4-(p-nitrophenylthio)aniline

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:

January 2019 - March 2019

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

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-(p-nitrophenylthio)aniline

EC Number:

202-957-1

EC Name:

4-(p-nitrophenylthio)aniline

Cas Number:

101-59-7

Molecular formula:

C12H10N2O2S

IUPAC Name:

4-(4-nitrophenyl)sulfanylaniline

Test material form:

solid: particulate/powder

Method

Target gene:

TA98:
his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA100:
his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA1535:
his G 46; rfa-; uvrB-: base-pair substitutions
TA1537:
his C 3076; rfa-; uvrB-: frame shift mutations
TA102:
his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
100 mM of ice-cold sodium-ortho-phosphate-buffer, pH 7.4, was added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of:
- 8 mM MgCl2
- 33 mM KCl
- 5 mM glucose-6-phosphate
- 4 mM NADP
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:
- co-factor solution 9.5 parts
- liver preparation 0.5 parts

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiment were chosen according to the results of the pre-experiment. Due to precipitation in the Pre-experiment 2500 μg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. The experiment was performed with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 μg/plate

Vehicle / solvent:

The test item was dissolved in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine; methylmethanesulfonate; 2-aminoanthracene

Details on test system and experimental conditions:

For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
- 100 μL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
- 500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
- 100 μL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
- 2000 μL Overlay agar.
Due to clear positive results in the first experiment of this study no independent repetition was performed.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Evaluation criteria:

A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, TA102)
- the negative control plates with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2016 -2018)
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analyzable.

Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

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

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test item was observed in all tester strains used at a concentration of 2500 μg/plate (with and without metabolic activation). The observed precipitation did not interfere with the scoring, thus it did not impact the results.
Toxic effects of the test item were noted in some tester strains used:
Toxic effects of the test item were noted in tester strain TA100 at a concentration of 2500 μg/plate (with and without metabolic activation). In tester strain TA1535 toxic effects of the test item were noted at concentrations of 1000 μg/plate and higher (without metabolic activation). The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in tester strain TA1535 in the negative control (without metabolic activation) was regarded as not biologically relevant due to the fact that the values of the negative control were within the historical control data and due to lack of concomitant clearing of the background lawn. The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in tester strain TA1537 at a concentration of 1000 μg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.
No biologically relevant increases in revertant colony numbers were noted in tester strains TA1537 and TA102.
Biologically relevant increases of revertant colony numbers were observed in tester strain TA98 at concentrations of 31.6 μg/plate and higher (without metabolic activation) and at concentrations of 10.0 μg/plate and higher (with metabolic activation). The threshold value of 2.0 was exceeded and a maximum mutation factor of 43.0 was reached at a concentration of 2500 μg/plate (with metabolic activation).
Biologically relevant increases of revertant colony numbers were also observed in tester strain TA100 at concentrations of 3.16 μg/plate to 1000 μg/plate (without metabolic activation) and at concentrations of 10.0 μg/plate to 1000 μg/plate (with metabolic activation). The threshold value of 2.0 was exceeded and a maximum mutation factor of 15.2 was reached at a concentration of 100 μg/plate (with metabolic activation).
In tester strain TA1535 biologically relevant increases of revertant colony numbers were observed at concentrations of 31.6 μg/plate to 100 μg/plate (with metabolic activation). The threshold value of 3.0 was exceeded and a maximum mutation factor of 4.0 was reached at a concentration of 100 μg/plate (with metabolic activation).
Moreover, a dose-response relationship was found in tester strains TA 100 and TA98 (with and without metabolic activation).
All criteria of validity were met

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, 4-(4-nitrophenylthio)-aniline - DAPEA caused gene mutations by base pair changes and frameshifts in the genomes of the tester strains TA98, TA100 and TA1535.
Therefore, 4-(4-nitrophenylthio)-aniline - DAPEA is considered to be mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of 4-(4-nitrophenylthio)-aniline - DAPEA for its ability to induce gene mutations the plate incorporation test was performed with theSalmonella typhimuriumstrains TA98, TA100, TA1535, TA1537 and TA102.

In the experiment several concentrations of the test item were used. The assay was conductedwithandwithoutmetabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared:

3.16, 10.0, 31.6, 100, 316, 1000 and 2500μg/plate

Precipitation was observed in all tester strains used (withandwithoutmetabolic activation).

Toxic effects of the test item were observed in some tester strains at concentrations of 1000μg/plate and higher (withoutmetabolic activation) and at a concentration of 2500μg/plate (withmetabolic activation), depending on the particular tester strain.

No biologically relevant increases in revertant colony numbers were noted in tester strains TA1537 and TA102.

Biologically relevant increases of revertant colony numbers were observed in tester strains TA98, TA100 and TA1535 at concentrations of 3.16μg/plate and higher (withoutmetabolic activation) and at 10.0μg/plate and higher, depending on the particular tester strain. The threshold value of 2.0 wasexceeded and a maximum mutation factor of 43.0 was reached at a concentration of 2500μg/plate (withmetabolic activation) in tester strain TA98.

Moreover, a dose-response relationship was found in tester strain TA98 and TA100 (withandwithoutmetabolic activation).

All criteria of validity were met.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, 4-(4-nitrophenylthio)-aniline - DAPEA caused gene mutations by base pair changes and frameshifts in the genomes of the tester strains TA98, TA100 and TA1535.

Therefore, 4-(4-nitrophenylthio)-aniline - DAPEA is considered to be mutagenic in this bacterial reverse mutation assay.