each of the cultures was centrifuged and the cells resuspended at 500,000 viable cells/ml in F10P. The concentrated cells were serially diluted and appropriate dilutions were plated in triplicate in cloning medium with and without TFT. Approximately 500,000 cells were plated on each of 3 selective medium plates containing 2 ug/ml TFT, and 100 cells were cloned on each of 3 non-selective plates for each test concentration and a control tube. The plates were incubated for 10-14 days. The mutant colonies (TK+/-) were counted on the selective TFT containing plates and the survivors (TK+/- and TK-/-) were counted on the non-selective medium plates. The daily cell counts were made using a microscope and hemocytometer; and the colony counts were obtained with an electronic colony counter. The number of mutants in the test and control groups was compared and their mutation frequencies determined, along with a determination of relative total survival based on the suspension cell growth and cloning efficiency.

Evaluation criteria:

Test substance considered positive if a dose-related response at two or more test concentrations (in the absence of severe toxicity) is at least two to three-fold higher than the mutation frequency of the solvent control.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without metabolic activation: Survival = 10.6% at 670 µg/ml and 3.0% at 1000 µg/ml; with metabolic activation: Survival = 10.9% at 1000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Exposure to eight graded doses of the test material in the presence of metabolic activation did not increase the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Exposure to the 202 and 1000 µg/ml dose levels of the test material in the absence of metabolic activation increased the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Under these conditions, t-butyl mercaptan did exhibit a positive response. However, the induction of increases in mutant frequency was not dose responsive and, thus, did not satisfy the stated criteria for a positive response; therefore, t-butyl mercaptan was not mutagenic under the conditions of this assay.

Treatment without S-9

	µg/ml	%Survival	MF	Fold Increase
DMSO	--	100	3.4	1.0
EMS	620	78.0	14.8	4.4
TBM	1000	3.0	7.6	2.2
	670	10.6	6.2	1.8
	449	60.0	5.3	1.6
	300	56.1	4.2	1.2
	202	93.5	7.4	2.2
	135	94.9	3.8	1.1
	90	126.8	2.3	0.7
	61	100.1	3.2	0.9

MF - mutation frequency [x10e-5]

Conclusions:

In an in vitro cell gene mutation mouse lymphoma assay, conducted in a similar way to OECD TG 476 and in compliance with GLP, concluded that 2-methylpropane-2-thiol did not induce any mutagenic response, neither in the presence nor in the absence of metabolic activation.

Executive summary:

An in vitro Mammalian Cell Gene Mutation (mouse lymphoma) assay was performed with 2 -methylpropane-2-thiol in a study similar to OECD Guideline 476. L5178Y TK (+/-) mouse lymphoma cells were exposed to eight concentrations of t-butyl mercaptan (61 to 10,000 µg/ml) in the presence and absence of a metabolic activation system. 2-methylpropane-2-thiol did not result in mutagenic responses in this assay. Positive controls provided the appropriate responses. 

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22-Jun2015 to 06-Aug-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1; direct plate
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2; pre incubation test:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9; 650 µg/plate in DMSO for TA100

Positive control substance:

2-nitrofluorene

Remarks:

without S9; 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Positive control substance:

other: ICR-191 2.5µg/plate in DMSO for TA1537 direct plate test

Remarks:

without S9

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9; 10 µg/plate in DMSO for WP2uvrA

Positive control substance:

sodium azide

Remarks:

without S9; 5 µg/plate in saline for TA1535

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation and pre incubation assay

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the pre incubation assay

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the pre incubation assay

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

MAIN 1 (direct plate assay):
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

MAIN 2 (direct plate assay):
- Toxicity was observed in all five tester strains

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative control values were within the laboratory historical control data ranges, except the response for TA1537 in the absence of S9-mix,
first experiment. However, this response was judged not to impact the reliability of the study. The deviation in the negative control value was
minor, the value (2) was just below the limit of the range (3).
 
- The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535, TA98 and TA100: with and without S9: 1600 µg/plate and above
TA1537 and WP2uvrA: without S9: with and without S9: 5000 µg/plate

Conclusions:

It is concluded that 2-methylpropane-2-thiol (Tertiary Butyl Mercaptan: TBM) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two repeated experiments.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo mouse chromosome aberration study, conducted according to OECD TG 474 and in compliance with GLP, reported that the test substance 2-methylpropane-2 -thiol did not induce chromosome mutations (Putman et al., 1995, Klimisch score = 1).

Link to relevant study records

Reference

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system 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

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility 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

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

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.

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 validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

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).

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)

Conclusions:

2-methylpropane-2-thiol is negative in the mouse micronucleus assay.

Executive summary:

A mouse micronucleus assay 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.

This study received a Klimisch Score of 1 and is classified as reliable without restriction because it is a GLP guideline study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

2-methylpropane-2-thiol was not mutagenic in two in vitro bacterial reverse mutation assay (OECD TG 471) (CP Chem, 1982a and Verspeek-Rip, 2015) and an in vitro mammalian cell gene mutation (mouse lymphoma) assay (OECD TG 476) (CP Chem, 1982b), both with and without metabolic activation. In an in vitro sister chromatid exchange assay (OECD 479) (CP Chem, 1982c), 2-methylpropane-2-thiol induced, in the presence of metabolic activation, a statistically significant increase in the number of SCEs per cell and SCEs per chromosome; but no significant increase was seen in the remaining dose levels, and no dose level showed a two-fold increase in SCEs. The increases in SCEs ranged from 0.9 to 1.2-fold in the absence of metabolic activation and 1.0 to 1.5-fold in the presence of activation. Therefore, 2-methylpropane-2-thiol was not considered to be positive in this test system. In an in vivo mouse micronucleus assay (OECD 474) (Putman et al., 1995), 2-methylpropane-2-thiol exposure resulted in slight reductions in the ratio of polychromatic erythrocytes to total erythrocytes but did not induce chromosomal mutations. 

Justification for classification or non-classification

According to REGULATION (EC) No 1272/2008 and the available in vitro and in vivo genotoxicity data, germ cell mutagenicity is not classified. 2-methylpropane-2-thiol is not genotoxic.