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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-1 (2000)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Purity: 97.04%
Impurities: Not reported

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-induced rat liver S9
Test concentrations with justification for top dose:
In the toxicity-mutation test, 66.7, 100, 333, 667, 1000, 3000, 4000 and 5000 μg/plate and in the Confirmatory test: 667, 1000, 3000, 4000, and 5000 μg/plate
Vehicle / solvent:
DMSO
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 4-nitroquinoline N-oxide, acridine mutagen ICR-191, and 2-aminoanthracene
Evaluation criteria:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.

A data set may be judged equivocal if there is a biologically relevant increased response that only partially meets criteria for a positive response. A response will be evaluated as negative if it is neither positive nor equivocal.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 4000 and 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 4000 and 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 4000 and 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Table 1 - Mutagenicity test in Salmonella typhimurium TA98 without S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

21

5

Positive Control*

306

46

    667

18

1

1000

26

4

3000

22

2

4000

37

7

5000

23

5

 

* 1.0 μg/plate 2-Nitrofluorene

 

Table 2 - Mutagenicity test in Salmonella typhimurium TA100 without S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

94

15

Positive Control*

1145

45

    667

78

19

1000

90

12

3000

71

5

4000

90

6

5000

83

17

 

* 2.0 μg/plate Sodium azide

 

Table 3 - Mutagenicity test in Salmonella typhimurium TA1535 without S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

12

3

Positive Control*

965

56

    667

13

6

1000

13

1

3000

12

3

4000

12

1

5000

12

3

 

* 2.0 μg/plate sodium azide

 

Table 4 - Mutagenicity test in Salmonella typhimurium TA1537 without S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

9

3

Positive Control*

1321

148

    667

7

3

1000

10

4

3000

7

3

4000

7

3

5000

5

3

 

* 2.0 μg/plate ICR-191

 

Table 5 - Mutagenicity test in Escherichia coli WP2uvrA without S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

21

1

Positive Control*

698

76

    667

19

1

1000

17

5

3000

19

4

4000

18

5

5000

12

1

 

* 1.0 μg/plate 4-Nitroquinoline-N-oxide

 

Table 6 - Mutagenicity test in Salmonella typhimurium TA98 with S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

26

5

Positive Control*

473

12

    667

21

9

1000

34

8

3000

30

6

4000

24

2

5000

26

8

* 2.5 μg/plate Benzo(a)pyrene

 

Table 7 - Mutagenicity test in Salmonella typhimurium TA100 with S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

98

14

Positive Control*

1445

101

    667

88

5

1000

85

11

3000

95

3

4000

91

10

5000

86

8

 

* 2.5 μg/plate 2-Aminoanthracene

 

Table 8 - Mutagenicity test in Salmonella typhimurium TA1535 with S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

7

1

Positive Control*

147

10

    667

10

6

1000

12

5

3000

7

4

4000

10

4

5000

8

3

 

* 2.5 μg/plate 2-Aminoanthracene

 

Table 9 - Mutagenicity test in Salmonella typhimurium TA1537 with S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

8

1

Positive Control*

76

5

    667

5

1

1000

8

2

3000

6

2

4000

6

2

5000

8

6

 

* 2.5 μg/plate 2-Aminoanthracene

 

Table 10 - Mutagenicity test in Escherichia coli WP2uvrA with S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

19

5

Positive Control*

145

22

    667

19

9

1000

20

4

3000

24

4

4000

16

4

5000

18

7

 

* 25 μg/plate 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.
Executive summary:

The test substance was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the plate incorporation method. Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9) (OECD Section 4 (Part 471), Guideline for the Testing of Chemicals (1997); U.S. EPA OPPTS Guideline 870.5100 (1998); EC Directive 440/2008/EC Method B.13/14; and MAFF Japan 12-Nousan-8147 Guideline Number 2-1-19-1 (2000)).

 

The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the dose range for the mutagenicity test and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test substance.

 

Dimethyl sulfoxide (DMSO) was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in DMSO at 50 mg/mL, the highest stock concentration that was prepared for use on this study.

 

In the toxicity-mutation test, the maximum dose evaluated was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in this test were 66.7, 100, 333, 667, 1000, 3000, 4000 and 5000 μg/plate. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation. No appreciable toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 4000 μg/plate with tester strains TA98, TA1535, and TA1537 in the presence of S9 activation; and at 5000 μg/plate with tester strains TA100 and WP2uvrA in the presence of S9 activation and tester strains TA1535 and TA1537 in the absence of S9 activation.

 

Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in this test were 667, 1000, 3000, 4000, and 5000 μg/plate for all tester strains. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation (see Tables 1 -10 in the "Any other information on results incl. tables" section for details). No appreciable toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 4000 μg/plate for all tester strains in both test condition with the exception of TA1535 without S9 activation where precipitation was observed only at 5000 μg/plate and for WP2uvrA without S9 activation where no test substance precipitation was observed.

 

All criteria for a valid study were met. Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.