Propane-1-thiol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: negative with and without activation in S. typhimurium strains TA 98, TA100, TA1535, TA1537 and E. coli WP2uvrA (OECD Test Guideline 471) (Charles River, 2017).

Chromosome aberration: negative with and without metabolic activation in human lymphocytes (OECD Test Guideline 473) (RTC, 2018).

Mammalian mutagenicity: negative with and without metabolic activation in mouse lymphoma L5178Y in read-across from isopropyl mercaptan (mixture of 84.3% 2-propanethiol + 15.2% 1-propanethiol) (OECD Test Guideline 490) (Citoxlab, 2018).

Mammalian mutagenicity: negative with and without metabolic activation in mouse lymphoma L5178Y in read-across from ethanethiol (OECD Test Guideline 476, Version 1984) (Pence, 1985).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 May 2017 to 31 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The vehicle of the test item was dimethyl sulfoxide.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in dimethyl sulfoxide at a concentration of 50 mg/ml. Test item concentrations were used within 1 hour after preparation. Any residual volumes were discarded.
- Preliminary purification step (if any): No correction factor required.
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and the WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. This experiment formed part of the main plate incorporation assay. The selected concentrations for the remaining strains in the plate incorporation assay with and for all strains in the preincubation assay were: 52, 164, 512, 1600 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on solubility test

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

TA1537 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA1537 and TA98 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA100 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline N-oxide

Remarks:

WP2uvrA without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

ACTIVATION: . S9-mix contained per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml Milli-Q water; 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution; 1 ml 0.33 M KCl solution.

DURATION
- Plate incorporation: strains TA1535, TA1537 and TA98 were incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. The preliminary toxicity study tested strains TA100 and WP2uvrA in the plate incorporation assay.
- Preincubation period: All strains were incubated for 30 minutes by 70 rpm at 37°C
- Exposure duration: at 37.0 ± 1.0°C for 48 ± 4 h

NUMBER OF REPLICATIONS: triplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: number of revertants; background lawn; presence of microcolonies
- Any supplementary information relevant to cytotoxicity: none

Rationale for test conditions:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and the WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.

Evaluation criteria:

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
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 follow up experiment.

Statistics:

Statistical significance

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at all tester strain with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at all tester strain with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at all tester strain with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at all tester strain with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at all tester strain with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not specified
- Effects of osmolality: not specified
- Water solubility: not specified
- Precipitation: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.
- Definition of acceptable cells for analysis: not applicable

RANGE-FINDING/SCREENING STUDIES: Propane-1-thiol was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

HISTORICAL CONTROL DATA
- Positive historical control data: within the ranges of positive historical control data
- Negative (solvent/vehicle) historical control data: within the ranges of negative historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Remarks on result:

other: preincubation

Table 1: Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and one Escherichia coli strain.

Concentration μL/plate	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA
Positive control	51	780	115	94	454	1357	786	816	655	133
Negative control	8	10	5	6	26	14	91	119	34	26
52	7	7	5	4	18	17	91	130	29	15
164	6	12	4	4	21	24	72	129	25	22
512	6	7	5	5	15	10	74	104	26	24
1600	MC	MC	3	3	14	9	63	69	22	13
5000	NP MC	NP MC	NP MC	NP MC	NP MC	NP MC	NP MC	NP MC	NP MC	NP MC

MC Microcolonies

NP No precipitate

Table 2: Positive controls, without metabolic activation

Strain	Chemical	Solvent	Concentration/plate Direct plate assay	Concentration/plate Pre-incubation assay
TA1535	sodium azide (SA) (Sigma Aldrich Chemie, Steinheim, Germany)	Saline	5  µg	5  µg
TA1537	ICR-191 (Sigma)	DMSO	2.5 µg
TA1537	2-nitrofluorene (NF) (Sigma)	DMSO		15  µg
TA98	2-nitrofluorene (NF)	DMSO	10  µg	10  µg
TA100	methylmethanesulfonate (MMS) (Sigma)	DMSO	650  µg	650  µg
WP2uvrA	4-nitroquinoline N-oxide (4-NQO) (Sigma)	DMSO	10  µg	10 µg

