N-[[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]carbamoyl]-2,6-difluorobenzamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 February 2001 - 02 March 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

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:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Japan MAFF Revised Mutagenicity Guidelines, 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]-3-(2,6-difluorobenzoyl)urea

EC Number:

601-779-5

Cas Number:

121451-02-3

Molecular formula:

C17H7Cl2F9N2O3

IUPAC Name:

1-[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]-3-(2,6-difluorobenzoyl)urea

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: White powder
- Storage condition of test material: Ambient temperature

Method

Target gene:

Histidine and tryptophan loci.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

- Type and identity of media: The broth used to grow overnight cultures of the tester strains was Vogel- Bonner salt solution (Vogel and Bonner, 1956) supplemented with 2.5 % (w/v) Oxoid Nutrient Broth No. 2 (dry powder).
Bottom agar (25 mL per 15 x 100 mm petri dish) was Vogel- Bonner minimal medium E (Vogel and Bonner, 1956), supplemented with 1.5 % (w/v) agar and 0.2 % (w/v) glucose.
Top (overlay) agar was prepared with 0.7 % agar (w/v) and 0.5 % NaCl (w/v) and was supplemented with 10 mL of 1) 0.5 mM histidine/biotin solution per 100 mL agar for selection of histidine revertants, or 2) 0.5 mM tryptophan solution per 100 mL of agar for selection of tryptophan revertants. For an agar overlay, 2.0 mL of the supplemented top agar was used.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
- Periodically checked for genotype: Tester strain cultures were checked for the following genetic markers on the day of their use in the mutagenicity assay; rfa Wall Mutation and pKM101 Plasmid.

Additional strain / cell type characteristics:

other: All Salmonella typhimurium strains; uvrB and rfa. Salmonella typhimurium stains TA98 and TA100; pKM101. Escherichia coli WP2; uvrA.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose range finding assay: 6.67, 10, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate
Mutagenicity assay: 33.3, 100 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene, ICR-191

Details on test system and experimental conditions:

Two independent assays were performed, an initial mutagenicity assay followed by a confirmation assay.

METHOD OF APPLICATION: preincubation method. S9 mix (or phosphate buffer, where appropriate), the tester strain, and the test article were preincubated prior to the addition of molten agar. The agar and the preincubation reaction mixture were mixed and then overlaid onto a minimal agar plate.

DURATION
- Preincubation period: 20 ± 2 minutes at 37 ± 2 °C.
- Exposure duration: 52 ± 4 hours at 37 ± 2 °C.

NUMBER OF REPLICATIONS: All concentrations of the test article, the vehicle controls and the positive controls were plated in triplicate.

REVERTANT COLONY COUNTING: The number of revertant colonies per plate for the vehicle controls and all plates containing test article were counted manually. The number of revertant colonies per plate for the positive controls were counted by automated colony counter.

DETERMINATION OF CYTOTOXICITY
- Method: Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that concentration level. Lawns were scored as 1) normal, 2) slightly reduced, 3) moderately reduced, 4) extremely reduced, 5) absent, or 6) obscured by precipitate.

Evaluation criteria:

- Tester Strain TA100: For a test material to be considered positive, it had to produce at least a 2-fold dose related and reproducible increase in the mean revertants per plate over the mean revertants per plate of the appropriate vehicle control. A response that did not meet all three of the above criteria (magnitude, dose-responsiveness, reproducibility) was not evaluated as positive.
- Tester Strains TA98, TA1535, TA1537, and WP2uvrA: For a test material to be considered positive, it had to produce at least a 3-fold dose related and reproducible increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. A response that did not meet all three of the above criteria (magnitude, dose-responsiveness, reproducibility) was not evaluated as positive.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose Rangefinding Assay
Cytotoxicity was observed with tester strain TA100 in the absence of S9 mix at the maximum concentration tested (5000 µg per plate) as evidenced by a concentration-related decrease in the number of revertants per plate. No cytotoxicity was observed with either tester strain TA100 in the presence of S9 mix, as evidenced by no concentration-related decrease in the number of revertants per plate and a normal bacterial background lawn. No cytotoxicity was observed with tester strain WP2uvrA in the presence or absence of S9 mix, as evidenced by no concentration-related decrease in the number of revertants per plate and a normal bacterial background lawn.

