Triflumezopyrim

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

Reference substance 002

Cas Number:

1263133-33-0

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Test results 6

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

Test results 7

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.