Propane-1-thiol

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Propane-1-thiol

EC Number:

203-455-5

EC Name:

Propane-1-thiol

Cas Number:

107-03-9

Molecular formula:

C3H8S

IUPAC Name:

propane-1-thiol

Test material form:

liquid

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

Method

Species / strain

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

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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

Controls 6

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.