N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-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:

04 Feb 1988 to 14 Apr 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

yes

Remarks:

E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 is missing

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

410-690-9

EC Name:

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

Cas Number:

103055-07-8

Molecular formula:

C17 H8 Cl2 F8 N2 O3

IUPAC Name:

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

Method

Target gene:

his- (S. typhimurium)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Aroclor 1254 induced rat liver S9
- Method of preparation of S9 mix: The S9 is prepared from rats (Tif: RAIf(SPF)) induced with Aroclor 1254 and 0.7 mL of a solution of co-factors.

Test concentrations with justification for top dose:

Pre-Experiment: 0.08 - 5000 μg/0.1 mL
Main experiment: 20, 78, 313, 1250 and 5000 μg/0.1 mL

Vehicle / solvent:

- Solvent used: acetone.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

cyclophosphamide

other: Daunorubicin-HCl

Details on test system and experimental conditions:

PRELIMINARY TEST FOR TOXICITY
A preliminary toxicity test was carried out with strain TA 100 without activation with 9 concentrations ranging from 0.08 to 5000 μg/0.1 mL. The protocol used is the same as in the mutagenicity test. Accordingly, the concentration of 5000 µg/0.1 mL was used as the highest in the mutagenicity test and the tests were performed with the following concentrations of the trial substance without and with microsomal activation: 20, 78, 313, 1250 and 5000 µg /0.1 mL.

EXPERIMENTAL PERFORMANCE
The substance is dissolved in acetone. Acetone alone is used for the negative controls (the substances and vehicles used for the positive controls are indicated below). Each Petridish contains:
- approx. 20 mL of minimum agar (agar bacterial grade, plus salts and glucose).
- 0.1 mL of a solution of the test substance or the vehicle and 0.1 mL of a bacterial culture (in nutrient broth, 2.5 %) in 2.0 mL of soft agar.
- The soft agar is composed of: 100 mL of 0.6 % agar solution with 0.6 % NaCl and 10 mL of a solution of L-histidine, 0.5 mM and + biotin, 0.5 mM.
- In the experiments in which the substance is metabolically activated, 0.5 mL of an activation mixture is added also.
- 1 mL activation mixture contains: 0.3 mL S9 fraction of liver from rats induced with Aroclor 1254 and 0.7 mL of a solution of co-factors.
- In the experiments without and with the addition of microsomal activation mixture three petri dishes are prepared per strain and per group (i.e. per concentration or per control group). The plates are incubated for about 48 hours at 37 ±1.5 °C in darkness.

DATA PRESENTATION
The data reported include individual numbers of revertants per plate, mean values and standard deviation for each bacterial strain and each concentration. Historical values of negative controls obtained with each strain without and with microsomal activation are listed in a separate table, which contains mean values of controls covering an experimental period of 1 year as well as standard deviations and acceptable minimal and maximal values of spontaneous revertants for each bacterial strain.

ANALYTICAL CONTROL
To prove that the indicator organisms are really exposed to the intended concentrations of the test substance, determination of the substance in solution is performed by the analytical unit. This determination is performed with the lowest concentration of the solutions used in the mutagenicity tests.

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE
The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the following strains: TA 98, TA 1535 and TA 1537,
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100. Generally a concentration-related effect should be demonstrable.

ACCEPTABILITY OF THE ASSAY
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision has to be based on scientific judgement.

Results and discussion

Test results

Key result

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TOXICITY TEST
Nine concentrations of the test substance ranging from 0.08 to 5000 µg/0.1 mL were tested to determine the highest concentration to be used in the mutagenicity assay. From the results obtained, the highest concentration suitable for the mutagenicity test was found to be 5000 µg/0.1 mL

MUTAGENICITY TEST
In two separate experiments performed without and with microsomal activation, none of the tested concentrations of test substance led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. At the concentrations of 313 μg/0.1 mL and above the substance precipitated in soft agar. The sensitivity of the test system, and the metabolic activity of the S9-mix, was clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances.

Applicant's summary and conclusion

Conclusions:

No induction of point mutation by the test substance or by its metabolites was detectable in the tested strains. It was concluded that the test substance showed no evidence of genotoxic activity in this assay system.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test and the pre-incubation test. For this purpose, the Salmonella typhimurium histidine-auxotrophic strains TA 1535, TA 1537, TA 98 and TA 100 and Aroclor 1254 pre-treated liver microsomes of rats (Tif: RAIf(SPF)) were used according to GLP principles and OECD TG 471.

A preliminary toxicity test was carried out with strain TA 100 without activation with 9 concentrations ranging from 0.08 to 5000 μg/0.1 mL. The protocol used is the same as in the mutagenicity test. The investigations were performed on Salmonella strains with 20, 78, 313, 1250 and 5000 μg/0.1 mL trial substance without and with microsomal activation (S9-mix) using liver microsomes of rats (Tif: RAIf(SPF)) pre-treated with Aroclor 1254. The substance was dissolved in acetone. Acetone alone was used for the negative controls. In the experiments without and with the addition of microsomal activation mixture 3 Petri dishes are prepared per strain and per group (i.e. per concentration or per control group). The plates are incubated for about 48 hours at 37 ± 1.5 °C in darkness.

The test material in solution was analysed to confirm the intended concentrations to be used in the mutagenicity tests. The values of the lowest concentrations to be analysed were found to be somewhat above the calculated concentrations, mainly due to the fast evaporation of the vehicle acetone. This, however, did not affect the test results.

From the results obtained in the toxicity test, the highest concentration suitable for the mutagenicity test was found to be 5000 μg/0.1 mL. In two separate experiments performed without and with microsomal activation, none of the tested concentrations of test substance led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. At the concentrations of 313 μg/0.1 mL and above the substance precipitated in soft agar. The sensitivity of the test system, and the metabolic activity of the S9-mix, was clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances.

To conclude, no induction of point mutation by the test substance or by its metabolites was detectable in the tested strains and this assay system did not show no evidence of genotoxic activity.