Mutagenicity Assay
In the initial mutagenicity assay, and in the confirmatory assay, all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mutagenicity Assay – Summary

Substance	Concentration µg/plate	Tester Strain										Background Lawn*
		TA98		TA100		TA1535		TA1537		WP2uvrA
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
With External Metabolic Activation System S9 mix
Vehicle Control		27	8	111	8	18	3	9	2	25	5	1
Test Material	33.3	26	7	91	4	14	2	8	4	34	5	1
	100	29	7	109	7	13	2	11	5	26	3	1
	333	32	4	98	5	16	2	9	1	27	2	1sp
	1000	31	4	106	5	13	2	8	2	23	4	1sp
	3330	28	3	104	17	12	2	9	4	22	7	1sp
	5000	27	5	97	19	12	2	7	3	24	6	1mp
Positive Control**		373	32	809	69	136	7	116	13	422	78	1
Without External Metabolic Activation
Vehicle Control		18	7	105	3	18	6	7	2	28	2	1
Test Material	33.3	21	1	101	12	13	6	7	3	23	4	1
	100	23	11	95	6	13	6	10	5	26	5	1
	333	18	5	100	11	13	0	6	4	28	6	1sp
	1000	17	7	88	8	12	4	6	1	21	6	1mp
	3330	12	3	98	3	9	1	4	2	16	4	1mp
	5000	13	0	86	16	13	1	6	1	18	4	1mp
Positive Control***		367	32	687	42	559	20	1170	63	545	28	1

 

Table 2: Confirmation Mutagenicity Assay – Summary

Substance	Concentration µg/plate	Tester Strain										Background Lawn*
		TA98		TA100		TA1535		TA1537		WP2uvrA
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
With External Metabolic Activation System S9 mix
Vehicle Control		33	4	108	7	14	3	11	1	23	4	1
Test Material	33.3	33	2	123	5	10	5	8	1	22	2	1
	100	31	3	115	17	13	3	9	5	22	7	1
	333	30	8	108	10	14	2	9	2	29	4	1sp
	1000	38	7	113	10	13	1	4	1	22	4	1sp
	3330	27	3	118	12	11	3	6	2	15	8	1mp
	5000	31	5	128	5	13	2	7	3	13	2	1mp
Positive Control**		371	52	1016	32	121	2	117	15	494	28	1
Without External Metabolic Activation
Vehicle Control		17	7	106	3	13	6	7	3	17	6	1
Test Material	33.3	27	4	116	14	13	2	4	1	13	6	1
	100	24	3	105	1	11	3	6	3	24	11	1
	333	21	4	101	7	14	3	5	1	21	3	1sp
	1000	22	10	103	6	10	2	6	2	17	2	1sp
	3330	23	7	107	6	14	5	5	1	11	3	1mp
	5000	17	5	111	5	11	1	6	5	13	5	1mp
Positive Control***		396	21	916	25	640	19	1828	19	351	23	1

 

Vehicle control DMSO 50 µL

** TA98, benzo[a]pyrene 2.5 µg/plate

TA100, 2-aminoanthracene 2.5 µg/plate

TA1535, 2-aminoanthracene 2.5 µg/plate

TA1537, 2-aminoanthracene 2.5 µg/plate

WP2uvrA, 2-aminoanthracene 25.0 µg/plate

 

*** TA98, 2-nitrofluorene 1.0 µg/plate

TA100, sodium azide 2.0 µg/plate

TA1535, sodium azide 2.0 µg/plate

TA1537, ICR-191 2.0 µg/plate

WP2uvrA, 4-nitroquinoline-N-oxide 0.4 µg/plate

 

* Background Lawn Evaluation Codes:

1 = normal; 2 = slightly reduced; 3 = moderately reduced; 4 = extremely reduced; 5 = absent; 6 = obscured by precipitate

sp = slight precipitate; mp = moderate precipitate (requires hand count); hp = heavy precipitate (requires hand count)

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Under the conditions of the test, the test material did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of S9 mix. The test material is therefore determined to be non-mutagenic in this assay.

Executive summary:

The genetic toxicity of the test material was determined in an in vitro bacterial reverse mutation assay, an ames test. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 471, EPA OPPTS 870.5100 and Japan MAFF Revised Mutagenicity Guidelines.

The concentrations tested in the mutagenicity assay were selected based on the results of a dose range finding assay using tester strains TA100 and WP2uvrA and ten concentrations of test article ranging from 6.67 to 5000 µg per plate.

The tester strain used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA 1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted with six concentration levels of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per concentration. The concentrations tested were 33.3, 100, 333, 1000, 3330, and 5000 µg per plate in both the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in the independent experiment.

Under the conditions of the test, the test material did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™ induced rat liver (S9). The test material is therefore determined to be non-mutagenic in this assay.