Ethanethiol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Two studies identified to evaluate the in vitro genetic toxicity potential in bacteria demonstrated that ethanethiol does not increase the induction of revertant colonies in S. typhimurium strains TA98, 100, 1535, 1537, and 1538 (Dechariaux, 1993; Pence, 1983a) . An in vitro mammalian cell gene mutation study was identified for ethanethiol and the result was ambiguous (Pence, 1985). Re-evaluation of the results of this study against current OECD test guideline (490) criteria for a positive result have changed this result to negative. 2-Methylpropane-2-thiol was observed to be negative in a key read across in vivo mouse micronucleus assay (Putman et al., 1995). One in vitro cytogenicity assay indicated that ethanethiol positively induces an increase in the number of sister chromatid exchanges in the absence and presence of metabolic activation (Pence, 1984). This study has been disregarded as the Sister Chromatid Exchange Assay is no longer regarded as a study on which genotoxic potential decisions can be based.

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:

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.

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: 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

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:

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In Vitro Genetic Toxicity

Multiple key studies were identified to evaluate the in vitro genetic toxicity potential of ethanethiol. In addition, key in vivo data from the related substance 2-methylpropane-2-thiol was used to support the conclusions of the in vitro genetic toxicity assays on ethanethiol.

In Vitro Genetic Toxicity in Bacteria:

In the key in vitro bacterial reverse mutation assay (Dechariaux, 1993; Klimisch Score = 2), five strains of S. typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) were exposed to ethanethiol in DMSO and phosphate buffer via plate-incorporation at concentrations of 0.05, 0.1, 0.5, 1, or 2.5% (v/v) with and without metabolic activation. Ethanethiol was tested up to cytotoxic concentrations in this study. In the preliminary toxicity assay a clear toxic effect was observed after 24 hours in the in the histidine positive colonies, primarily in TA98 at ≥ 5% and TA 100 at ≥1%, with and without metabolic activation (S9 mix). The positive controls induced the appropriate responses in the corresponding strains.  There was no evidence of induced S. typhimurium mutant colonies over background in the absence or presence of metabolic activation (±S9), over background in the reverse gene mutation assay in bacteria.

 

In a supporting in vitro bacterial reverse mutation assay (Pence, 1983a; Klimisch Score = 2), five strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) were exposed to ethanethiol in dimethylsulfoxide (DMSO) via plate-incorporation at 123.5; 370.4; 1111; 3333; or 10,000.0 micrograms per plate. The 10,000 micrograms per plate dose exhibited cytotoxicity. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced S. typhimurium mutant colonies over background, in the absence or presence of metabolic activation (±S9) in the reverse gene mutation assay in bacteria.

No data are available for a strain of bacteria able to detect cross-linking or oxidising mutagens. Data are available from anin vitromammalian mutagenicity study, and from anin vivomicronucleus assay, both of which would be expected to detect cross-linking or oxidising mutagens. Ethanethiol does not contain structural alerts for mutagenicity (Benigni and Bossa, 2006, Benigniet al, 2008), so is unlikely to cause cross-linking or oxidation of DNA. It is therefore considered that no further information would be obtained from additional testing in bacteria.

 

In Vitro Cytogenicity in Mammalian Cells:

One in vitro study was identified to evaluate the cytogenicity potential of ethanethiol in mammalian cells.

 

In an in vitro mammalian sister chromatid exchange assay (Pence, 1984; Klimisch Score = 2), Chinese hamster Ovary (CHO) cell cultures were exposed to ethanethiol in dimethylsulfoxide (DMSO) at concentrations of 25, 84, 250, 840 or 2,500 µg/mL with and without metabolic activation (S-9 rat liver) for 4 hours. Ethanethiol was tested up to cytotoxic concentrations. A statistically significant increase was seen in the number of SCEs per chromosome at the 840 µg/mL dose without metabolic activation (0.62 [1.2-fold] vs. 0.50 in DMSO). Cells recovered at the 2,500 µg/mL dose were first division metaphases and could not be analyzed. This dose was repeated and the cells were recovered after 43 hours to allow for two cell divisions. In this repeated test a statistically significant increase of SCEs per cell and per chromosome was seen at 2,500 µg/mL with (SCEs per cell: 20.64 [2.4-fold] vs. 8.62 in DMSO; SCEs per chromosome: 1.04 [2.4-fold] vs. 0.44 in DMSO) and without (SCEs per cell: 21.00 [2.5-fold] vs. 8.50 in DMSO; SCEs per chromosome: 1.09 [2.5-fold] vs. 0.43 in DMSO) metabolic activation, and showed a two-fold increase needed to confirm a positive response. Positive, negative, and solvent controls were appropriate. There was positive evidence of SCE induced over background, with and without metabolic activation.

