Sodium 2(or 5)-[[1-amino-4-[[3-[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxo-2-anthryl]oxy]-5(or 2)-(tert-butyl)benzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Acid Violet 109 was not mutagenic in the bacterial reverse mutation assays.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 November 1994 to 11 April, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: P.4
- Expiration date of the lot/batch: February 1999

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Target gene:

Histidine auxotrophic Salmonella strains

Species / strain / cell type:

other: S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 and TA102

Metabolic activation:

with and without

Metabolic activation system:

S9 from liver of rats treated with AROCLOR 1254.
Supplier: MOLTOX Inc., Annapolis, MD, USA
Batch number (by testing facility): 142/94 and 36/95

Test concentrations with justification for top dose:

5000, 1600, 512, 164 and 52 µg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water for injections

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: As positive control articles, t-Butyl hydroperoxide (t-BHP) at 100 ug per plate and 2-amino-anthracene (2-A) at 10 ug per plate were used in the absence and presence of a metabolic activation, respectively.

Evaluation criteria:

Evaluation Criteria:
The test article will be considered to be clearly mutagenic if all the following criteria are met:
1) The assay is valid (Acceptance criteria)
2) Dunnctt's test gives a statistically significant response (p < 0.05) with evidence of a dose relationship
3) The positive effects described in (2) are reproducible.

ACCEPTANCE CRITERIA
The assay will usually be considered valid if the following criteria are met :
1) The mean negative control counts fall within the normal range
2) The positive control chemicals induce clear increases in revenant numbers confirming discrimination between different strains and an active S9 Mix preparation
3) No more than 5 % of the plates in the assay are lost through contamination or some other unforeseen event.

Statistics:

After completion of scoring, mean of revenant colonies and standard deviation for each dose are determined. Dunnett's test is used to compare the counts at each dose level with the negative control. If positive responses are obtained, then the presence of a dose response will be checked by a linear regression analysis.

Species / strain:

other: Salmonella typhinmrium TA98, TA100, TA1535, TA1537, TA1538 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2500 µg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

PRELIMINARY STUDY:

No precipitate was noted after incorporation of the dosing solution into the overlay (top) agar with any of the dose levels tested. After incorporation of the dosing solution, a blue staining of the overlay (top) agar made the observation of the bacterial lawn difficult. However, signs of toxicity such as the clear decrease in the number of revertants was observed from 512 µg per plate in the absence of metabolic activation and at 5000 µg per plate in the presence of metabolic activation.

All the criteria for a valid study were

MAIN STUDY #1

The results of the preliminary study constitute the actual mutagenicity data for TA100 for Experiment 1 and the same dose levels (5000 - 1600 - 512 - 164 and 52 µg per plate) were selected for the first experiment of the mutagenicity assay in TA98 - TA 1535 - TA 1537 - TA1538 and TA102.

After incorporation of the dosing solutions, no precipitate was noted with any of the dose levels but a blue staining of the overlay (top) agar was observed. For this reason, the colonies (revertants) were counted manually from the dose level of 164 µg/plate up to 5000 µg/plate

. • Without metabolic activation - In the absence of obvious signs of toxicity in the bacterial lawn, variable decreases in the number of revertants were observed at 5000 µg per plate with all the tester strains. The decrease was more marked in the tester strain TA 100 with evidence of a dose-effect relationship from 512 µg per plate. All data were acceptable and all criteria for a valid study were met. A statistically significant increase in the number of revertants without evidence of a dose effect relationship was observed at 52 µg per plate with TA 1537.

. • With metabolic activation - As signs of toxicity, variable decreases in the number of revertants were observed at 5000 µg per plate with TA 98 and TA100, mainly. All data were acceptable and all criteria for a valid study were met. Slight statistically significant increases in the number of revertants were noted with TA 100 (512 and 1600 µg per plate) and with TA 102 (164 and 512 µg per plate).

MAIN STUDY #2

From the results obtained in the first study, a closer range of dose levels near to the top limit dose level was selected in this study : 2500 - 1400 - 784 - 439 and 246 µg/plate in both the presence and absence of a metabolic activation system.

After incorporation of the dosing solutions, no precipitate was noted with any of the dose levels, but a blue staining of the overlay (top) agar was observed. For this reason, the revertants were counted manually from the dose level of 246 µg per plate.

• Without metabolic activation - Variable decreases in the number of revertants were observed at 2500 µg depending upon the tester strain. The change was more marked in the tester strains TA100 and TA98 with evidence of a dose-effect relationship. All data were acceptable and no statistically significant increases in the number of revertants per plate containing the test article were observed with any of the tester strains. All criteria for a valid study were met.

• With metabolic activation - Variable decreases in the number of revertants were observed at 2500 µg per plate with all the tester strains. All data were acceptable and no statistically significant increases in the number of revertants per plate containing the test article were observed with any of the tester strains.

All criteria for a valid study were met.

Conclusions:

FAT 20200/B was not mutagenic in the Salmonella reverse mutation assay.

Executive summary:

The test article FAT 20200/B was tested on 6 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538 and TA102), with and without metabolic activation. A range of sub-toxic concentrations was determined in a preliminary study on the strain TA100 without and with metabolic activation. Five concentrations (5000, 1600, 512, 164 and 52 µg/plate) were tested in triplicate on the strains mentioned above with and without metabolic activation. The results were confirmed in a second independent study, using a closer range of dose levels near to the top limit dose level (2500, 1400, 784, 439 and 246 µg/plate). A negative control (vehicle) and a positive control (specific standard mutagen) were included in each study. The test article did not cause reproducible statistically significant increases in the number of revenants per plate in any of the tester strains used TA98, TA100, TA1535, TA1537, TA1538 and TA102 in the presence or absence of a metabolic activation system in both independent studies performed. Under the experimental conditions and according to the criteria of the test protocol, it is concluded that the test article FAT 20200/B induces no mutagenic effects in the Ames test.