Table 3: Positive controls, with metabolic activation

Strain	Chemical	Solvent	Concentration/plate Direct plate assay	Concentration/plate Pre-incubation assay
TA1535	2-aminoanthracene (2AA) (Sigma)	DMSO	2.5  µg	2.5  µg
TA1537	2-aminoanthracene (2AA)	DMSO	2.5  µg	2.5  µg
TA98	2-aminoanthracene (2AA)	DMSO	1  µg	1  µg
TA100	2-aminoanthracene (2AA)	DMSO	1  µg	5  µg
WP2uvrA	2-aminoanthracene (2AA)	DMSO	15 µg	15  µg

Conclusions:

Propane-1-thiol has been tested in a valid bacterial reverse mutation assay, according to OECD TG 471 (1997), and in compliance with GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA. No increase in the number of revertants was observed in any test strain, with or without metabolic activation. Appropriate positive and solvent controls were used and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimental phase: 11 September 2017; End of experimental phase: 13 December 2017; Study completion: 12 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

S9 liver fraction from rats treated with phenobarbitone and betanaphtoflavone (mixed induction).

Test concentrations with justification for top dose:

The maximum dose level of 762 µg/mL (corresponding to 10 mM) and the lower dose levels of 508, 339, 226, 151, 100, 66.9, 44.6 and 29.7 µg/mL were used for all treatment series. For the continuous treatment in the absence of S9, the additional dose level of 19.8 µg/mL was included.

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Lymphocyte cultures were prepared using fresh venous blood drawn from healthy, non smoking individuals with no known recent exposures to genotoxic chemicals or radiation.
Treatment conditions:
3 hour treatment time in the absence of S9 metabolic activation and harvest time at 24 hours after beginning of treatment
3 hour treatment time in the presence of S9 metabolic activation and harvest time at 24 hours after beginning of treatment
24 hour treatment time in the absence S9 metabolic activation until harvest

Rationale for test conditions:

Lymphocytes in whole blood cultures were stimulated to divide by exposure to phytohaemagglutinin. After approximately 48 hours, a large proportion of the cells were completing their first division; at this point they were treated with the test item or control solutions. Post treatment mitosis were harvested for analysis at a time corresponding to approximately 1.5 cell cycle lengths (24 hours) to allow detection of chemicals which may cause cell cycle delays.

Evaluation criteria:

The test item is considered as clearly positive if the following criteria are met:
Any dose level shows a statistically significant increase in aberration-bearing cells (excluding gaps)
The incidence of cells bearing aberrations is outside the normal distribution of historical control values
The increase of cells bearing aberration is dose-related when evaluated with an appropriate trend test
The test item is considered clearly negative in this assay, if none of the above criteria is met.

Statistics:

Fisher’s Exact Test was used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures.
Bonferroni’s corrections were applied for multiple comparisons. The analysis was performed using sets of data either including or excluding gaps.
Cochran-Armitage trend test (one-sided) was performed to aid determination of concentration response relationship.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Solubility: no opacity nor precipitation of the medium was observed at the beginning or end of treatment, in the absence or presence of S9 metabolism, at any dose level, in any treatment series.

Effects on pH and osmolality: no remarkable variation of pH over the concurrent control was observed at any dose level or treatment conditions.

Cytotoxicity results: Following the short term treatment in the absence of S9 metabolism, severe toxicity was observed at the highest dose level of 762 µg/mL, where few metaphases were recovered, marked toxicity was observed at the lower dose levels of 508 and 339 µg/mL reducing the mitotic index to 36% and 40% of the concurrent negative control, respectively. Moderate toxicity was observed at the dose levels of 226, 151, 100 and 66.9 µg/mL where the relative mitotic index ranged between 46% and 55%. Mild and no remarkable toxicity was observed at the two lower dose levels of 44.6 and 29.7 µg/mL. Following the short termtreatment in the presence of S9 metabolsism, severe toxicity was observed at the highest dose level of 762 µg/mL, where few metaphases were recovered. Marked toxicity was observed at the lower dose levels of 508, 339 and 226 µg/mL reducing the mitotic index between 20% and 32% of the concurrent negative control. Moderate to mild cytotoxicity was observed over the remaining dose range.

