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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
AMES test
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4-nitrobenzoic acid
EC Number:
200-526-2
EC Name:
4-nitrobenzoic acid
Cas Number:
62-23-7
Molecular formula:
C7H5NO4
IUPAC Name:
4-nitrobenzoic acid
Test material form:
solid: crystalline

Method

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:
The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH, Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
Test concentrations with justification for top dose:
The 4-Nitrobenzic acid concentrations investigated in the Initial Mutation Test:
in S. typhimurium TA98, TA1535, TA1537 and E. coli WP2 uvrA:
±S9: 5000, 1600, 1000, 500, 160, 50 and 16 µg/plate;
in S. typhimurium TA100:
±S9: 1000, 750, 500, 400, 320, 160 and 50 µg/plate.

The Confirmatory Mutation Test was performed with S. typhimurium TA100 and TA1535 strains and the following concentration levels were investigated:
±S9: 1000, 750, 500, 400, 320, 160 and 50 µg/plate.

Recommended maximum concentration according to the OECD 471 guidance is 5000 µg/plate.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylenediamine, NPD, Sodium azide, SAZ, 9-Aminoacridine, 9AA, Methyl methanesulfonate, MMS
Details on test system and experimental conditions:
Tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia
coli WP2 uvrA were obtained from:
Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures. The identification codes, arrival
date and expiry dates of the actual applied stock cultures are summarized in Table 4.
Rationale for test conditions:
Based on the results of the preliminary tests, a stock solution with a concentration of 50 mg/mL was prepared from the test item with dimethyl sulfoxide (DMSO), which was diluted by serial dilutions to obtain seven dosing solutions for lower doses. The maximum test concentration was 5000 µg/plate (±S9) in S. typhimurium TA98, TA1535, TA1537 and E. coli WP2 uvrA and 1000 µg/plate (±S9) in S. typhimurium TA100. At the concentration choice the cytotoxicity, the solubility and a possible positive effect of the test item were taken into consideration .
As indicated in section 6.1.2, this top concentration was a non-precipitating concentration.
Evaluation criteria:
The colony numbers on the controls (untreated, vehicle, positive) and the test item plates were determined (counted manually, evaluated by unaided eye), the mean values and appropriate standard deviations and mutation rates were calculated.

* : untreated, vehicle or positive control

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control;
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
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 specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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 specified
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item induce gene mutations by base pair changes in the genome of the investigated Salmonella typhimurium TA100 and TA1535 strains.
In conclusion, the test item 4-Nitrobenzic acid has mutagenic activity on the applied Salmonella typhimurium TA100 and TA1535 strains in the absence and presence of exogenous metabolic activation, under the test conditions used in this study.
It is classifed as Muta 2 . H341 based on CLP criterium.

Executive summary:

It is classifed as Muta 2 . H341 based on CLP criterium.

The test item 4-Nitrobenzic acidwas tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

An Initial Mutation Test (Plate Incorporation Test) was carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. In the subsequent Confirmatory Mutation Test (Plate Incorporation Test) theSalmonella typhimuriumTA100 and TA1535 strains were investigated; the strains:Salmonella typhimuriumTA98, TA1537 andEscherichia coliWP2uvrAwere not further examined.

The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Plate Incorporation Test, whereSalmonella typhimuriumTA100 and TA1535 strains were investigated, see above).

Based on the results of the Solubility Test and the Concentration Range Finding Test the test item was dissolved in dimethyl sulfoxide (DMSO).

At the concentration choice the cytotoxicity, the solubility of the test item (obtained in the Concentration Range Finding Test) and a possible positive effect of the test item were taken into considerationand based on the recommendations of OECD 471 guideline [6] the following concentrations of the test item were prepared and investigated in the Initial Mutation TestinS. typhimuriumTA98, TA1535, TA1537 andE. coliWP2uvrA:

±S9: 5000, 1600, 1000, 500, 160, 50 and 16 µg/plate;

inS. typhimuriumTA100:

±S9: 1000, 750, 500, 400, 320, 160 and 50 µg/plate.

Because of the positive results obtained in the Initial Mutation Test, modification of the originally planned testing procedure was considered as necessary.

To confirm and to investigate the reproducibility of these positive results in the Confirmatory Mutation Test the following concentration levels were investigated:

±S9: 1000, 750, 500, 400, 320, 160 and 50 µg/plate.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study.

In the performed experiments inhibitory, cytotoxic effects of the test item were noticed. The inhibitory effect was indicated by absentrevertants or revertant colony numbers below the historical control data ranges and/or vehicle control data ranges or by higher number of characteristic pinpoint colonies.

In general, 750 µg/plate (noticed following the plate incorporation procedures in
S. typhimuriumTA100 strain in the absence of exogenous metabolic activation) was considered as lowest concentration showing unequivocal cytotoxicity.


The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO) plates with and without S9 demonstratedthe characteristic mean number of spontaneous revertantsthat was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

In the Initial Mutation Test, following the plate incorporation procedure, positive results, significant, biologically relevant, dose-related changedrevertant colony number increases, revertant colony numbers above the vehicle control data and above the historical control data ranges were obtained inS. typhimuriumTA100 and TA1535 strains in the absence and presence of exogenous metabolic activation (±S9).

In the Confirmatory Mutation Test the Initial Mutation Test results were adequately repeated in the case ofS. typhimuriumTA100 and repeated and further refined (due to the changed concentration levels) in the case of TA1535.

In summary unequivocal biologically relevant increases in revertant colony numbers were observed inSalmonella typhimuriumTA100 and TA1535 strains following treatment with4-Nitrobenzic acid, in the absence and presence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test iteminduce gene mutations by base pair changes in the genome of the investigatedSalmonella typhimuriumTA100 and TA1535 strains.

In conclusion, the test item4-Nitrobenzic acidhas mutagenic activity on the appliedSalmonella typhimuriumTA100 and TA1535 strainsin the absence and presence of exogenous metabolic activation, under the test conditions used in this study.