5',5'''-[ethylenebis(p-phenyleneazo)]bis[1',2'-dihydro-6'-hydroxy-4'-methyl-2'-oxo-1,3'-bipyridinium] dilactate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Ames BN, Mc Cann J, Yamasaki E, Mutation Res. (1975) 31, 347 - 364
Green M.H.L. and Muriel W.J. Mutation Res. (1976) 38, 3 -32

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Basic Yellow 094

IUPAC Name:

Basic Yellow 094

Method

Species / strain

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

The Escherichia coli was obtained from "National Collection of Industrial Bacteries", Torrey Research station P.O., Aberdeen, Scotland, UK.
The strains 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.
The test strains were checked at regular intervals for their genetic markers.

Starting experiment the bacteria were grown overnight in a shaking waterbath for 16 h at 37 °C using 2.5 % nutrient broth no. 2 (source: Oxoid). After centrifugation the bacteria were resuspended to a titer of about 5 × 10E8 – 3 × 10E9 cells per milliliter in 0.16 % nutrient broth and 0.5 % NaCl. This titer was controlled photometricallly and determined in an experimental test with histidine-rich KCl solution on minimal agar plate.

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction S9 mix

Test concentrations with justification for top dose:

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

Controls

Untreated negative controls:

yes

Remarks:

aqua bidest.

True negative controls:

yes

Remarks:

aseptic control with test material and S-9 mix

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).
Immediately before the experiment the highest dose solution was prepared by adding the test material to the appropriate solvent. The other doses were dilutions of the high dose with the solvent. The following materials were mixed in a test tube and poured onto minimal agar plates:
100 µl tests solution or control solvent or positive control solution
500 µl S-9 mix (for tests with metabolic activation) or Na2HPO4, 0.15 M (for tests without metabolic activation)
100 µl Bacteria suspension (1 × 10E8 – 2 × 10E9 cells/ml)
2 ml Molten agar consisting of 0.6 % Bacto agar and 0.6 % NaCl supplemented with 10 % 0.5 mM L-histidine and 0.5 biotine solution.
A pourmatic was used to fill the plates automatically with 22 ml of 1.5 % Bacto agar in Vogel Bonner medium E with 2 % glucose.

DURATION
Exposure duration: plates were incubated in the dark for 3 days.

NUMBER OF REPLICATIONS
Each compound concentration, including controls, was tested in triplicate.

NUMBER OF CELLS EVALUATED
1.6 - 3.1 × 10E9 cells/ml

ASEPTRIC CONTROL
For the aseptic control experiments 100 µl of the solution of the test compound or 100 µl of S-9 mix were added to 2 ml molten agar and treated as described above.

DETERMINATION OF CYTOTOXICITY
The toxicity of the test material may be determined by a reduction of the number of spontaneous revertants and by an examination of the background lawn of bacterial growth resulting from traces of tryptophane added to the top agar. Toxicity reduces the sensitivity to testing of mutagenicity in a bacterial test. Therefore, the toxicity estimation is required to validate the collected data.

OTHER EXAMINATIONS
The his+ revertant colonies were counted with a Fisher counter 880 (Fisher, Comp).

TEST CONDITIONS
All experimentation was carried out under sterile conditions.
In order to avoid any light effects on labile test compounds, all experimentation was carried out under yellow light.

LIVER MICROSOMAL FRACTION S-9 MIX
For the study fresh liver preparations from animals, sacrificed on the day of the experiment, were used.
Specific pathogen-free male Wistar rats (180 - 250 g outbred) were obtained from Kleintierfarm Madoerin AG, Fuellinsdorf/BL, Switzerland. After acclimatization the rats received five days before the experiment a single ip. injection of Aroclor 1254 (source: Analabs) dissolved in oleum arachidis (200 mg/ml) at a dosage of 500 mg/kg body weight to induce liver microsomal enzyme activity. The rats were killed on the fifth day p. appl. after a 14 - 16 hour 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 degrees centigrade. The supernatant fraction (S-9 fraction) was collected for the preparation of S-9 mix.
Composition of 1 ml S-9 mix
Na2HPO4100 µmoles
MgCl28 µmoles
KCl 33 µmoles
NaDP+4 µmoles
G-6-P 5 µmoles
S-9 fraction 0.3 ml

Evaluation criteria:

A mutagenic activity was assumed if at least a two fold increase of the number of induced revertants was obtained in comparison with the spontaneous revertants of the corresponding controls.

Results and discussion

Test results

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A slight precipitation of the test material was observed after pouring the content of the test tube onto the surface of the selective agar plate at the concentration above 158 µg/plate. The revertants were easily recognized, therefore the plates could be evaluated (counting by hand).
Up to the highest tested dose, with and without microsomal activation, no relevant increase of the revertant colony numbers was obtained in comparison with the corresponding controls.

TOXICITY OF THE TEST MATERIAL
No toxic effects of the test item were observed.

RESULTS OF CONTROL EXPERIMENTS
The control plates with the solvent (negative control) showed numbers of spontaneous revertant colonies per plate within the normal range of the testing laboratory experience.
The control plates with reference mutagens (positive controls) showed a distinct elevation of the revertant colonies with the tester strain. The positive results of the mutagen 2-aminoanthracene indicate that the metabolizing system was functioning.

The aseptic control showed no contamination for either the test material solution or for the S-9 mix.

Applicant's summary and conclusion

Conclusions:

In the experiments performed, no relevant increase of the revertant colony numbers was observed in Echerichia coli strain tested, in the presence and in the absence of S-9 mix.

Executive summary:

The compound was tested for detecting its potential gene mutagenic activity according to the plate incorporation method described by Green and Muriel (1976) and Ames et al (1975), using the Escherichia coli strain WP2 uvrA. The tests was performed with and without metabolic activation. The compound was examined in triplicate for 8 concentrations from 1.58 to 5000 µg/plate.

A slight precipitation of the test material was observed after pouring the content of the test tube onto the surface of the selective agar plate at the concentration above 158 µg/plate. The revertants were easily recognized, therefore the plates could be evaluated.

Up to the highest tested dose, with and without microsomal activation, no relevant increase of the revertant colony numbers was obtained in comparison with the corresponding controls.

No toxic effects of the test material were observed.

Conclusion

In the experiments performed, no relevant increase of the revertant colony numbers was observed in Echerichia coli strain tested, in the presence and in the absence of S-9 mix.

REFERENCE

Ames BN, Mc Cann J, Yamasaki E, Mutation Res. (1975) 31, 347 - 364

Green M.H.L. and Muriel W.J. Mutation Res. (1976) 38, 3 -32