sodium 4-[(4-chlorobenzoyl)amino]benzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

other: USA, EPA (TSCA) OCSPP harmonized guideline - Bacterial Reverse Mutation Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

sodium 4-[(4-chlorobenzoyl)amino]benzoate

EC Number:

807-461-4

Cas Number:

1489170-67-3

Molecular formula:

C14H9ClNNaO3

IUPAC Name:

sodium 4-[(4-chlorobenzoyl)amino]benzoate

Test material form:

not specified

Details on test material:

- Storage condition of test material: room temperature in the dark

Method

Target gene:

Salmonella typhimurium
TA1537: his C 3067; rfa-; uvrB-
TA98: his D 3052; rfa-; uvrB-; R-factor
TA1535: his G 46; rfa-; uvrB-
TA100: his G46; rfa-; uvrB-; R-factor

Escherichia coli
WP2uvrA: trp-; uvrA-

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:

rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Experiment 1: Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed (with and without metabolic activation) in triplicate against each tester strain, using the direct plate incorporation method.

Experiment 2: The dose range used for experiment 2 was determined by the results of Experiment 1 and was 5 to 5000 jig/plate.
Additional dose levels and an expanded dose range were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: suspension in sterile distilled water at 12.5 mg/mL
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water (12.5, 25 and 50 mg/mL), dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in-house. The test item formed the best doseable suspension in sterile distilled water at 12.5 mg/mL, therefore, this solvent was selected as the vehicle.

Controls

Untreated negative controls:

yes

Remarks:

2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non-mutagenic in the absence of metabolizing enzymes, were used

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period (only in Experiment 2): incubation at 37 °C± 3 °C for 20 minutes (with shaking)
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3 plates per dose level, in two independent experiments

OTHER: A test item precipitate (opaque white and powdery in appearance) was observed at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

with and without

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. Although some slightly lowered revertant counts were noted at 5000 µg/plate to several of the tester strains (1500 µg/plate to TA1537 in the presence of S9-mix), there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the first mutation test and consequently the same maximum dose level was used in the second mutation test. In Experiment 2 (pre-incubation method) the test item caused a visible reduction in the growth of the bacterial background lawn and/or a substantial reduction in revertant colony frequency of several tester strains, initially from 1500 µg/plate in the presence and absence of metabolic activation. A test item precipitate (opaque white and powdery in appearance) was observed at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.
There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1. A small, statistically significant increase in TA98 revertant colony frequency was observed in the absence of S9-mix at 1.5 µg/plate in the first experiment. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship. Furthermore, the individual revertant colony counts at 1.5 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method).
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test substance was considered to be non-mutagenic under the conditions of this test. The study was performed under GLP according to OECD guideline 471 and EU Method B.13/14 and therefore reliability of Klimisch 1 has been assigned.