Following the continuous treatment in the absence of S9, severe toxicity was observed at the highest dose levels of 762, 508 and 339 µg/mL, where no methaphases or few metaphases were recovered. Moderate toxicity was seen at the dose levels of 226 and 151 µg/mL (relative mitotic index 52% and 50% respectively). Mild to no cytotoxicity was seen over the remaining dose range.

Dose levels selected for scoring:

3 hour treatment -S9 Mix	226, 100 and 44.6 µg/mL
3 hour treatment +S9 Mix	151, 66.9 and 29.7 µg/mL
24 hour treatment -S9 Mix	226, 100 and 44.6 µg/mL

Results show that the proportion of cells with structural aberrations (excluding gaps) in vehicle control cultures fell within the normal range based on historical control data (confidence interval: mean value ± 2 standard deviations). Adequate number of cells (at least 300 at each test point) and test item concentrations were analysable. The positive control items, Mitomycin-C and Cyclophosphamide, induced statistically significant increases in the incidence of cells with structural aberrations compared with the concurrent

negative control and the responses were compatible with the historical control range. The study was accepted as valid.

Test item treatments did not induce statistically significant increases in aberration-bearing cells and no statistically significant dose-effect relationship was indicated. The incidences of cells bearing aberrations were within the distribution of the historical data for negative

controls.

Conclusions:

Based on the results obtained, it is concluded that 1-PROPANETHIOL does not induce chromosome aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Executive summary:

The test item 1-PROPANETHIOL was assayed for the ability to induce chromosomal damage in cultured human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation. Three treatment series were included in the study. A short termtreatment was performed where the cells were treated for 3 hours in the presence and absence of S9 metabolism. The harvest time of 24 hours corresponding to approximately 1.5 cell cycle was used. A long term (continuous) treatment was also performed in the absence of S9 metabolism until harvest at 24 hours.

Solutions of the test item were prepared in dimethylsulfoxide (DMSO).

The maximum dose level of 762 µg/mL (corresponding to 10 mM) and the lower dose levels of 508, 339, 226, 151, 100, 66.9, 44.6 and 29.7 µg/mL were used for all treatment series. For the continuous treatment in the absence of S9, the additional dose level of 19.8 µg/mL was included. Appropriate negative and positive controls were included. Two replicate cell cultures were prepared at each test point.

For all treatment series, dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments (as determined by the reduction in mitotic index).

Dose levels selected for scoring were:

S9	Treatment time (hours)	Harvest time (hours)	Dose level (µg/mL)	Relative mitotic index (%)
-	3	24	226, 100 and 44.6	46, 55 and 75
+	3	24	151, 66.9 and 29.7	46, 55 and 61
-	24	24	226, 100 and 44.6	52, 63 and 79

 

For each culture, 150 well spread metaphases were scored to assess the frequency of aberrant cells.

Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any dose level and treatment condition.

No dose related increase of the incidence of cells bearing aberration was observed and all the incidences were within the distribution of the historical data for negative controls.

Statistically significant increases in the incidence of cells bearing aberrations (both including and excluding gaps) were seen following positive control treatments with Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.

It is concluded that 1-PROPANETHIOL does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 October 2017 - 06 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine Kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: obtained from ATCC (American Type Culture Collection, Manassas, USA), through Biovalley (Marne-La-Vallée, France)
- Suitability of cells: recommended by guideline
- For cell lines:
- Absence of Mycoplasma contamination: yes, checked
- Methods for maintenance in cell culture: in flasks as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 10% v/v, in a 37°C, 5% CO2 humidified incubator.
- Cell cycle length, doubling time or proliferation index : 10-12 hours
- Modal number of chromosomes:40
MEDIA USED
- Type and identity of media: The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

The selected dose levels were 0.31, 0.63, 1.25, 2.5, 3.8, 5, 7.5 and 10 mM, both with and without S9 mix.
The dose were selected based on a preliminary test where no precipitate was observed in the culture medium at the end of the 3-hour treatment period, at any dose levels but a severe cytotoxicity was induced at the highest recommended dose level (10 mM). 10 mM was therefore selected as the highest dose level for the main experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO).

