Reaction products of diazotised m-phenylendiamine, subsequently coupled with diazotised 4-aminobenzene-1-sulfonic acid, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

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:

2020

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Direct Brown 44

IUPAC Name:

Direct Brown 44

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Target gene:

his +/- for Salmonella T.
triptofan locus for E. Coli

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 hamster liver (Prival test)

Test concentrations with justification for top dose:

The highest dose level for the mutation assays was selected as a concentration which elicits moderate toxicity.The toxicity was assessed on the basis of a decline in the number of spontaneous revertants or a thinning of the background lawn. Solubility of the test item was evaluated in a preliminary trial using sterile water for injection. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble at 50.0mg/mL. This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test
Since no evidence of toxicity following treatment with the test item was observed, then the highest dose level is 5 mg/plate or 5 μL/plate. Following doses are at four lower concentrations spaced at approximately half-log interval:
1580, 500, 158 and 50.0 µg/plate.

Vehicle / solvent:

sterile water for injection (( Eurospital, batch 20B0703)

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate
- Number of independent experiments : one

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: none
- Exposure duration/duration of treatment: exposure was the same as imcubation time, i.e. 72h at 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
The toxicity will be assessed on the basis of a decline in the number of spontaneous revertants or a thinning of the background lawn.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
count of the number of revertants for each plate manually

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

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

Positive controls validity:

valid

Additional information on results:

Ames test:
- Signs of toxicity : no sgin of toxicity at any dose, with and without metabolic activaton
- Individual plate counts : see tables attached
- Mean number of revertant colonies per plate and standard deviation : see tables attached

A Main Assay was performed. Individual plate counts for this test and the mean and standard error of the mean for each test point, together with a statistical analysis are presented in Table 2.
The maximum concentration of the test item to be used in the Main Assay should be determined taking into consideration solubility in the final treatment mixture and cytotoxicity results obtained in the preliminary toxicity test.
Since no precipitation of the test item or toxicity was observed, 5000 µg/plate was selected as the top concentration for the Main Assay. The test item was asayed at the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate.
No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.
Plates treated with the test item showed a dose dependent brown color of the agar, which did not interfere with the scoring of colonies or the evaluation of thinning of the background lawn.
The test item induced dose related increases in the number of revertant colonies with TA1537, TA98 and TA100 tester strains both in the absence and presence of S9 metabolism.
The number of revertant colonies at higher dose levels fell out the historical control range (95% confidence limits).
Since these increases were considered a clear evidence of mutagenicity, no further experiment was performed.
The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Applicant's summary and conclusion

Conclusions:

The substance was tested for in vitro gene mutation study in bacteria following OECD 471. Under the experimental conditions the test item induced reverse mutation in bacteria with and wihtout metabolic activation in the strain TA98, TA100 and TA 1537.

Executive summary:

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.

The test item was used as a solution in sterile water for injection and was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration tested, in the absence or presence of S9 metabolic activation. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.

Dose related increases in revertant numbers were observed with the TA1537, TA98 and TA100 tester strains both in the absence and presence of S9 metabolic activation.

On the basis of toxicity test results, in the Main Assay using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate.

Neither precipitation of the test item, nor toxicity was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of

S9 metabolism. The test item induced dose related increases in the number of revertant colonies with TA1537, TA98 and TA100 tester strains both in the absence and presence of

S9 metabolism. These increases were greater than twice the concurrent negative control values and so can be considered a clear evidence of mutation induction. Since a clear

positive response was observed, no further experiment was undertaken.