Reaction products of 4'-amino-4-nitrodiphenylamine-2-sulfonic acid diazo, 4-nitroaniline diazo, aniline-2,5-disulfonic acid diazo and resorcinol, sodium salts

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

Study period:

From the 8th to the 29th of July, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From the 8th to the 29th of July, 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from supporting substance (structural analogue or surrogate)

Remarks:

Study conducted according to internationally accepted testing guideline

Justification for type of information:

Justification for Read Across is detailed in the endpoint summary and it is further detailed in the report attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Principles of method if other than guideline:

The test has been conducted following the Environmental Protection Agency, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicity, Revision July 1st, 1986 "The salmonella Typhimurium Reverse Mutation Assay".

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

10.0 µg/plate; 100.0 µg/plate; 333.3 µg/plate; 1000.0 µg/plate; 5000.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water bidest- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative nontoxicity for the bacteria.

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: NaN3 (TA 1535, TA 100); 4-NOPD (TA 1537, TA 1538, TA 98); With metabolic activation: DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
Preincubation period: 6 hours at 37 °C in a shaking water bath
Exposure duration: After solidification the plates wew incubated upside down for 72 hours at 37 °C in the dark
NUMBER OF REPLICATIONS: Two indipendent study, three replicates for each concentration

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
Corresponding background growth on both negative control and test plates
Normal range of spontaneous rerversion tates.
Range of spontaneous reversion frequencies (5,9)TA 1535 (3 - 37); TA 1537 (4 - 31); TA 1538 (12 - 37); TA 98 (15 - 60); TA 100 (75 - 200)
Due to the international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.
A test article is considered as positive if either a significant dose
- related increase in the number of revertants nor a significant and reproducible increase for at least one test concentration is induced.A test article producing neither a significant dose
-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non mutagenic in this system.
A positive response is described as follows:
A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.Also a dose dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the teest article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No appropriate statistical method is available

Species / strain:

other: S. typhimurium TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: S. typhimurium TA 1537, TA 1538 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Independent test n. 2

In Strain TA 98 a distinct mutagenic effect occurred without S9 -mix in both experiments. In the presence of metabolic activation no positive response was obtained in Experiment 1. In te experiment 2 a weak increase in the number of revertants was found at 1000 and 5000 µg/plate.

Conclusions:

Positive with and without metabolic activation. Under the test condition the substance did induce point mutation by base pair change or frameshifts in the genome of the strains TA 1537, TA 1538, TA 98 and TA 100

Executive summary:

Method

The test has been conducted on the Similar substance 01, according to the OECD Guideline 471 and to the EU Method B.14 in order to evaluate the potential of the test item to induce gene mutations according to the plate incorporation test.

During the test the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 have been used. The assay has been performed in two independent experiments, using identical procedures, both with and without microsomal activation. Each concentration, including the controls, has been tested in triplicate. The substance has been tested at the concentrations: 10, 100, 333.3, 1000 and 5000 µg/plate.

Observations

No toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in any of the test groups with and without metabolic activation.

The plates incubated showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, a significant and reproducible dose-dependent increase in revertant colony numbers was obtained with the strains TA 1537, TA 1538, TA 98 and TA 100. The presence of liver microsomal activation did not influence the genotoxic effect, However the positive response in strain TA 98 was not reproduced in the independent experiment in the presence of S9 mix.

Results

Under the test condition the substance did induce point mutation by base pair change or frameshifts in the genome of the strains TA 1537, TA 1538, TA 98 and TA 100. The test article is considered to be mutagenic in this assay.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Principles of method if other than guideline:

The test has been conducted following the Environmental Protection Agency, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicity, Revision July 1st, 1986 "The salmonella Typhimurium Reverse Mutation Assay".

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar substance 01 of Acid Brown 314:1

IUPAC Name:

Similar substance 01 of Acid Brown 314:1

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

10.0 µg/plate; 100.0 µg/plate; 333.3 µg/plate; 1000.0 µg/plate; 5000.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water bidest- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative nontoxicity for the bacteria.

Controls

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: NaN3 (TA 1535, TA 100); 4-NOPD (TA 1537, TA 1538, TA 98); With metabolic activation: DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
Preincubation period: 6 hours at 37 °C in a shaking water bath
Exposure duration: After solidification the plates wew incubated upside down for 72 hours at 37 °C in the dark
NUMBER OF REPLICATIONS: Two indipendent study, three replicates for each concentration

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
Corresponding background growth on both negative control and test plates
Normal range of spontaneous rerversion tates.
Range of spontaneous reversion frequencies (5,9)TA 1535 (3 - 37); TA 1537 (4 - 31); TA 1538 (12 - 37); TA 98 (15 - 60); TA 100 (75 - 200)
Due to the international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.
A test article is considered as positive if either a significant dose
- related increase in the number of revertants nor a significant and reproducible increase for at least one test concentration is induced.A test article producing neither a significant dose
-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non mutagenic in this system.
A positive response is described as follows:
A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.Also a dose dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the teest article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No appropriate statistical method is available

Results and discussion

Test results

Test results 1

Species / strain:

other: S. typhimurium TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

other: S. typhimurium TA 1537, TA 1538 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Independent test n. 2

Any other information on results incl. tables

In Strain TA 98 a distinct mutagenic effect occurred without S9 -mix in both experiments. In the presence of metabolic activation no positive response was obtained in Experiment 1. In te experiment 2 a weak increase in the number of revertants was found at 1000 and 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:

Positive with and without metabolic activation.
Under the test condition the substance did induce point mutation by base pair change or frameshifts in the genome of the strains TA 1537, TA 1538, TA 98 and TA 100

Executive summary:

Method

The test has been conducted according to the OECD Guideline 471 and to the EU Method B.14 in order to evaluate the potential of the test item to induce gene mutations according to the plate incorporation test.

During the test the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 have been used. The assay has been performed in two independent experiments, using identical procedures, both with and without microsomal activation. Each concentration, including the controls, has been tested in triplicate. The substance has been tested at the concentrations: 10, 100, 333.3, 1000 and 5000 µg/plate.

Observations

No toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in any of the test groups with and without metabolic activation.

The plates incubated showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, a significant and reproducible dose-dependent increase in revertant colony numbers was obtained with the strains TA 1537, TA 1538, TA 98 and TA 100. The presence of liver microsomal activation did not influence the genotoxic effect, However the positive response in strain TA 98 was not reproduced in the independent experiment in the presence of S9 mix.

Results

Under the test condition the substance did induce point mutation by base pair change or frameshifts in the genome of the strains TA 1537, TA 1538, TA 98 and TA 100. The test article is considered to be mutagenic in this assay.