- Justification for choice of solvent/vehicle: available solubility data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

cultures treated with the vehicle DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): two cultures/dose level
- Number of independent experiments: a single mutagenicity experiment

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1.6 cells/well were seeded in two 96-well plates/culture (four plates/dose level)
- Test substance added in medium

TREATMENT:
- Exposure duration/duration of treatment: 3 h treatment with and without S9 mix

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 hours
- Selection time (if incubation with a selective agent): 11-12 days
- Method used: microwell plates
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: trifluorothymidine (TFT). To select the TFTR (trifluorothymidine resistant) mutant cells (for the determination of CEmutant), 2000 cells/well were seeded in four 96-well plates/culture (eight plates/dose level). After 11-12 days of incubation in a 37°C, 5% CO2 humidified incubator in the presence of 4 µg TFT/mL of culture medium, the clones were counted, differentiating small and large colonies.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 1.6 cells/well were seeded in two 96-well plates/culture (four plates/dose level)
- Criteria for small (slow growing) and large (fast growing) colonies: size of small colonies: < 25% of the diameter of the well; size of large colonies: > 25% of the diameter of the well.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).

METHODS FOR MEASUREMENTS OF GENOTOXICIY: CEmutant (cloning efficiency in selective medium), relative Mutant Frequency (MF) and the Induced Mutation Frequency (IMF)

Rationale for test conditions:

Preliminary cytotoxicity test: To assess the cytotoxicity of the test item, eight dose levels (one culture/dose level) were tested for 3 hours both with and without metabolic activation. Since the test item was found freely soluble in the final treatment medium and induced a severe cytotoxicity only at the highest recommended dose level (i.e. 10 mM) in the preliminary cytotoxicity test, 10 mM was selected as the highest dose level for the main experiment, according to the criteria specified in the international guidelines.

Evaluation criteria:

Evaluation of a positive response:
Based on IWGT recommendations, a test item is considered clearly positive if, in any of the experimental conditions examined:
- at least at one dose level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10^-6,
- a dose-response relationship is demonstrated by a statistically significant trend test.
Evaluation of a negative response:
A test item is considered clearly negative if, in all experimental conditions, no dose-response relationship is demonstrated or, if there is an increase in MF, it does not exceed the GEF.
Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose levels with Adj. RTG between 10 and 20%, are not considered as positive results.
A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if:
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG

Statistics:

To assess the dose-response relationship, a linear regression was performed between dose levels and individual mutation frequencies obtained at dose levels showing a mean Adj. RTG ≥ 10%. This statistical analysis was performed using SAS Enterprise Guide software.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 5 mM without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no
- Data on osmolality: no
- Water solubility: the test item was found freely soluble in the final treatment medium
- Possibility of evaporation from medium: Due to the volatile characteristic of the test item, tightly sealed tubes were used during the 3-hour treatment period.
- Precipitation and time of the determination: No precipitate was observed in the culture medium at the end of the 3-hour treatment period, at any dose levels.


RANGE-FINDING/SCREENING STUDIES (if applicable): Using a test item concentration of 152.32 mg/mL and a treatment volume of 0.5 % (v/v) in the culture medium (i.e. 100 µL/20 mL culture medium), the highest recommended dose level of 10 mM (corresponding to 761.6 µg/mL) was achievable. Thus the dose levels selected for the treatment of the preliminary test were: 0.005, 0.01, 0.04, 0.12, 0.37, 1.11, 3.33 and 10 mM.
At the highest tested dose level of 10 mM, the pH of the culture medium was approximately 7.7 (7.4 for the vehicle control) and the osmolality was 365 mOsm/kg H2O (380 mOsm/kg H2O for the vehicle control). Thus none of the selected dose levels was considered to produce extreme culture conditions and the highest recommended dose level of 10 mM could be selected as the highest dose level for the main mutagenicity experiment.
No precipitate was observed in the culture medium at the end of the 3-hour treatment period, at any dose levels.
A 40 to 93% decrease in the Adj. RTG was observed at dose levels = 3.33 mM, both with and without S9 mix.
Since the test item was found freely soluble in the final treatment medium and induced a severe cytotoxicity only at the highest recommended dose level (i.e. 10 mM) in the preliminary cytotoxicity test, 10 mM was selected as the highest dose level for the main experiment, according to the criteria specified in the international guidelines.
The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.
For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid.


