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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-05-12 to 1993-07-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study received a Klimisch score of 2 and was classified as reliable with restrictions because it closely follows OECD 471 guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Unlike what is recommended in OECD 471, some samples were incubated for 24 hours, instead of the recommended 48-72 hours. Study does not include E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102, as recommended.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ethyl mercaptan (ethanethiol)

Method

Target gene:
Histidine
Species / strain
Species / strain:
other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
- Type and identity of media: Nutrient agar
- Properly maintained: yes
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from Arcolor 1254- induced rat liver (5%)
Test concentrations with justification for top dose:
0, 0.05, 0.1, 0.5, 1, and 2.5% (V/V)
2.5 % = 65 µl/plate
1 % = 26 µl/plate
0.5 % = 13 µl/plate
0.1 % = 2.6 µl/plate
0.05 % = 1.3 µl/plate
Vehicle:
No data reported
- Vehicle(s)/solvent(s) used: DMSO and phosphate buffer
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Without metabolic activation- Na azide (TA100, TA1535); 2- nitrofluorene (TA98); 9-aminoacridine (TA1537). With metabolic activation- 2-aminoanthracene (TA100, TA1538, TA1535, TA1537, TA98)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data reported
- Exposure duration: 24 and 48 hours
- Expression time (cells in growth medium): 24 hours after 24-hour exposure; the cultures were incubated for 48 hours with the test material during the 48-hour exposure
- Selection time (if incubation with a selection agent): No data reported
- Fixation time (start of exposure up to fixation or harvest of cells): No data reported


SELECTION AGENT (mutation assays): No data reported
SPINDLE INHIBITOR (cytogenetic assays): No data reported
STAIN (for cytogenetic assays): No data reported


NUMBER OF REPLICATIONS: triplicate


NUMBER OF CELLS EVALUATED:No data reported


DETERMINATION OF CYTOTOXICITY
Bacterial background lawn and number of Histidine revertant colonies per plate

OTHER EXAMINATIONS:
No data reported
Evaluation criteria:
Revertant colonies were counted and compared to the number of spontaneous revertant colonies on control plates.
Statistics:
No data reported

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
No data reported

RANGE-FINDING/SCREENING STUDIES: A range finding toxicity study was performed using TA98 and TA100 with and without metabolic activation to determine the metabolic activation to determine the concentration range to be used in the main genotoxicity study.

Any other information on results incl. tables

Table 1. Mean number of histidine revertant colonies per plate, with and without metabolic activation

Concentration

%

TA 98

TA 100

TA 1535

TA 1537

TA 1538

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

DMSO

35.7

31.3

137.3

172.3

10.7

13.3

7.5

6.7

19.0

11.3

Spontaneous revertants

42.7

31.7

169.3

173.3

11.0

9.3

11.3

8.3

12.7

13.0

Dichloromethane 7.5 %

 

487.3

 

2277.3

 

35.3

 

6.7

 

4.0

Positive control

959.5

464.5

538.0

639.0

309.0

825.0

50.5

1158.5

192.5

223.5

Ethyl mercaptan 0.05 %

39.7

29.0

148.3

131.0

10.7

9.3

6.7

7.0

13.7

6.7

Ethyl mercaptan 0.1 %

32.3

22.0

157.0

154.7

7.3

7.7

11.3

9.3

15

12.0

Ethyl mercaptan 0.5 %

34.7

32.7

114.3

18.7

8.7

0.7

5.0

1.7

7.7

7.3

Ethyl mercaptan 1 %

31.0

23.3

0.0

0.3

0.0

0.0

0.0

0.0

2.7

5.3

Ethyl mercaptan 2.5 %

0.0

0.0

0.0 +

0.0+

0.0+

0.0+

0.0

0.0+

0.0+

0.0

MA - metabolic activation

+ - reduced background lawn growth

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

There was no evidence of induced S. typhimurium mutant colonies over background in the reverse gene mutation assay in bacteria.
Executive summary:

In a reverse gene mutation assay in bacteria, five strains of S. typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) were exposed to ethanethiol at 0.05, 0.1, 0.5, 1, and 2.5% concentrations.

Ethanethiol was tested up to cytotoxic concentrations. In a preliminary toxicity assay, a clear toxic effect was observed after 24 hours in the in the histidine positive colonies, primarily in TA98 at greater than and equal to 5% and TA 100 at greater then or equal to 1%, with and without metabolic activation. There was no evidence of induced S. typhimurium mutant colonies, with or without metabolic activation, over background in the reverse gene mutation assay in bacteria. The positive controls induced the appropriate responses in the corresponding strains.

This study received a Klimisch score of 2 and was classified as reliable with restrictions because it closely follows OECD 471 guidelines.