Benzoic acid, 2-([1,1'-biphenyl]-4-ylcarbonyl)-

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:

From November 05, 2012 to November 29, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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:

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

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:

2-{[1,1'-biphenyl]-4-carbonyl}benzoic acid

EC Number:

700-862-4

Cas Number:

42797-18-2

Molecular formula:

C20 H14 O3

IUPAC Name:

2-{[1,1'-biphenyl]-4-carbonyl}benzoic acid

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): 2-(4Phenylbenzoyl)benzoic acid
- Physical state: white powder
- Analytical purity: 99.9%
- Lot/batch No.: 5307
- Expiration date of the lot/batch: 01 May 2015
- Storage condition of test material: room temperature in the dark

Specific details on test material used for the study:

Batch No.: 5307; Purity: >99.9%

Method

Target gene:

Histidine and tryptophan

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:

S9 fraction

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

2µg/plate for WP2uvrA, 3µg/plate for TA100, 5µg/plate for TA 1535

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

9-aminoacridine

Remarks:

80µg/plate for TA1537

Controls 3

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2µg/plate for TA98

Controls 4

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-amino anthracene

Remarks:

10µg/plate for WP2uvrA, 1µg/plate for TA100, 2µg/plate for TA1535 and TA1537

Controls 5

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

benzo(a)pyrene

Remarks:

5µg/plate for TA98

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 48 h at 37°C
- Exposure duration: 20 min with shaking

NUMBER OF REPLICATS: 3

DETERMINATION OF CYTOTOXICITY:
- Method: microscopic evaluation of thinning

EXPERIMENT I - Plate incomporation method

Eight concentrations of the test subsance 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate were assayed in tripliate against each tester strain, using the direct plate incomporation method.

Without metabolic activation:
0.1 mL of appropriate concentration of the test substance, vehicle or appropriate positive control was added to 2 mL of trace amino-acid supplemented media (at approximately 45°C) containing 1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were than mixed and overlayed onto a Vogel-Boner agar plate. Negative (untreated) controles were also performed on the same day as the mutation test. Each concentration of the test substance,m appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With metabolic activation:
Same procedure was applied as for study without metabolic activation except that following the addition of the test substance formulation and bacterial culture, 0.5 mL of S9-mix was added to the trace amino-acid supplemented media intead of phosphate buffer.

Incubation and scoring:
All of the plates were incubated at 37 °C ± 3°C for approximately 48 h and scored for the presence revertant colonies using an automated colony counting system. The plates were viewd microscopically for evidence of thinning (toxicology).

EXPERIMENT II - test for mutagenicity - Pre-Incubation Method

As experiment I was deemed negative. Experiment 2 was performed using the pre-incubation method in the presence and absece of metabolic ativation.

Without metabolic activation:
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test substance formulation, vehicle or appropriate positive control were incubated at 37 ± 3°C for 20 min (with shaking) prior to addition of 2 mL of amino-acid supplemented media and subsequent planting onto a Vogel-Boner plates. Negative (untreated) controles were also performed on the same day as the mutation test. All testing for this experiment was performed in triplicates.

With metabolic activation:
Same procedure was applied as for study without metabolic activation except that following the addition of the test substance formulation and bacterial culture, 0.5 mL of S9-mix was added to the trace amino-acid supplemented media intead of phosphate buffer, prior to incubation at 37 ± 3°C for 20 min (with shaking) and addition of amino-acid supllemented media. All testing for this experiment was performed in triplicate.

Incubation and scoring:
All of the plates were incubated at 37 ± 3°C for approximately 48 h and scored for the presence revertaant colonies using an automated colony counting system. The plates were viewd microscopically for evidence of thinning (toxicology).

Evaluation criteria:

There are several criteria for determining positive result. Any one or all of the following can be used to determine overall result of the study:
1. 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. Statystical analysis of data as determined by UKEMS (Mahon et al. 1989).
5. Fold increase greater than two times the concurent solvent control for any tester strain (especially if acompanied by out-of-historical range response (Cariello and Piegorsch, 1996)).
A test substance 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, is some instances the data generated will prohibit making a definite judgment about test substance activity. Results of this type will be reported as equivocal.

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

INFORMATION ON CYTOTOXICITY: cytotoxicity was observed at 5000 µg/plate with and without metabolic activation for all strains.

In Experiment I (plate incorporation method) the test substance induced a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the revertant colony frequency of all of the tester strais from 1500 µg/plate (Sallmonella strains) and at 5000 µg/plate (Escherichia Coli strain WP2uvrA) both in the presence and absence of metabolic activation. Consequently the same maximum dose level was used in the second mutation test of Escherichia Coli and the toxic limit of the test substance was selected for the Salmonella typhimurium strains. The toxic response of the test substance in Experiment II (pre-incubation method) closely followed the results of the first experiment with weakened lawns noted to the Sallmonella strains from 1500 µg/plate and to Escherichia Coli strain WP2uvrA 5000 µg/plate. No test substance precipitate was observed on the plates at any of the doses tested in either presence or absence of the S9-mix.

There were no significant increases in the frequency of relevant colonies recorded for any of the bacterial strain, with any dose of the test substance, either with or withourt metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency revertant colonies thus confirming the activity of the S9-mix and sensitivity of the bacterial strains. Results for the negative controls are also considered to be acceptable.

There were no 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 (plate incorporation method). 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).

Remarks on result:

other: not mutagenic

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was not found to be mutagenic.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance PBBA, according to OECD Guideline 471, in compliance with GLP. The test substance was examined using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA 100) and Escherichia coli strain WP2uvrA in the Ames plate incorporation and pre-incubation methods up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors). The dose range for Experiment I was pre-determined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test substance formulations. Additional dose levels and an expanded dose range were selected in Experiment 2 in order to achieve both non-toxic dose levels and the toxic limit of the test substance. Results for the positive and negative controls were in line with the historical laboratory data. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation in both the experiment. Cytotoxicity was observed at 5000 µg/plate with and without metabolic activation for all strain. Under the study conditions of the in vitro Ames test, the test substance was considered to be non-mutagenic to S typhimurium strains, both with and without metabolic activation (Thompson, 2013).