Reference 2

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:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four tester strains tested, no tester strain to detect cross-linking mutagens was included

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

December 29, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

July 13, 1987

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 400047.32
- Expiration date of the lot/batch: December 1998

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Target gene:

Histidine auxotrophic Salmonella strains

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

5000, 1666.67, 555.56, 185.19 and 61.73 µg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylformamide
- Justification for choice of solvent/vehicle: Dimethylformamide was found to be the best suited vehicle for the formation of a suspension of the test material at room temperature.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA100, TA 98, TA 1537 with Metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

TA 1535 with Metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 without Metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without Metabolic activation

Details on test system and experimental conditions:

Setting up of the test plates:
0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the positive control or the solvent as a negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20.0 ml of minimal agar (1.5 % agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2 % glucose). The top agar was composed of 0.6 % agar and 0.6 % NaCl and was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM (+)biotin dissolved in water.

Preliminary range finding test:
Six concentrations of FAT 21016/C (Irganol Brillant violett base FLS trocken) ranging from 20.6 to 5000 µg/plate were tested with strain Salmonella typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation.The plates were inverted and incubated for about 48 hours at 37±1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration and negative control was used.

Mutagenicity test:
The mutagenicity test was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration and controls. The highest concentration applied was determined in the preliminary range finding test and the four lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37±1.5 °C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

Colony counting and scoring of the plates:
Colonies were counted electronically using an Artek Colony Counter (Fisher Scientific), or manually where minor agar damage or test chemical precipitates or strong coloration of the agar plates might have interfered with automating counting. The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means for all mutagenicity assays were calculated.

Evaluation criteria:

Assay acceptance criteria:
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.

Criteria for a positive response:
The test substance will be considered to be positive in the test system if the following condition is met:
At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the strains tested.
Generally a concentration-related effect should be demonstrable.

Statistics:

A statistical analysis of the test data was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.

Species / strain:

other: TA 98, TA 100, TA 1535 and TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

Range finding test:

Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate with and without metabolic activation.

Mutagenicity test:

In the original experiments performed with and without metabolic activation, with one exception, treatment of strains TA 98, TA 100, TA 1535 and TA 1537 with FAT 21016/C (Irganol Bnllantviolettbase FLS trocken) did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control. In the experiment with activation carried out on strain TA 1537, a marginally increased number of revertant counts was registered at the highest concentration. Since no such effect occurred in the repeat experiment with activation on this strain, the increase in the number of back-mutants is attributed to fluctuations of the spontaneous rate of back-mutants and not due to action of the test substance.

In the confirmatory experiments performed with and without metabolic activation, again after treatment of strains TA 98, TA 100, TA 1535 and TA 1537 with FAT 21016/C (Irganol Brillantviolettbase FLS trocken) no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control.

In the mutagenicity tests, normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced with increasing concentrations. Therefore, the test substance exerted no toxic effect on the growth of the bacteria.

Conclusions:

FAT 21016/C did not induce gene mutations in the Salmonella reverse mutation assay.

Executive summary:

FAT 21016/C was assesed for mutagenic activity in bacterial test systems in the presence and absence of a rat liver metabolic activation system as per the OECD guideline 471, under Good Laboratory Practice conditions.

The test was conducted using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was suspended in dimethylformamide and tested at five concentrations in the range of 61.7 to 5000.0 ug/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

In both experiments, performed with and without metabolic activation, none of the tested concentrations of FAT 21016/C led to a biologically relevant increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

Based on the results of these experiments and on standard evaluation criteria, it is concluded that Acid violet 109 and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acid Violet 109 was assessed for mutagenic potential in two Salmonella reverse mutation assays. In the study, considered as key study, FAT 21016/C was assessed for mutagenic activity in bacterial test systems in the presence and absence of a rat liver metabolic activation system as per the OECD guideline 471, under Good Laboratory Practice conditions. The test was conducted using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was suspended in dimethylformamide and tested at five concentrations in the range of 61.7 to 5000 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations.

In both experiments, performed with and without metabolic activation, none of the tested concentrations of FAT 21016/C led to a biologically relevant increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. Based on the results of these experiments and on standard evaluation criteria, it is concluded that Acid Violet 109 and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.

In a supporting study, FAT 20200/B was tested on 6 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538 and TA102). Five concentrations (5000, 1600, 512, 164 and 52 µg/plate) were tested in triplicate on the strains mentioned above with and without metabolic activation. The results were confirmed in a second independent study, using a closer range of dose levels near to the top limit dose level (2500, 1400, 784, 439 and 246 µg/plate). The test article did not cause reproducible statistically significant increases in the number of revenants per plate in any of the tester strains used TA98, TA100, TA1535, TA1537, TA1538 and TA102 in the presence or absence of a metabolic activation system in both independent studies performed.

Both Salmonella reverse mutation assays returned negative results, indicating that Acid Violet 109 is not mutagenic in bacteria.

Justification for classification or non-classification

Based on the available bacterial reverse mutation assays, Acid Violet 109 does not need to be classified for genotoxicity according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.