STUDY RESULTS
- Concurrent vehicle negative and positive control data : The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : yes
- Statistical analysis: p<0.005


Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: In the absence of S9 mix, a 72 to 89% decrease in the Adj. RTG was observed at dose levels ≥ 3.8 mM. The recommended level of cytotoxicity was reached at dose levels ≥ 5 mM (means Adj. RTG between 11 and 19%). In the presence of S9 mix, a 46 to 100% decrease in the Adj. RTG was observed at dose levels ≥ 3.8 mM. None of the selected dose levels induced the recommended level of cytotoxicity. However, considering the narrow dose levels spacing used, this selection was considered as suitable to allow a reliable interpretation.


- Genotoxicity results:
No noteworthy increase in the mutation frequency was noted relative to the corresponding vehicle control, at any of the tested dose levels. In the absence of S9 mix only, a dose-response relationship in the MF was demonstrated by the linear regression (p<0.005). Since all the Induced Mutation Frequencies (IMF) remained substantially below the GEF of 126 x 10^-6, this linear trend was considered as meaningless and these results were considered to be a negative response.
These overall results did not meet the criteria of a positive response.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see attachment
- Negative (solvent/vehicle) historical control data: see attachment

See attachment for data tables.

Conclusions:

Isopropyl mercaptan (mixture of 84.3% propane-2-thiol + 15.2% propane-1-thiol) has been tested for mutagenicity in L5178Y TK+/- mouse lymphoma cells in a valid in vitro mammalian cell gene mutation study with the microwell method, conducted according to OECD Test Guideline 490 and in compliance with GLP. The test material did not induce significant increase in the mutation frequency relative to the corresponding solvent control, at any of the tested dose levels. In the absence of metabolic activation only, a dose-response relationship in the MF was demonstrated by the linear regression (p<0.005). Since all the Induced Mutation Frequencies (IMF) remained substantially below the GEF of 126 x 10⁻⁶, this linear trend was considered as meaningless and these results were considered to be a negative response. Therefore, the test material was concluded to be negative for mutagenicity to mammalian cells when tested up to cyto toxic concentration with and without metabolic activation. Positive and vehicle controls were included and gave the expected results.

Executive summary:

In a mammalian cell gene mutation study (thymidine kinase locus), isopropyl mercaptan (2-propanethiol) was evaluated for its ability to induce mutations at the TK (thymidine kinase) locus in L5178Y TK+/- mouse lymphoma cells. The study was performed according to OECD Test Guideline 490 and in compliance with GLP.

After a preliminary cytotoxicity test, 2-propanethiol in DMSO was tested in a single experiment (3-hour treatment), with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 10⁵cells/mL were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% in a 37°C, 5% CO₂humidified incubator.

Due to the volatile characteristics of 2-propanethiol, tightly sealed tubes were used during the 3-hour treatment period. Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

Since the 2-propanethiol was freely soluble in the final treatment medium and induced a severe cytotoxicity only at the highest recommended dose level (10 mM, 762µg/ml) in the preliminary cytotoxicity test, 10 mM was selected as the highest dose level for the main experiment, according to the criteria specified in the international guidelines.

The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. For the positive control cultures, the increase in the mutation frequencies also met the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid.

The selected dose levels were 0.31, 0.63, 1.25, 2.5, 3.8, 5, 7.5 and 10 mM (24, 48, 95, 190, 289, 381, 571 and 762µg/ml) both with and without S9 mix.

