Benzeneacetic acid, α-ethylidene-4-nitro-

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 2017-07-11 to 2017-07-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzeneacetic acid, α-ethylidene-4-nitro-

Cas Number:

90924-63-3

Molecular formula:

C10 H9 N1 O4

IUPAC Name:

Benzeneacetic acid, α-ethylidene-4-nitro-

Specific details on test material used for the study:

Batch: 16012R71A
Purity: not specified

Method

Target gene:

histidine locus in several strains of Salmonella typhimurium and tryptophan locus of Escherichia coli (E. coli) strain

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver metabolic activation system (S9 homogenate)

Test concentrations with justification for top dose:

-Dose-range Finding Test:
TA100 and the WP2uvrA, both with and without S9-mix: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate.
Experiment:
TA1535, TA1537 and TA98, both with and without S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Through solubility test based on visual assessment, the test item was dissolved in dimethyl sulfoxide.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For TA1535, without metabolic activation.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

For TA1537, without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

For TA98, without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

For TA100, without metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

For WP2uvrA, wthout metabolic activation

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

For TA1535, TA1537, TA98, TA100 and WP2uvrA, with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Mutation assay: plate incorporation
- Cell density at seeding (if applicable): the optical density of 1.0 ± 0.1 at 700 nm (1E+09 cells/ml)

DURATION
- Exposure duration: 48 ± 4 h

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies whencompared against relevant historical control data generated at the lab.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the xperiment will be repeated.

Statistics:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants intester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment. A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Precipitate: No precipitation of the test item on the plates was observed at the start and end of the incubation period at all tested concentrations.

Toxicity: Cytotoxicity, as evidenced by reduction of the revertant colonies, was observed at the highest concentration in tester strain TA1535 in absence of S9-mix and TA1535 and TA100 in presence of S9-mix.

Mutagenicity: In the absence of S9-mix, the test item induced 6.8, 5.0 and 3.3-fold dose-related increases in the number of revertant colonies compared to the solvent control in the tester strains TA100, WP2uvrA and TA98, respectively.

In the presence of S9-mix, the test item induced up to 9.5, 3.7 and 2.2-fold dose-related increases in the number of revertant colonies compared to the solvent control in the tester strains TA100, WP2uvrA and TA98, respectively. These increases were above the historical data, except in tester strain TA98.

Applicant's summary and conclusion

Conclusions:

Test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The study procedures described in this report were based on OECD 471 guidelines (1997). The objective of this study was to determine the potential of the test item and/or its metabolites to induce reverse mutations at the histidine locus in several strains of

Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli(E. coli) strain WP2uvrAin the presence or absence of an exogenous mammalian metabolic activation system (S9). Since the test results of the mutation assay showed clear positive responses, a follow-up experiment was not performed.

 

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrAin the direct plate assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 in the presence of S9-mix at the highest tested concentration.

 

In the mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA1535 in the absence and presence of S9-mix at the highest tested concentration.

 

In the absence of S9-mix, the test item induced 6.8, 5.0 and 3.3-fold dose-related increases in the number of revertant colonies compared to the solvent control in the tester strains TA100, WP2uvrAand TA98, respectively. These increases were above the historical data and were two-fold (tester strains TA100 and WP2uvrA) or three-fold (tester strain TA98) the concurrent vehicle control values, therefore these increases are considered biologically relevant.

 

In the presence of S9-mix, the test item induced up to 9.5, 3.7 and 2.2-fold dose-related increases in the number of revertant colonies compared to the solvent control in the tester strains TA100, WP2uvrAand TA98, respectively. The increases in the tester strains TA100 and WP2uvrA were above the historical data and two-fold the concurrent vehicle control values, therefore these increases are considered biologically relevant. The increases observed in tester strain TA98 were within the laboratory historical control data range and were not three-fold.

 

Taken together, since 2.2- to 9.5-fold increases were observed both in the absence and presence of S9-mix in the tester strains TA100, WP2uvrAand TA98, and the results were above the laboratory historical control data range in all three tester strains in the absence of S9-mix and in the tester strains TA100 and WP2uvrAin the presence of S9-mix, these increases are considered biologically relevant and the test item is mutagenic in the Salmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.

 

In the tester strains TA1535 and TA1537, no increases in the number of revertants were observed upon treatment with the test item.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

In conclusion, based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay