Sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate

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:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only four strains were tested)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate

EC Number:

251-257-2

EC Name:

Sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate

Cas Number:

32846-21-2

Molecular formula:

C23H26N4O6S2.Na

IUPAC Name:

sodium 6-amino-5-({2-[cyclohexyl(methyl)sulfamoyl]phenyl}diazenyl)-4-hydroxynaphthalene-2-sulfonate

Test material form:

other: solid

Specific details on test material used for the study:

Name: FAT 20'003/I
Batch No.: not indicated by the sponsor
Vers. No.: 9-5-97
Aggregate State at Room Temperature: Solid
Colour: red
Purity: approx 90%
Stability in Solvent: Stable for atleast 24 hours at room temperature and under test conditions at 37°C in water, PEG 400, saline, FCA/NaCl-solution and CMC solution.
Storage: room tempearture
Expiration Date: Feb 2001.

On the day of the experiment, the test article FAT 20003/I was dissolved in DMSO. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Method

Target gene:

Histidine gene

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the uvrB"-mutation. In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. Regular checking of the properties of the strains regarding the membrane permeability, ampicillin- and tetracycline-resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(for TA 1535 and TA100 without metabolic activation)

Controls 2

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

(for TA 1537 and TA98 without metabolic activation)

Controls 3

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(for all strains with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and the pre-incubation test (experiment II)

For each strain and dose level, including the controls, a minimum of three plates were used.
Experiment 1: The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL: Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),
- 500 µL: S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
- 100 µL: Bacteria suspension (cf. test system, pre-culture of the strains),
- 2,000 µL: Overlay agar

Experiment 2: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix /S9 mix substitution buffer and 100 µL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 h at 37 °C in the dark.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are: corresponding background growth on both negative control and test plates as well as normal range of spontaneous reversion rates.
A test substance is considered as positive if either a dose related or reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test substance producing neither a dose related and reproducible 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 biologically relevant response is described as follows:
A test substance is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test substance regardless whether the highest dose induced the criteria described above or not.

Statistics:

A statistical analysis of the data is not required.

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(only in plate incorporation method)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. The frequency of spontaneous revertant colony formation in strain TA 1535 in the second experiment slightly exceeded the historical control range (36 colonies versus 10 - 29 colonies) in the absence of metabolic activation. This increase is most likely based upon a slight increase in the bacterial cell density during pre-incubation and does not represent statistical fluctuations since all test points are at or somewhat above the upper limit of the historical range of negative controls. A slight or moderate increase in the bacterial cell density is not uncommon during pre-incubation since small amounts of histidine containing medium is transferred together with the bacteria and the overall dilution during the preincubation step is considerably lower as compared to the direct plate incorporation method. Therefore, this slightly elevated frequency of spontaneous revertants was judged as biologically irrelevant within the scope of the assay.
No substantial increase in revertant colony numbers of any of the four tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Applicant's summary and conclusion

Conclusions:

The test substance was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance (at ca. 90 % purity) to induce gene mutations according to OECD Guideline 471 in compliance with GLP with deviation (i.e., only four strains were tested).

The assay was performed in two independent experiments both with and without liver microsomal activation using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. Experiment I was performed as a plate incorporation assay and experiment II was performed as a pre-incubation assay. Each concentration, including the controls, was tested in triplicate. The substance was tested up to 5000 µg/plate.

Slight toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 98 with and without S9 mix at 5000 µg/plate in Experiment I. In Experiment II, no toxic effects were observed. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increase in revertant colony numbers of any of the four tester strains was observed at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Taking into consideration the above findings, the test substance was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.