Alkali salt of (sulfonato oxyethane sulfonyl) arylamino triazinylamino sulfonato (sulfonato arylazo) aryl sulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

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:

From July 18, 2006 to August 03, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 471, EU Method B.13/14, EPA OPPTS 870.5100 and Prival et al in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Prival et al (Prival MJ and Mitchell VD. Analysis of a method for testing azo dyes for mutagenicity in Salmonella typhimurium in the presence of flavine mononucleotide and hamster liver S9. Mutation research, 97: 103-116; 1982)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver (plate incorporation test) and S9-mix from hamster liver (preincubation test)

Test concentrations with justification for top dose:

Concentrations in the initial mutation test and confirmatory mutation tests: 5,000; 4,000; 2,000; 800; 320 and 128 μg/plate.

Vehicle / solvent:

Distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals)

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (i.e., 4-NPD)

Remarks:

without metabolic activation for strain TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals)

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation for strain TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals)

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation for strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals)

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation for strain WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals)

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (i.e., 10% rat liver) for all strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals)

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (i.e., 30% hamster liver) for strain TA 100, TA 1535, TA 1537 and WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(two solvent control (i.e., DMSO solvent and distilled water) groups were used depending on the solubility of the test substance and the solubility of strain specific positive chemicals)

Positive controls:

yes

Positive control substance:

congo red

Remarks:

with metabolic activation (i.e., 30% hamster liver) for strain TA 98

Details on test system and experimental conditions:

ASSAY PROCEDURE

Test substance concentrations and controls in the main tests (i.e., initial and confirmatory mutation tests)

Based on the results of the preliminary tests, a 100 mg/mL stock solution was prepared from the test substance with the solvent, which was diluted in 5 steps. In the main tests six different amounts of the test substance were tested with mainly 0.4 intervals between test points. The highest concentration of the test substance in these tests was 5,000 μg test substance/plate.

Test substance concentrations tested:

No. of concentration (μg/plate)
1 5,000
2 4,000
3 2,000
4 800
5 320
6 128

Test solutions were freshly prepared at the beginning of the experiments.

Control groups used in the main tests (i.e., initial and confirmatory mutation tests)
Strain-specific positive and negative (i.e., solvent) controls, both with and without metabolic activation were included in each test. In addition, untreated control was used demonstrating that the chosen solvent induced no deleterious or mutagenic effects.

Experimental Method
The experimental methods were essentially the same as those described by Ames et al. and Maron and Ames, Kier et al., Venitt and Parry, Prival and Mitchell, OECD Guideline No 471, 1997, EPA Guidelines, OPPTS 870.5100, 1998, 1996 and EEC Directive B13/14, 2000.

Procedure for exposition in the initial mutation test
A standard plate incorporation procedure was performed, as an initial mutation test. Bacteria (i.e., cultured in Nutrient broth No.2.) were exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (i.e., 3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test substance and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
Top agar: .....................................................................................................2,000 μL
Solvent or solution of test substance or positive controls:.................50 μL
Overnight culture of tester strain:.......................................................... 100 μL
Phosphate buffer (pH:7.4) or S9 mix:......................................................500 μL
This solution was mixed up and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activation and an activation test conditions and each of them with the addition of negative and positive controls. After preparation the plates were incubated at 37°C for 48 h.

Procedure for exposition in the confirmatory mutation test
A pre-incubation procedure was performed as a confirmatory mutation test since the result of the initial mutation test was negative.
In the pre-incubation procedure the test substance, S9, and bacteria were incubated at higher concentrations than in the standard plate incorporation method. Pre-incubation procedures have equal or greater sensitivity than the plate incorporation assays.
Before the overlaying of the test substance, the bacterial culture and the S9 mix (i.e., containing hamster liver S9) or phosphate buffer were added into appropriate tubes to provide direct contact between bacteria and the test substance (i.e., in its solvent). These tubes were mixed and incubated for 20 minutes at 30ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes, the content mixed and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of a non-activation and activation test conditions and each of them with the addition of negative and positive controls. After preparation the plates were incubated at 37°C for 48 h.

Evaluation criteria:

EVALUATION OF EXPERIMENTAL DATA
The colony numbers on the control, positive control and the test plates were determined. The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test substance and for the controls using Microsoft ExcelTM software.

A test substance is considered as mutagenic if:
- a dose–related increase in the number of revertants occur and/or
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the solvent control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results; a statistical evaluation of the results is not regarded as necessary.

