Sodium 1-amino-4-[[3,5-bis[[(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

Acid Blue 225 was found to be non-mutagenic in the bacterial reverse mutation assay as well as the in vitro mammalian cell gene mutation assay (HPRT). It was also negative in the in vitro mammalian chromosomal aberration assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 July 1993 to 04 Oct 1993

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 OTS 798.5265 (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:

Name: FAT 21036/C
Purity: Approx 90 %

Target gene:

Histidine for Salmonella

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: 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.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction): Rat-liver microsomal fraction S9 was prepared in advance from male RAI rats (Tif: RAIf[SPF]), reared at the Animal Farm of CIBA GEIGY, 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.

Test concentrations with justification for top dose:

Range in the cytotoxicity test; 20.6, 61.7, 185.1, 555.5, 1666.6, 5000 µg/plate
Range in the mutagenicity test: 61.7, 185.1, 555.5, 1666.6, 5000 µg/plate

Vehicle / solvent:

Bidistilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For TA 100 & TA 1535 without microsomal activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

For TA 102 without microsomal activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

For TA 98 without microsomal activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For TA 1537 without microsomal activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2- aminoanthracene

Remarks:

For TA 100, TA 102, TA 98& TA 1537 with microsomal activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

For TA 1535 with microsomal activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: in agar (plate incorporation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY : Background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Colonies were counted electronically with an Artek counter

Evaluation criteria:

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. In either case the final decision is based on the scientific judgement of the study director.

Criteria for a positive response:
The test substance is considered to be mutagenic in this 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.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

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

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

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

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

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

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

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

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

RANGE-FINDING/SCREENING STUDIES

Six concentrations of Polar blau RLS roh trocken (FAT 21036/C) ranging from 20.6 to 5000 µg/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. 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. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with activation.

 

Mutagenicity test, original experiment

In the original experiment carried out without metabolic activation, treatment of strain TA 102 with Polar blau RLS roh trocken (FAT 2103 6/C) 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.

 

Mutagenicity test, confirmatory experiment

In the confirmatory experiment performed without microsomal activation, after treatment with Polar blau RLS roh trocken (FAT 21036/C), 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 our established limits. There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the data.

 

SUMMARY OF THE MUTAGENICITY EXPERIMENTS

Original experiments without metabolic activation

 

Strain	Treatment	Mean counts
TA 100	Negative control	126.3
	61.7284 µg/plate	141.3
	185.1852 µg/plate	155.0
	555.5556 µg/plate	143.0
	1666.6667 µg/plate	144.3
	5000.0000 µg/plate	108.7
	Positive control	1320.3

 

Strain	Treatment	Mean counts
TA 1535	Negative control	13.7
	61.7284 µg/plate	14.7
	185.1852 µg/plate	11.7
	555.5556 µg/plate	11.7
	1666.6667 µg/plate	14.7
	5000.0000 µg/plate	11.33
	Positive control	1100.7

 

 

Strain	Treatment	Mean counts
TA 98	Negative control	15.0
	61.7284 µg/plate	16.0
	185.1852 µg/plate	15.3
	555.5556 µg/plate	21.0
	1666.6667 µg/plate	14.7
	5000.0000 µg/plate	13.0
	Positive control	1582.7

 

Strain	Treatment	Mean counts
TA 1537	Negative control	9.7
	61.7284 µg/plate	8.0
	185.1852 µg/plate	9.0
	555.5556 µg/plate	4.3
	1666.6667 µg/plate	5.0
	5000.0000 µg/plate	5.0
	Positive control	2091.7

 

Strain	Treatment	Mean counts
TA 102	Negative control	237.0
	61.7284 µg/plate	240.0
	185.1852 µg/plate	273.3
	555.5556 µg/plate	327.3
	1666.6667 µg/plate	394.3
	5000.0000 µg/plate	378.0
	Positive control	1130.3

 

 

