Reaction products of 2,4,6-trichloro-1,3,5-triazine copuled with 3-aminopropyldiethylamine, coupled with 3-acetamidoanilinium chloride, diazotised, coupled with 2,5-Xylidine, coupled with 3-amino-4-hydroxybenzenesulphonamide, further coupled with resorcinol and metallized with iron chloride.

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 July 27th to September 28th, 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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:

Basic Brown 022

IUPAC Name:

Basic Brown 022

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Prof. B.M. AMES, University of California Berkeley, California 94720 USA.
- Storage: the strain cultures were kept in sterile 0.5 ml ampoules (0.45 ml bacterial culture + 0.05 ml dimethylsulfoxide) at -70 °C and in liquid nitrogen.
- Preparation of inoculum: starting an experiment the bacteria were grown overnight in a shaking waterbath for 15 hours at 37 °C, using 2.5 nutrient broth No 2 Fa. Oxoid.
- Periodical check: the test strains are checked at regular intervals for their genetic markers.

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsomal fraction S9 mix

Test concentrations with justification for top dose:

1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 µg per plate

Vehicle / solvent:

- Vehicle: aqua dest.

Controls

Untreated negative controls:

yes

Remarks:

aqua dest.

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test
- Preparation of inoculum: after centrifugation the bacteria are resuspended to a titer of about 5 × 10^8 - 2 × 10^9 cells per millilitre in 0.16 % nutrient broth and 0.5 % NaCl. The titer was controlled photometrically and determined in the experimental test with histidin rich KCl solution on minimal Agar plate.
- Test tube: 100 µl test solution, or control solvent, or positive control solution + 500 µl S-9 mix or KCl 0.15 M + 100 µl bacteria suspension (5 × 10^8 - 2 × 10^9 cells/ml) + 2 ml Molten Agar (i.e. 0.6 % Bacto Agar and 0.6 % NaCl supplemented with 10 % 0.5 mM L-Histidine and 0.5 mM Biotine solution).
- Incubation: in the dark for 3 days.

NUMBER OF REPLICATIONS: each compound concentration including control experiment was tested in triplicate.

DETERMINATION OF CYTOTOXICITY
To estimate the toxicity of the test compound prototrophe bacteria (His+) (spontaneous revertants from TA 1537) were added as an internal standard to plates together with the bacteria strain TA 1537, which gives low numbers of revertant colonies and their survival was determined. The ratio of the differences in the numbers of colonies of the RTA and the TA 1537 plates for each substance concentration and solvent control gives the relative survival rate.
In addition, the toxicity of the compound may be evidenced by a reduction of the number of spontaneous revertants in the tests with the inserted strains and by an examination of the background lawn of bacterial growth resulting from traces of Histidine added to the top Agar. Toxicity reduces the sensitivity for testing of mutagenicity in a bacterial test. Therefore, the toxicity estimation is required to validate the collected data.

LIVER MICROSOMAL FRACTION S-9 MIX
Fresh liver preparations from animals, sacrificed on day of the experiment, were used. Specific pathogen free male Wistar rats (180 - 250 g) were obtained from Kleintierefarm Madoerin AG, Fuellinsdorf/BL, Switzerland.
After acclimatization the rats received five days before the experiment a single i.p. injection of Aroclor 1254, dissolved in Oleum Arachidis (200 mg/ml) at a dosage of 500 mg/kg bw to induce liver microsomal enzyme activity.
The rats were killed on the fifth day p. appl. after a 14 - 16 hours starvation period.
The livers were removed under aseptic conditions and homogenised with 0.15 molar, ice cold KCl (5 g of liver to 15 g of KCl). The homogenates were centrifuged at 9000 g for 10 minutes at 0 to 2 °C. The supernatant fraction (S-9 fraction) was collected for the preparation of S-9 mix.

Composition of 1 ml S-9 mix: Na2HPO4 100 µmol, MgCl2 8 µmol, KCl 33 µmol, NADP+ 4 µmol, glucose.-6-phosphate 5 µmol S-9 fraction 0.3 ml.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate without S9 mix

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The compound precipitated after pouring the content of the test tube onto minimal agar plates, at the concentrations as of 500 µg/plate in the experiment without metabolic activation and as for 1580 µg/plate in the experiment with metabolic activation. The precipitate was fine granular and homogene. The revertant colonies were good recognizable and were evaluated manual with a colony counter.

In the described bacterial mutagenicity tests, the compound showed no relevant differences, i.e. less than a two fold increase of revertant colony numbers in any Salmonella typhimurium strain inserted and in the non toxic tested doses i.e. up to 1580 µg/plate in comparison with the corresponding controls.

NEGATIVE CONTROL
The control plates with the solvent (negative control) showed numbers of spontaneous revertant colonies per plate within the normal range of testing facility experience and similar to those described in literature.

POSITIVE CONTROLS
The control plates with reference mutagens (positive controls) showed a distinct elevation of the revertant colonies by the tester strains. This confirmed the reversion properties of each strain. The positive results of the mutagens 2-aminoanthracene and Benzo[a]pyrene indicate that the metabolizing system was functioning.

ASEPTIC CONTROL
The aseptic control showed no contamination for either the compound solution or for the S-9 mix.

CYTOTOXICITY
The compound showed a toxic effect at the concentration of 5000 µg per plate in the experiment without metabolic activation.

Applicant's summary and conclusion

Conclusions:

In the described bacterial test no mutagenic activity was observed with the test item, under the experimental conditions reported.

Executive summary:

The test compound was examined for mutagenic activity in a plate incorporation test, employing Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. The compound was tested in the presence and absence of liver microsomal activation system. The compound was tested at 8 concentrations: 1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 µg per plate. Each compound concentration including control experiment was tested in triplicate.

The compound showed a toxic effect at the concentration of 5000 µg per plate in the experiment without metabolic activation.

Up to the highest non toxic tested dose 1580 µg per plate, no relevant differences, i.e. less than a two fold increase of revertant colony numbers in any Salmonella t. strain inserted, were observed with the compound in comparison with the corresponding controls.

Conclusion

In the described bacterial test, no mutagenic activity was observed with the test item, under the experimental conditions reported.