Disodium 7-[[4,6-bis[(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

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

Study period:

From August 31st to September 20th, 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The combination of strains recommended in the currently adopted OECD guideline was not assayed: none of the E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was tested.

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 and Yamasaki E, Mutation Res. (1975) 31, 347 - 364

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Direct Red 253

IUPAC Name:

Direct Red 253

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA1535, TA1537 and TA1538

Details on mammalian cell type (if applicable):

All strains contain a histidine mutation and two additional mutations which increase their sensitivity to mutagens. One mutation causes loss of the excision repair system uvrB, the other causes loss of the lipopolysaccharide barrier that coats the surface of the bacteria (rfa). The two strains TA 98 and TA 100 contain a resistance transfer factor (r-factor) which renders both extremely sensitive in detecting several mutagens. The strains TA 1535 and TA 100 are sensitive in detecting mutagens causing base-pair substitutions, the strains TA 1537, TA 1538 and TA 98 are sensitive in detecting mutagens causing frameshift mutations.

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.
The test strains were checked at regular intervals for their genetic markers according to the directions in the literature (Ames BN, Mc Cann J, Yamasaki E, Mutation Res. (1975) 31, 347 - 364).

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 1 × 10E8 – 2 × 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

Vehicle / solvent:

- Vehicle used: aqua bidest.

Controls

Untreated negative controls:

yes

Remarks:

aqua bidest.

Negative solvent / vehicle 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:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

methylmethanesulfonate

other: 2-aminoanthracene

Remarks:

Without S9: MMS for TA100, 9-AA for TA1537, 2-NF for TA 98, TA 100, TA1537, TA1538. With S9: 2-AA for TA 98, TA 100, TA1538, TA1535, TA1537; B(a)P for TA 98, TA 100, TA1537, TA1538.

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
9.4 × 10E8 – 1.9 × 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
To estimate the toxicity of the test material, prototrophic bacteria (his+ spontaneous revertants from TA 1537) were used. These bacteria were added as an internal standard to plates together with the bacteria strain TA 1537 which gives low numbers of revertant colonies (this mixture is referred to as RTA) 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 test material may be determined 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 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 (for TA 100: one and half 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:

S. typhimurium, other: TA 98, TA 100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the experiments performed, in the presence and in the absence of S-9 mix., no relevant increase of the revertant colony numbers (cf. evaluation of the assay) was obtained in any Salmonella typhimurium strain and at any dose level tested in comparison with the corresponding controls.

TOXICITY OF THE TEST MATERIAL
The toxicity of the compound was tested over a series of 8 concentrations and expressed as relative survival rate. Neither quantitative nor qualitative evidence of a toxic effect of the compound was 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 and similar to those described in literature (Ames BN, Mc Cann J, Yamasaki E, Mutation Res. (1975) 31, 347 - 364). The control plates with reference mutagens (positive controls) showed a distinct elevation of the revertant colonies with 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.

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 any Salmonella typhimurium 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 of Ames et al. (1975), using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. The tests were performed with and without metabolic activation. The compound was examined in triplicate at 8 concentrations from 1.53 to 5000 µg/plate. In the experiments performed, in the presence and in the absence of S-9 mix., no relevant increase of the revertant colony numbers (cf. evaluation of the assay) was obtained in any Salmonella typhimurium strain and at any dose level tested in comparison with the corresponding controls.

The toxicity of the compound was tested over a series of 8 concentrations and expressed as relative survival rate. Neither quantitative nor qualitative evidence of a toxic effect of the compound was observed. The control plates with the solvent (negative control) showed numbers of spontaneous revertant colonies per plate within the normal range of our laboratory experience and similar to those described in literature (Ames BN, Mc Cann J, Yamasaki E, Mutation Res. (1975) 31, 347 - 364). The control plates with reference mutagens (positive controls) showed a distinct elevation of the revertant colonies with 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.

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

Conclusion

In the experiments performed, no relevant increase of the revertant colony numbers was observed in any Salmonella typhimurium 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