This study has been disregarded as the Sister Chromatid Exchange Assay is no longer regarded as a study on which genotoxic potential decisions can be based.

 

In Vitro Gene Mutation in Mammalian Cells:

One key in vitro mammalian cell gene mutation studies was identified for ethanethiol.

In a key mammalian cell gene mutation study (thymidine kinase locus) (Pence, 1985; Klimisch score = 2), mouse lymphoma L5178Y cells cultured in vitro were exposed to ethanethiol in dimethylsulfoxide at 60.6, 90.5, 135.0, 201.5, 300.8, 448.9, 670, and 1000 micrograms per plate in the presence and absence of metabolic activation (Aroclor-induced rat liver microsomal fraction) for four hours. Ethanethiol was tested up to the solubility limit (1000 µg/ml). Exposure resulted in induced mutation frequencies of 4.0 to 6.3 per 10^-5cells as compared to 3.6 per 10-5 cells exposed to the solvent control. The exception was 90.5 µg/ml, without activation, which resulted in a mutation frequency of 13.3 per 10^-5cells. Positive controls did induce the appropriate response. Assessment of the results according to the criteria in the guideline for mutagenicity at the thymidine kinase locus (OECD 490, 2015), reveals that only one test concentration, 90.5 μg/ml, showed an increase above the Global Evaluation Factor for the agar version of the assay, and this increase was slight. According to OECD 490,“a test chemical is considered to be clearly positive if, in any of the experimental conditions examined (see paragraph 33), the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test).”Therefore it is considered that in view of the lack of a dose response, and with the deficiencies of the study which did not include replicate concentrations, the result of the study should be considered negative.

In Vivo Genetic Toxicity:

No key in vivo genetic toxicity studies were identified for ethanethiol. However, one key read across study was identified for 2-methylpropane-2-thiol.

In a key read across in vivo micronucleus assay (Putman et al., 1995; Klimisch score = 2) ICR mice (15/sex –first three dose groups; 20/sex - highest dose group) were administered a single dose (via oral gavage) of 2-methylpropane-2-thiol at 0, 1250, 2500, 0r 5000 mg/kg bw. The clastogenic potential of the test material to increase the incidence of micronucleated polychromatic erythrocytes in the mouse bone marrow was subsequently evaluated at 24, 48, and 72 hours.

No mortality was observed in either male or female mice through the study period. Male and female mice dosed at 1250, 2500, and 5000 mg/kg bw exhibited signs of lethargy. Compared to controls, a slight reduction in the ratio of polychromatic erythrocytes to total erythrocytes was observed in some of the treated animals. However, no significant increase in the number of micronucleated polychromatic erythrocytes was observed in male or female mice at 24, 48, and 72 hours post-exposure. Based on the results of this study, 2-methylpropane-2-thiol was considered to be negative in the mouse micronucleus assay.

Read-Across Justification

To reduce animal testing REACH recommends to make use of a read-across approach where appropriate based on the high accordance in properties relevant for the specific endpoint.

There are no data on the in vivo genetic toxicity for ethanethiol, therefore a good quality study on the related substance 2-methylpropane-2-thiol has been read-across to assess the potential for ethanethiol to cause genetic toxicity in vivo.

The low molecular weight aliphatic alkylthiols are volatile organic liquids of moderate aqueous solubility and low n-octanol-water partition coefficient. The substances all contain a single thiol (-SH) functional group. The acid dissociation constant (pKa) of the thiol group is approximately 10 therefore the substances can be considered as weak acids which will not be significantly ionised at physiologically relevant pH. Ethanethiol was negative in two in vitro bacterial gene mutation assays, which is consistent with findings for butane-1-thiol and 2-methylpropane-2-thiol. It was also negative in an in vitro gene mutation test in mammalian cells. An in vivo micronucleus study for 2-methylpropane-2-thiol was negative. Therefore based on structural similarities, comparable metabolic pathways and negative in vitro genetic toxicity studies in all substances with data, it is predicted that ethanethiol would also be negative for genetic toxicity in vivo.

Benigni and Bossa (2006). Current Computer-Aided Drug Design 2, (2), 169-176.

Benigni et al. (2008). The Benigni/Bossa rule base for mutagenicity and carcinogenicity JR Scientific report EUR 23241 EN.

Justification for classification or non-classification

Ethanethiol was not observed to be mutagenic in two bacterial reverse mutation assays and negative in the mouse lymphoma assay. Read across substance 2 -methylpropane-2 -thiol was negative in the in vivo mouse micronucleus test. Taking into consideration the weight of evidence from in vitro data on ethanethiol and in vivo data on a closely related substance, ethanethiol does not meet the criteria for classification as mutagenic according to Regulation (EC) No 1272/2008.