Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test article name : Sodium methylmercaptide
CAS RN°: 5188-07-8
Origin: Elf Atochem, Rotterdam
Batch: 462

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 102, TA 98, TA 100
Additional strain / cell type characteristics:
other: test organisms were properly maintained and were checked for appropriate genetic markers (rfa mutation, R factor)
Metabolic activation:
with and without
Metabolic activation system:
S9 derived from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254 (500 mg/kg)
Test concentrations with justification for top dose:
Plate incorporation assay: 
0, 312.5, 625, 1250, 2500 or 5000 µg/plate for all test strains with and without metabolic activation. 
Pre-incubation assay: 
0, 312.5, 625, 1250, 2500 or 5000 µg/plate for TA1535, TA1537 and TA100 or 0, 125, 250, 500, 1000 or 2000 µg/plate for TA 102 and TA 98, without metabolic activation. 
0, 312.5, 625, 1250, 2500 or 5000 µg/plate for TA1535, TA1537, TA100 and TA102 or 0, 312.5, 625, 1250, 2500 or 4000 µg/plate for TA 102, with metabolic activation.
Vehicle / solvent:
Distilled water
Controls
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
distilled water (50 mg/mL)
True negative controls:
no
Positive controls:
yes
Remarks:
All positive controls were diluted in dimethyl sulfoxide.
Positive control substance:
other: With S9:  2-anthramine 2.0 µg/plate (TA98-TA100-TA1535-TA1537 ), Danthron 30 µg/plate ( TA102 ). Without S9: 2-nitrofluorene 0.5 µg/plate(TA98 ), Sodium azide 1 µg/plate (TA100-TA1535), 9-aminoacridine 50 µg/plate (TA1537), Mitomycin 0.5 µg/plate (TA102)
Details on test system and experimental conditions:
PRELIMINARY TOXICITY ASSAY
The preliminary toxicity assay was used to establish the dose range over which the test article would be assayed. 

MUTAGENICITY ASSAY
Five dose levels of test article along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and TA102 on selective agar in the presence and absence of Aroclor induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.

TEST PROCEDURE
Plate incorporation method:
One half (0.5) milliliter of S9 or Sham mix, 100 µL of tester strain and 100 µL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45°C. After vortexing, the mixture was overlaid onto the surface of minimal bottom agar. 
Preincubation method:
One-half (0.5) milliliter of S9 or sham mix, 100 µL of  tester strain and 100 µL of vehicle or test article dilution were added to glass culture tubes. Alter vortexing, these mixtures were incubated with shaking for 60 minutes at 37°C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of minimal bottom agar.  When plating the positive controls, the test article aliquot was replaced by the of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37°C.  Revertant colonies for a given tester strain and activation condition were counted by automated colony counter.
Evaluation criteria:
Data sets for a tester strain was judged positive if the increase in mean revertants at one or more of the tested concentrations is equal to or greater than 2.0-times the mean vehicle control value.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 102, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Without S9: >= 1000 µg/plate. With S9: >= 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The negative and solvent control results were equivalent to those usually obtained in the Laboratory. The number of revertants induced by the positive controls was higher than the spontaneous one, which demonstrated the sensitivity of
this test and the efficacy of the S9 mix throughout this study.

The test substance SODIUM METHYL MERCAPTIDE did not induce any significant increase in the revertant number with or
without S9 mix in any of the 5 strains.

Mean Number of Revertants Per Plate

Activation:  None

Dose (µg/plate)

TA98

TA100

TA102

TA1535

TA1537

Ctrl

2

7

370*

14

15

312.5

5

5

478

16

16

625

3

7

400

13

17

1250

18(+++)

5

388

16

16

2500

5(++)

5

347(+++)

19

13

5000

18 (+++)

5

229(+++)

20(+)

15

Positive Control

103

43

1996

490

348

*Spontaneous revertants rather than solvent control, study error

Dose (µg/plate)

TA100

TA1535

TA1537

Ctrl

102

16

12

312.5

93

24

18

625

97

21

14

1250

94(+)

19

9

2500

90(++)

20

17

5000

64(+++)

18(+)

8

Positive Control

658

461

180

Dose (µg/plate)

TA98

TA102

Ctrl

32

284

125

43

294

250

34

320

500

27

362

1000

34(++)

325(++)

2000

13(++)

242(+++)

Positive Control

1173

1335

Activation:  S9, Direct plate incorporation

Dose (µg/plate)

TA98

TA100

TA102

TA1535

TA1537

Ctrl

33

122

505

14

14

312.5

28

139

607

20

20

625

35

128

551

21

19

1250

41

139

457

16

20

2500

36

131

578

16

16

5000

35

120

446

16

15

Positive Control

1211

1765

984

263

115

Activation:  S9, Plate - preincubation

Dose (µg/plate)

TA98

TA100

TA102

TA1535

TA1537

Ctrl

42

113

375

23

15

312.5

48

94

431

21

24

625

38

97

489

21

20

1250

47

99(+)

513

27

24

2500

47(++)

94(++)

530

26

21

5000

0(+++)

0(+++)

409(+)

0(+++)

0(+++)

Positive Control

952

1217

1040

338

148

 ( ) Parentheses denote degree of toxicity noted at these concentration: +, ++ , +++


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

A Salmonella typhimurium reverse mutation assay was performed with sodium methanethiolate according to OECD Guideline 471. S. typhimuriumstrains, TA98, TA100, TA102, TA1535 and TA1537, were exposed to five concentrations of sodium methanethiolate in the presence and absence of a metabolic activation system using the plate incorporation and the preincubation methods. All doses of sodium methanethiolate, vehicle control and positive controls were plated in triplicate and incubated for 48-72 hours at 37 ºC. The positive controls gave expected responses. Sodium methanethiolate was cytotoxic at ≥1000 and ≥2500 µg/plate, in the absence and presence of a metabolic activation system, respectively. Treatment of the S. typhimurium strains with sodium methanethiolate in the presence or absence of metabolic activation did not induce any significant increases in the revertant numbers.