In the absence of S9 mix, a 72% to 89% decrease in the Adj. RTG was observed at dose levels ≥ 3.8 mM. The recommended level of cytotoxicity was reached at dose levels ≥ 5 mM (means Adj. RTG between 11 and 19%). In the presence of S9 mix, a 46 to 100% decrease in the Adj. RTG was observed at dose levels ≥ 3.8 mM. None of the selected dose levels induced the recommended level of cytotoxicity. However, considering the narrow dose levels spacing used, this selection was considered as suitable to allow a reliable interpretation.

No significant increase in the mutation frequency was noted relative to the corresponding solvent control, at any of the tested dose levels.

In the absence of S9 mix only, a dose-response relationship in the mutation frequency (MF) was demonstrated by the linear regression (p<0.005). Since all the Induced Mutation Frequencies (IMF) remained substantially below the GEF of 126 x 10^-6, this linear trend was considered as meaningless and these results were considered to be a negative response as the overall results did not meet the criteria of a positive response.

Under the experimental conditions of this study, the test item, 2-propanethiol, did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1985-03-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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, all dose levels showed significant toxicity, individual culture data were not provided, historical control data were not provided and large vs small colonies were not determined.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y heterozygous mouse lymphoma cells, subline 3.7.2C.

Additional strain / cell type characteristics:

other: Diploid cells. The thymidine kinase heterozygous cells (TK+/-) are sensitive to 5-bromodeoxyuridine and trifluorothymidine and resistant to a solution of thymidine, hypoxanthine, methotrxate and glycine.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver microsomal fraction

Test concentrations with justification for top dose:

Eight graded concentrations were used. The maximum dose was 1000 microgram per millilitre because that was the limit of the test material's solubility. The doses in micrograms per millilitre were: 60.6, 90.5, 135.0, 201.5, 300.8, 448.9, 670, and 1000.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide

Untreated negative controls:

yes

Remarks:

Media

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 3-methylcholanthrene with metabolic activation, and methylmethanesulfonate without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in agar (plate incorporation)

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 plus or minus 2 days
- Fixation time (start of exposure up to fixation or harvest of cells): No data reported

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: No data reported

NUMBER OF CELLS EVALUATED: 6,000,000 cells per dose


DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth


OTHER EXAMINATIONS: None


OTHER:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

Evaluation criteria:

A chemical would be considered positive if a dose-related response is obtained in which the mutation frequencies at two or more test concentrations are at least two to three-fold higher than the mutation frequency in the solvent control.

Statistics:

No data reported.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The evidence of induced mutant colonies over background is unclear because only one sample showed a positive result.

Table 1. Summary of mouse lymphoma data for ethyl mercaptan (Ethanethiol), with and without metabolic activation

Concentration μg/ml	% Total Survival		Mutation Frequency		Fold Increase		Increase above GEF
	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA
+MA	-MA	-	4.0	5.1	-	-	n/a	n/a
DMSO	100	100	5.9	3.6	1.0	1.0	n/a	n/a
EMS (620 μg/ml)		53.5	-	41.7	-	8.2	-	381
MCA (3 μg/ml)	72.4	-	19.2	-	3.2	-	133	-
1000	NR	NR	NR	NR	NR	NR	-	-
670	NR	NR	NR	NR	NR	NR	-	-
488.9	0.9^a	0.9^a	8.8	5.4	1.5	1.5	29	18
300.8	1.3^a	1.2^a	7.2	5.1	1.2	1.4	13	15
201.5	0.5^a	1.1^a	6.9	7.4	1.2	2.1	10	38
135.0	16.0	15.6	4.4	6.3	0.7	1.8	-15	27
90.5	22.6	20.1	4.0	13.3	0.7	3.7	-19	97
60.6	10.0	30.8	5.2	5.6	0.9	1.6	-7	20

DMSO - Dimethylsulfoxide

EMS - Ethylmethanesulfinate

3 -MCA - 3 -Methylcholanthrene

NR - No result, toxic dose

a - Invalid dose, less than 10 % survival

Mutant frequency: per 10⁻⁵ viable cells

GEF Global evaluation frequency

n/a not applicable

Conclusions:

Ethanethiol has been tested in a valid in vitro mammalian mutagenicity study, conducted according to a guideline similar to OECD Test Guideline 476 (1984 Version) but without information on GLP compliance. Ethanethiol was tested up to its solubility limit (1000 µg/ml). Exposure resulted in induced mutation frequencies of 4.0 to 6.3 per 10⁵ cells as compared with 3.6 per 10⁵ 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 10⁵ cells. Positive controls induced the appropriate response. Assessment of the results according to the criteria in the current 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 (GEF) 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, the increase in mutation frequency above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test).” Therefore, it is considered by the reviewer 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.