SUMMARY OF THE MUTAGENICITY EXPERIMENTS

Original experiments with metabolic activation

 

Strain	Treatment	Mean counts
TA 100	Negative control	135.3
	61.7284 µg/plate	130.3
	185.1852 µg/plate	142.7
	555.5556 µg/plate	173.0
	1666.6667 µg/plate	159.3
	5000.0000 µg/plate	144.3
	Positive control	1389.7

 

Strain	Treatment	Mean counts
TA 1535	Negative control	17.0
	61.7284 µg/plate	17.7
	185.1852 µg/plate	14.7
	555.5556 µg/plate	15.3
	1666.6667 µg/plate	14.3
	5000.0000 µg/plate	10.7
	Positive control	339.7

 

 

Strain	Treatment	Mean counts
TA 98	Negative control	34.0
	61.7284 µg/plate	33.7
	185.1852 µg/plate	29.3
	555.5556 µg/plate	31.3
	1666.6667 µg/plate	23.3
	5000.0000 µg/plate	23.7
	Positive control	2207.3

 

Strain	Treatment	Mean counts
TA 1537	Negative control	7.3
	61.7284 µg/plate	9.0
	185.1852 µg/plate	12.0
	555.5556 µg/plate	11.3
	1666.6667 µg/plate	7.3
	5000.0000 µg/plate	4.0
	Positive control	205.7

 

Strain	Treatment	Mean counts
TA 102	Negative control	244.7
	61.7284 µg/plate	348.3
	185.1852 µg/plate	295.3
	555.5556 µg/plate	389.3
	1666.6667 µg/plate	349.0
	5000.0000 µg/plate	354.0
	Positive control	1431.7

 

 

SUMMARY OF THE MUTAGENICITY EXPERIMENTS

Confirmatory experiments without metabolic activation

 

Strain	Treatment	Mean counts
TA 100	Negative control	173.3
	61.7284 µg/plate	195.7
	185.1852 µg/plate	200.3
	555.5556 µg/plate	174.3
	1666.6667 µg/plate	194.3
	5000.0000 µg/plate	183.3
	Positive control	1345.0

 

Strain	Treatment	Mean counts
TA 1535	Negative control	19.0
	61.7284 µg/plate	14.7
	185.1852 µg/plate	20.7
	555.5556 µg/plate	18.0
	1666.6667 µg/plate	15.7
	5000.0000 µg/plate	15.0
	Positive control	1187.7

 

 

Strain	Treatment	Mean counts
TA 98	Negative control	28.3
	61.7284 µg/plate	28.7
	185.1852 µg/plate	24.7
	555.5556 µg/plate	24.7
	1666.6667 µg/plate	27.7
	5000.0000 µg/plate	23.0
	Positive control	1935.0

 

Strain	Treatment	Mean counts
TA 1537	Negative control	10.3
	61.7284 µg/plate	12.0
	185.1852 µg/plate	14.7
	555.5556 µg/plate	10.0
	1666.6667 µg/plate	9.7
	5000.0000 µg/plate	6.7
	Positive control	2315.0

 

Strain	Treatment	Mean counts
TA 102	Negative control	336.7
	61.7284 µg/plate	340.3
	185.1852 µg/plate	391.7
	555.5556 µg/plate	374.3
	1666.6667 µg/plate	419.3
	5000.0000 µg/plate	357.7
	Positive control	1961.0

 

 

SUMMARY OF THE MUTAGENICITY EXPERIMENTS

Confirmatory experiments with metabolic activation

 

Strain	Treatment	Mean counts
TA 100	Negative control	184.7
	61.7284 µg/plate	208.3
	185.1852 µg/plate	226.7
	555.5556 µg/plate	219.3
	1666.6667 µg/plate	226.0
	5000.0000 µg/plate	213.7
	Positive control	1881.0

 

Strain	Treatment	Mean counts
TA 1535	Negative control	17.3
	61.7284 µg/plate	24.3
	185.1852 µg/plate	20.7
	555.5556 µg/plate	16.3
	1666.6667 µg/plate	25.3
	5000.0000 µg/plate	19.7
	Positive control	378.7

