reaction mass of sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate and sodium 1-amino-4-[[3-[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non genotoxic

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: data on Acid Blue 225_constituent 1

Adequacy of study:

key study

Study period:

From July 27 to September 03, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

The experiment was conducted on one of the substance components. It should be noted that the lot tested was characterized by the Acid Blue 225_constituent 1 as main component; however, the impurity profile resulted to be closely similar to that characterizing the substance under assessment (details are given in the document attached to IUCLID section 13.2).

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

May 26, 1983

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

December 29, 1992

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 13, 1987

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Source of cells: the histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 102, TA 1535 and TA 1537) were obtained from Prof. B. Ames, Berkeley, CA., U.S.A. Strain TA 100 was obtained from Dr. M. Schiipbach, Hofmann-La Roche Ltd., Basle, Switzerland.

Preparation of the bacterial cultures
Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.

Control of the genotype of the strains
The characteristics of the strains were checked monthly. Histidine-auxotrophy of the strains was demonstrated by the requirement for 1-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene (strains TA 98, TA 100, TA 1535 and TA 1537) was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. Strain TA 102 additionally was checked for tetracycline resistence (presence of multicopy plasmid pAQl). The presence of the uvr+ gene was demonstrated by the resistence of strain TA 102 against UV-light.
Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).

Metabolic activation:

with and without

Metabolic activation system:

rat liver post-mitochondrial fraction

Test concentrations with justification for top dose:

Range finding: 20.5761, 61.7284, 185.1852, 555.5556, 1666.6667 and 5000.0000 µg/plate
Main experiments: 61.7284, 185.1852, 555.5556, 1666.6667 and 5000.0000 µg/plate, woth and withour metabolic activation

Vehicle / solvent:

- Vehicle: bidistilled water.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
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 substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 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. It was supplemented with 10 % of 0.5 mM 1-histidine and 0.5 mM (+)biotin dissolved in water.

REPLICATES: each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration as well as each positive and negative control with each tester strain. Two experiments were conducted.

INCUBATION: 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.

CYTOTOXICITY TEST
A toxicity test (check for reduction in the number of revertant colonies) was carried out with strain TA 100 without and with microsomal activation at six concentrations of the test substance and one negative control. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of 3. 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, as well as each negative control was used.

COLONY COUNTING AND SCORING
Colonies were counted electronically with an Artek counter. 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.

PREPARATION OF METABOLIC ACTIVATION MIXTURE
Rat-liver microsomal fraction S9 was prepared in advance from male RAI rats (Tif: RAIf[SPF]), reared at the Animal Farm of testing facility, Sisseln, Switzerland. The animals (150-250 g) were treated with Aroclor 1254 (500 mg/kg, i.p.) 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl and the 9000x g supernatant (S9) was stored at approximately -80 °C for no longer than one year. The protein contents of the S9 fractions were 33.6 and 30.2 mg/ml.
The activation mixture contained: Rat liver S9 fraction 100 µl/ml, NADP 4 µmol/ml, MgCl2 8 µmol/ml, KCl 33 µmol/ml, Na-phosphate-buffer pH 7.4 100 µmol/ml and glucose-6-phosphate 5 µmol/ml.

ASSAY ACCEPTANCE CRITERIA
A test is considered acceptable if the mean colony counts of the 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.

Evaluation criteria:

The test substance is considered to be mutagenic in the test system if the following conditions are 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 following strains S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537;
- generally a concentration-related effect should be demonstrable.

Statistics:

In deviation to the OECD guideline a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended. No appropriate statistical method is available

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the original experiment carried out without metabolic activation, treatment of strain TA 102 with the substance led to a marginal increase in the number of backmutants at the concentrations of 1666.7 and 5000 µg/plate. No effects were observed with the other strains. In the experiment with activation performed on strain TA 102, a marginal increase in the number of revertants occurred at the concentration of 555.6 µg/plate only. No effects were observed with the other strains.

In the confirmatory experiment performed without microsomal activation, after treatment with test item, no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. The effect observed in the original experiment on strain TA 102 could not be reproduced. In the experiment with activation performed on strain TA 102, a marginal increase in the number of back-mutants occurred at the concentration of 1666.7 µg/plate only. Again, no effects were observed with the other strains.
In the mutagenicity tests without and with metabolic activation, normal back-ground growth was observed. The numbers of revertant colonies was not reduced. The test material exerted no toxic effect on the growth of the bacteria.
The various mutagens, promutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within testing laboratory established limits.
There were no known circumstances or occurrences in the study that were considered to have affected the quality or integrity of the data.

CYTOTOXICITY TEST
The numbers of revertant colonies was not reduced. Normal back-ground growth was observed at all concentrations. The test material exerted no toxic effect on the growth of the bacteria.

Conclusions:

Under the experimental conditions reported, the test article did not induce gene mutations.

Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: in the cytotoxicity test in the range of 20.6 - 5000 µg/plate; in the mutagenicity test in the range of 61.7 - 5000 µg/plate.

The numbers of revertant colonies was not reduced. Normal back-ground growth was observed at all concentrations. The test material exerted no toxic effect on the growth of the bacteria.

In the original experiment carried out without metabolic activation, treatment of strain TA 102 with the substance led to a marginal increase in the number of backmutants at the concentrations of 1666.7 and 5000 µg/plate. No effects were observed with the other strains. In the experiment with activation performed on strain TA 102, a marginal increase in the number of revertants occurred at the concentration of 555.6 µg/plate only. No effects were observed with the other strains.

In the confirmatory experiment performed without microsomal activation, after treatment with test item, no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. The effect observed in the original experiment on strain TA 102 could not be reproduced. In the experiment with activation performed on strain TA 102, a marginal increase in the number of back-mutants occurred at the concentration of 1666.7 µg/plate only. Again, no effects were observed with the other strains.

In the mutagenicity tests without and with metabolic activation, normal back-ground growth was observed. The numbers of revertant colonies was not reduced. The test material exerted no toxic effect on the growth of the bacteria.

The various mutagens, promutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within testing laboratory established limits.

There were no known circumstances or occurrences in the study that were considered to have affected the quality or integrity of the data.

Discussion and conclusion

Based on the results of the experiments and on standard evaluation criteria, it was indicated that the metabolites of test item exerted a marginal mutagenic action on strain S. typhimurium TA 102. Considering that:

- the marginal increase observed in the number of revertants, observed only in strain TA 102, is not dose-dependent

- in both the experiments conducted with metabolic activation only one out of five concentrations showed significant increase (and not the highest one)

- the concentrations which showed an increase of revertants were not consistent between the two experiments

it can be concluded that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There is no specific data about the genetic toxicity potential of Acid Blue 225, therefore the available information on Acid Blue 225_constituent 1 has been taken into consideration; it should be noted that the lot tested was characterized by the Acid Blue 225_constituent 1 as main component. The data can be considered as adequate and the approach can be considered as suitable (details are given in the document attached to IUCLID section 13.2).

The study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: in the cytotoxicity test in the range of 20.6 - 5000µg/plate; in the mutagenicity test in the range of 61.7 - 5000µg/plate.

The numbers of revertant colonies was not reduced. Normal back-ground growth was observed at all concentrations. The test material exerted no toxic effect on the growth of the bacteria.

In the original experiment carried out without metabolic activation, treatment of strain TA 102 with the substance led to a marginal increase in the number of backmutants at the concentrations of 1666.7 and 5000 µg/plate. No effects were observed with the other strains. In the experiment with activation performed on strain TA 102, a marginal increase in the number of revertants occurred at the concentration of 555.6 µg/plate only. No effects were observed with the other strains.

In the confirmatory experiment performed without microsomal activation, after treatment with test item, no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. The effect observed in the original experiment on strain TA 102 could not be reproduced. In the experiment with activation performed on strain TA 102, a marginal increase in the number of back-mutants occurred at the concentration of 1666.7 µg/plate only. Again, no effects were observed with the other strains.

In the mutagenicity tests without and with metabolic activation, normal back-ground growth was observed. The numbers of revertant colonies was not reduced. The test material exerted no toxic effect on the growth of the bacteria.

The various mutagens, promutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within testing laboratory established limits.

There were no known circumstances or occurrences in the study that were considered to have affected the quality or integrity of the data.

Based on the results of the experiments and on standard evaluation criteria, it was indicated that the metabolites of test item exerted a marginal mutagenic action on strainS. typhimuriumTA 102.

Considering that:

- the marginal increase observed in the number of revertants, observed only in strain TA 102, is not dose-dependent

- in both the experiments conducted with metabolic activation only one out of five concentrations showed significant increase (and not the highest one)

- the concentrations which showed an increase of revertants were not consistent between the two experiments

it can be concluded that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations.

In order to support the conclusion reached during the review of the results obtained in the key study, available data on the structural analogous Similar Substance 01 has been taken into consideration.

The read across approach can be considered as adequate and suitable (details are given in the document attached to related endpoint record).

The substance was tested on 6 strains of Salmonella typhinmrium (TA 98, TA 100, TA 1535, TA 1537, TA 1538 and TA 102), with and without metabolic activation. A range of sub-toxic concentrations was determined in a preliminary study on the strain TA 100 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).

The test article did not cause reproducible statistically significant increases in the number of revenants per plate in any of the tester strains used TA 98, TA 100, TA 1535, TA 1537, TA 1538 and TA 102 in the presence or absence of a metabolic activation system in both independent studies performed.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggests that test substance did not show any reasons of concern from the genotoxicity point of view.

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC) No 1272/2008.