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Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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
Controlsopen allclose all
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

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium; TA98, TA100, TA1535, and TA1537 and E. coli WP2uvrA
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.