A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the test points is considered non-mutagenic in this system.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and peincubation test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and peincubation test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary range finding test (i.e., informatory toxicity test)

-In the preliminary range finding test plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 test strains under activation and non-activation conditions.

-In the concentration range finding test the concentrations examined were: 5,000; 2,500; 1,250; 625; 312.5; 156.25; 78.13; 39.06 and 19.53 µg/plate.

-The revertant colony numbers were slightly increased compared to the revertant colony numbers of the solvent control plates in the case of S. typhimurium TA100 with addition of metabolic activation in the concentration range of 5,000-1,250 µg/plate and additionally at the concentration of 78.13 µg/plate. Further slight revertant colony number increases were observed in the case of S. typhimurium TA98 without metabolic activation at the concentration of 625 µg/plate, with metabolic activation at the concentration of 19.53 µg/plate.

-Inhibitory effects of the test substance were not observed in the examined bacterium strains. The development of a background lawn and the number of revertant colonies were not reduced. The revertant colony numbers were slightly decreased compared to the revertant colony numbers of the solvent control plates in only one case, in the case of S. typhimurium TA98 at the concentration level of 78.13 µg/plate (+S9).

-The observed changes were considered as reflecting the biological variability in the test.

Initial and confirmatory mutation tests (i.e., main test)

-In the initial mutation test the plate incorporation method, in the confirmatory mutation test the pre-incubation method was used. The experiments were carried out using Salmonella typhimurium strains (i.e., TA98, TA100, TA1535, TA1537) and Escherichia coli WP2 uvrA strain, in presence and absence of metabolic activation (i.e., S9 Mix containing rat liver S9 for plate incorporation and hamster liver S9 for preincubation tests) with appropriate positive and negative controls.

-The examined concentrations were: 5,000; 4,000; 2,000; 800; 320 and 128 μg/plate. Sporadic changes were observed, such as non significant, not dose dependent increases in revertant colony numbers compared to the solvent control values in both independently performed experiments at different concentration levels in the examined bacterium strains.

-The highest increase was observed in the Confirmatory Mutation Test in S. typhimurium TA1537 at the concentration of 2,000 µg/plate (-S9) where the mutation factor value was: 1.52.

-The observed increases were of minor intensity, and without any biological significance. In addition, the thresholds for being positive were not reached in any case. The increases observed should thus be considered as reflecting the biological variability in the test.

-No inhibitory (i.e., cytotoxic) effect of the test substance could be observed. The revertant colony numbers were slightly decreased compared to the solvent control values in several cases in the confirmatory mutation test, but these decreases reflect the expected biological variability. The background lawn development was normal at every bacterium strain examined with and without S9 Mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471, EU Method B.13/14, EPA OPPTS 870.5100 and Privalet al, in compliance with GLP.

Two mutagenicity studies were conducted, one as the plate incorporation method (i.e. Initial Mutation Test) and the other one as a preincubation test (i.e., Confirmatory Mutation Test). The studies were performed in the absence and presence of a metabolizing system derived from rat or hamster liver homogenate. The substance was tested for mutagenic effects without and with metabolic activation at six concentrations in the range of 128-5,000 µg/plate in all assays.

In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence and presence of the metabolic activation (rat liver S9-mix (10% (v/v)). Also, in the preincubation test no relevant increase in the number of revertants was observed in any of the bacterial strains in the absence and presence of the metabolic activation (hamster liver S9 -mix (30% (v/v)). Further, the spontaneous revertant colony numbers of solvent control plates without S9 Mix were within the historical control data range. The reference mutagens showed the expected increase in induced revertant colonies

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471, EU Method B.13/14, EPA OPPTS 870.5100 and Prival et al, in compliance with GLP.

Two mutagenicity studies were conducted, one as the plate incorporation method (i.e., Initial Mutation Test) and the other one as a preincubation test (i.e., Confirmatory Mutation Test). The studies were performed in the absence and presence of a metabolizing system derived from rat or hamster liver homogenate. The substance was tested for mutagenic effects without and with metabolic activation at six concentrations in the range of 128-5,000 µg/plate in all assays. In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence and presence of the metabolic activation (rat liver S9-mix (10% (v/v)). Also, in the preincubation test no relevant increase in the number of revertants was observed in any of the bacterial strains in the absence and presence of the metabolic activation (hamster liver S9 -mix (30% (v/v)). Further, the spontaneous revertant colony numbers of solvent control plates without S9 mix were within the historical control data range. The reference mutagens showed the expected increase in induced revertant colonies. Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay (Vertesi, 2006).

Justification for selection of genetic toxicity endpoint
Guideline-compliant study conducted according to GLP.