 

 

Strain	Treatment	Mean counts
TA 98	Negative control	42.7
	61.7284 µg/plate	43.0
	185.1852 µg/plate	49.3
	555.5556 µg/plate	47.0
	1666.6667 µg/plate	33.7
	5000.0000 µg/plate	33.7
	Positive control	2659.0

 

Strain	Treatment	Mean counts
TA 1537	Negative control	14.7
	61.7284 µg/plate	9.3
	185.1852 µg/plate	12.3
	555.5556 µg/plate	15.3
	1666.6667 µg/plate	9.0
	5000.0000 µg/plate	6.7
	Positive control	414.7

 

Strain	Treatment	Mean counts
TA 102	Negative control	331.7
	61.7284 µg/plate	369.3
	185.1852 µg/plate	379.7
	555.5556 µg/plate	427.3
	1666.6667 µg/plate	496.3
	5000.0000 µg/plate	458.7
	Positive control	1723.3

 

Conclusions:

FAT 21036/C was not mutagenic in this bacterial reverse mutation assay.

Executive summary:

A study was performed to investigate the potential of FAT 21036/C to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. This test was conducted in accordance with OECD test guideline 471. 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:

Range in the cytotoxicity test; 20.6, 61.7, 185.1, 555.5, 1666.6, 5000 µg/plate

The concentration range of FAT 21036/C to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, the test material was tested for mutagenic effects without and with metabolic activation at five concentrations in the range 61.7, 185.1, 555.5, 1666.6, 5000 µg/plate.

 

Mutagenicity test, original experiment

In the original experiment carried out without metabolic activation, treatment of strain TA 102 with Polar blau RLS roh trocken (FAT 2103 6/C) 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.

 

Mutagenicity test, confirmatory experiment

In the confirmatory experiment performed without microsomal activation, after treatment with Polar blau RLS roh trocken (FAT 21036/C), 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 experiment without and with metabolic activation no toxic effect of the test material on the growth of the bacteria was observed. A marginal increase in backmutants was reported with strain TA 102 with metabolic activation at 555.6 µg/plate only in the original experiment and at 1666.7 µg/plate only in the confirmatory experiment. This marginal effect seen was regarded as not significant as the increase was seen at two different concentrations during original and confirmatory experiments, without being seen at higher concentrations in these experiments. Thus, the effect seen can not be considered as dose-dependent or concentration driven. Hence, based on the results of these experiments and on standard evaluation criteria, it is concluded that FAT 21036/C was not mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

human peripheral blood lymphocytes

Species / strain / cell type:

other: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

human peripheral blood lymphocytes

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Based on the results of the preliminary cytotoxicity test, the following concentrations were selected for testing in all the three experiments of the chromosomal aberration assay:

Experiments 1 & 2 (presence and absence of metabolic activation with 3-hour exposure) & Experiment 3 (absence of metabolic activation with 22-hour exposure)

a) 50 b) 100 and c) 200 µg/mL (factor of 2)

Vehicle / solvent:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Test approaches currently accepted under the OECD and ICH guidance for the assessment of mammalian cell clastogenicity involve the use of human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Human blood cultures were established and used as the test system

Rationale for test conditions:

Test approaches currently accepted under the OECD for the assessment of mammalian cell clastogenicity involve the use of human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Human blood cultures were established and used as the test system

Evaluation criteria:

When all the validity criteria are fulfilled:
1. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase in aberrant metaphases compared with the concurrent vehicle control.
• The increase is dose-dependent when evaluated with an appropriate trend test.
• Any of the results are outside the distribution of the historical vehicle control data.
When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.
2. A test chemical is considered to be clearly negative if, in all experimental conditions examined:
• None of the test concentrations exhibits a statistically significant increase in aberrant metaphases compared with the concurrent vehicle control.
• There is no concentration-related increase in aberrant metaphases when evaluated with an appropriate trend test.
• All results are inside the distribution of the historical vehicle control data.
3. The results will be considered equivocal if they do not meet the criteria specified for a positive or negative response.
4. An increase in the number of polyploid cells may indicate that the test substances have the potential to inhibit mitotic processes and to induce numerical chromosomal aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test substances have the potential to inhibit cell cycle progression.

