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Toxicological information

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:
2008-01-18 to 2008-02-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
published June 8, 2000
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
S. typhimurium: his operon
E. coli: trp operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
DNA polymerase A deficient
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
DNA polymerase A deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Based on the results of the dose range finding test, the substance was tested in the first mutation assay at a concentration range of 3 to 1000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains.
In an independent repeat of the assay with additional parameters, the substance was tested at a concentration range of 3 to 333 µg/plate in the absence and presence of 10% (v/v) S9-mix in tester strains TA 1535 and TA 100, at a concentration range of 1 to 100 µg/plate in the absence of S9-mix and at 3 to 333 µg/plate in thepresence of S9-mix in tester strains TA 1537 and TA 98 and at a concentration range of 10 to 1000 µg/plate in the absence and presence of S9-mix in the tester strain WP2uvrA. Toxicity was observed in all tester strains.
Vehicle / solvent:
dimethyl sulfoxide
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Saline, Milli-Q water or DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding (if applicable):1E+9 cells/mL

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction of background lawn, reduction of number of revertants or an increase in the size of the microcolonies

Rationale for test conditions:
The procedures described in this study were based on the most recent OECD and EEC guidelines.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the
concurrent control, and the total number of revertants in tester strains TA1535, TA1537,
TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case an positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:In the dose range finding test, no increase in the number of revertants was observed upon treatment with the substance under all conditions tested. Cytotoxicity by means of a reduced backgroud lawn and in the number of revertant colonies were found for TA 100 and WP2uvrA at the concentrations summarized in Table 1.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Table 2
- Negative (solvent/vehicle) historical control data: Table 3

Any other information on results incl. tables

Table 1: Toxicity of the test item in the dose range finding test

Strain

without S9-mix

with S9-mix

 

Dose (µg/plate)

Bacterial backgroud lawn

Revertant colonies

Dose (µg/plate)

Bacterial backgroud lawn

Revertant colonies

TA 100

100

moderate

-1

100

moderate

-2

 

333-5000

absent

complete

333

extreme

microcolonies

 

 

 

 

1000-5000

absent

complete

WP2uvrA

1000-3330

extreme

microcolonies

1000-3330

extreme

microcolonies

 

5000

absent

complete

5000

absent

complete

-1Reduction in the number of revertant colonies, but not less than the minimal value of the historical control data range

-2No reduction in the number of revertant colonies

Table 2: Historical positive control data

Strain

 

Minimum value

Maximum value

Mean

± 3 x S.D.

TA1535

- S9-mix

231

1923

1145

 819

 

+ S9-mix

58

538

183

 233

TA1537

- S9-mix

79

927

315

 415

 

+ S9-mix

57

833

334

 433

TA98

- S9-mix

147

1790

1053

 748

 

+ S9-mix

154

1703

648

 966

TAI OO

- S9-mix

452

1593

1038

 596

 

+ S9-mix

223

2061

1094

 996

WP2uvrA

- S9-mix

67

1280

643

  565

 

+ S9-mix

56

887

254

 343

Table 3: Historical negative control data

Strain

 

Minimum value

Maximum value

Mean

± 3xS.D.

TA 1535

- S9-mix

3

     28                

13

14

 

+ S9-mix

3

29

12

14

TA1537

- S9-mix

3

17

6

8

 

+ S9-mix

3

 21

6

9

TA98

- S9-mix

12

45

20

19

 

+ S9-mix

12

51

25

21

TAI OO

- S9-mix

63

194

126

79

 

+ S9-mix

60

195

116

83

WP2uvrA

- S9-mix

4

39

14

17

 

+ S9-mix

4

44

15

18

Applicant's summary and conclusion

Conclusions:
In the present study conducted according to OECD guideline 471, all bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, thus, the substance does not need to be classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).