Executive summary:

In a mammalian cell gene mutation study (thymadine kinase locus) (Pence, 1985), 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^-5 cells 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^-5 cells. 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 by the reviewer 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.

The study was classified as reliable with restrictions because it was similar to guideline study but only 0.5E06 cells were plated for selection of mutants, all dose levels showed significant toxicity, individual culture data were not provided, historical control data were not provided and large vs small colonies were not determined.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

1-Propanethiol has been tested in a valid bacterial reverse mutation assay, according to OECD Test Guideline 471 (1997), and in compliance with GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA (Charles River, 2017). No increase in the number of revertants was observed in any test strain, with or without metabolic activation. Appropriate positive and solvent controls were used and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

1-Propanethiol has been tested for ability to cause chromosome aberrations in human lymphocytes according to OECD Test Guideline 473 and in compliance with GLP (RTC, 2018). No increase in the number of cells with aberrations was observed either with or without metabolic activation in human lymphocytes when tested up to cytotoxic concentration. Appropriate solvent and positive controls were included and gave the expected results.

There are no available measured data for the in vitro mammalian cell mutagenicity endpoint for the registration substance, 1-propanethiol (CAS 107-03-9; n-propylmercaptan, NPM), therefore data were read-across from the structurally analogous substances ethanethiol (CAS 75-08-1) and 2-propanethiol (CAS 75-33-2, IPM). 1-Propanethiol (NPM, target), ethanethiol (Source 1) and 2-propanethiol (IPM, Source 2) all contain a thiol (-SH) functional group with a linear aliphatic carbon chain. See attachment to Section 13 for justification of read-across.

Isopropyl mercaptan (mixture of 84.3% 2-propanethiol + 15.2% 1-propanethiol) has been tested for mutagenicity in L5178Y TK+/- mouse lymphoma cells in a valid in vitro mammalian cell gene mutation study (thymidine kinase locus), conducted according to OECD Test Guideline 490 and in compliance with GLP. The test material did not induce significant increase in the mutation frequency relative to the corresponding solvent control, at any of the tested dose levels. In the absence of metabolic activation only, a dose-response relationship in the MF was demonstrated by the linear regression (p<0.005). Since all the Induced Mutation Frequencies (IMF) remained substantially below the GEF of 126 x 10⁻⁶, this linear trend was considered as meaningless and these results were considered to be a negative response. Therefore, the test material was concluded to be negative for mutagenicity to mammalian cells when tested up to cyto toxic concentration with and without metabolic activation. Positive and vehicle controls were included and gave the expected results (Citoxlab, 2018).

Ethanethiol has been tested in a valid in vitro mammalian mutagenicity study, conducted according to a guideline similar to OECD Test Guideline 476 (1984 Version) but without information on GLP compliance. Ethanethiol was tested up to its solubility limit (1000 µg/ml). Exposure resulted in induced mutation frequencies of 4.0 to 6.3 per 10⁵ cells as compared with 3.6 per 10⁵ 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 10⁵ cells. Positive controls induced the appropriate response. Assessment of the results according to the criteria in the current 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 (GEF) 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, the increase in mutation frequency above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test).” Therefore, it is considered by the reviewer 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 (Pence, 1985).

Justification for classification or non-classification

Based on the available data for 1-propanethiol and the structural analogues ethanethiol (CAS 75-08-1) and 2-propanethiol (CAS 75-33-2, IPM), no classification for genetic toxicity is required for 1-propanethiol according to Regulation (EC) No 1272/2008.