Statistics:

The statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analysed for proportions of aberrant metaphases in each sample, excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p <0.05) level.

Key result

Species / strain:

lymphocytes: primary culture

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tested up to highest concentration exhibiting 50 % cytotoxicity.

Positive controls validity:

valid

S9 Characterization:
a) Sterility Check:
The S9 homogenate was found to be sterile.

b) Metabolic Activation:
The S9 homogenate was found to be active.

c) Protein Content:
The protein content of the S9 homogenate was 31.931 mg/mL.

Preliminary Solubility Test:
Test item was soluble in DMSO at 160 mg/mL.

Preliminary Cytotoxicity Test and Justification for the Selection of Test Concentrations:
At the end of the 3-hour exposure period, FAT 21036/H TE did not cause precipitation in the test media at any of the tested concentrations till 1600 μg/mL both, in the presence and absence of metabolic activation, at the beginning and end of treatment. In the presence of metabolic activation, at the end of 3-hour exposure to the test item, the pH of the test medium ranged between 7.10 and 7.23 with a pH value of 7.27 in the DMSO control. In the absence of metabolic activation, the pH ranged between 7.12 and 7.24 with a pH value of 7.24 in the DMSO control. At the end of 3-hour exposure, osmolality of the test medium at the highest test item treatment condition (1600 μg/mL) was 0.471 and 0.477 OSMOL/kg in the presence and absence of metabolic activation, respectively. The corresponding osmolality in the DMSO control was 0.503 and 0.511 OSMOL/kg in the presence and absence of metabolic activation, respectively. FAT 21036/H exhibited the required level of cytotoxicity (reduction in the mitotic index by 45±5 % of the concurrent vehicle control) at 200 μg/mL both in the presence and absence of metabolic activation with 3-hour exposure and 22-hour exposure. Based on these observations, in the chromosomal aberration assay, a top concentration of 200 μg/mL was tested in the 3-hour exposure, in the presence and absence of metabolic activation and in the absence of metabolic activation with 22-hour exposure. Test item showed 42 %, 44 % and 48 % cytotoxicity at 200 μg/mL, in the presence of metabolic activation at 3-hour exposure and absence of metabolic activation at 3-hour exposure and 22-hour exposure compared to the vehicle control, respectively.

Chromosomal Aberration Assay:

Experiment 1:
At the highest concentration tested (200 μg/mL), the mitotic inhibition was 43 % compared to the vehicle control. The incidence of aberrations in the vehicle control was within the range of the in-house historical control data. There was no statistically significant increase in the number of aberrant metaphases in any of the test concentrations when compared to the vehicle control. No incidences of polyploidy and endoreduplication were seen. The positive control, cyclophosphamide monohydrate, caused a statistically significant (p <0.05) increase in the aberrant metaphases excluding gaps.

Experiment 2:

At the highest concentration tested (200 μg/mL), the mitotic inhibition was 45 % compared to the vehicle control.
The incidence of aberrations in the vehicle control was within the range of the in-house historical control data. There was no statistically significant increase in the number of aberrant metaphases in any of the test concentrations when compared to the vehicle control. There were no incidences of polyploidy and endoreduplication.

Experiment 3:

At the highest concentration tested (200 μg/mL), the mitotic inhibition was 49 % compared to the vehicle control.
The incidence of aberrations in the vehicle control was within the range of the in-house historical control data. There was no statistically significant increase in the number of aberrant metaphases in any of the test concentrations when compared to the vehicle control. No incidences of polyploidy and endoreduplication were seen. The positive control, Ethylmethanesulfonate caused a statistically significant (p <0.05) increase in the aberrant metaphases excluding gaps.

DISCUSSION:
No evidence of induction of chromosome aberrations excluding gaps was obtained in any of the experiments at any test concentrations either in the presence or in the absence of metabolic activation at 3-hour and 22-hour exposure, as compared to vehicle control. Similarly, there were no incidences of polyploidy and endoreduplication. The respective positive controls tested produced a statistically significant increase in aberrant metaphases, demonstrating that the system was able to detect the effect of known mutagens.

Conclusions:

FAT 21036/H is not clastogenic under the conditions of testing employed.

Executive summary:

The clastogenic potential of the test item, FAT 21036/H, to induce chromosomal aberrations in mammalian cells was evaluated using cultured human peripheral blood lymphocytes. Human lymphocytes in whole blood culture, stimulated to divide by addition of Phytohaemagglutinin (PHA) 48 hours prior to treatment, were exposed to the test item in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver). The study consisted of a preliminary cytotoxicity test and a chromosomal aberration assay. Chromosomal aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 22-hour exposure. In a preliminary cytotoxicity test, blood cells were exposed to FAT 21036/H at the concentrations of 50, 100, 200, 400, 800 and 1600 µg/mL along with a DMSO control. At the end of the 3-hour exposure period, FAT 21036/H did not cause precipitation in the test media at any of the tested concentrations till 1600 µg/mL both, in the presence and absence of metabolic activation, at the beginning and end of treatment. FAT 21036/H exhibited the required level of cytotoxicity (reduction in the mitotic index by 45±5 % of the concurrent vehicle control) at 200 µg/mL both in the presence and absence of metabolic activation with 3-hour exposure and absence of metabolic activation with 22-hour exposure. Test item showed 42 %, 44 % and 48 % cytotoxicity at 200 µg/mL in the presence and absence of metabolic activation at 3-hour exposure and absence of metabolic activation at 22-hour exposure respectively, compared to the concurrent vehicle control. Based on these observations, in the chromosomal aberration assay, a top concentration of 200 µg/mL was tested in the 3-hour exposure, in the presence and absence of metabolic activation and in the absence of metabolic activation with 22-hour exposure. In the chromosomal aberration assay, blood cultures were exposed to the test item in duplicate at the concentrations of  50, 100 and 200 mg/mL in Experiments 1 and 2 (presence and absence of metabolic activation with 3-hour test item exposure) and Experiment 3 (absence of metabolic activation with 22-hour test item exposure). In a similar way, concurrent vehicle control (DMSO) and the positive controls (cyclophosphamide monohydrate in the presence of metabolic activation in Experiment 1 and ethyl methanesulfonate in the absence of metabolic activation in Experiment 3) were tested in duplicate cultures. In each case, the cells in the C-metaphase were harvested at approximately 22 hours after the start of the treatment from the vehicle control, test item concentrations and the positive control groups. At the highest concentration tested, the reduction in mitotic index was 43, 45 and 49% in Experiments 1, 2 and 3, respectively, compared to the vehicle control. A total of 300 metaphases from duplicate cultures from each of the controls and three treatment levels were evaluated for chromosomal aberrations. The data from the treatment groups and the positive controls were statistically compared with the vehicle control. There were no statistically significant increase in the incidence of structurally aberrant metaphases, either at 3 hours in the presence and absence of metabolic activation and 22 hours exposure, in the absence of metabolic activation in any of the tested concentrations. Under identical conditions, the respective positive control substances produced statistically significant (p <0.05) increase in aberrant metaphases confirming the sensitivity of the test system and the activity of the S9 mix.

The study indicated that, under the conditions of this study, FAT 21036/H was not clastogenic in human peripheral blood lymphocytes up to the respective highest concentration of 200 µg/mL in the presence and absence of metabolic activation with 3-hour exposure and absence of metabolic activation with 22-hour exposure.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Based on the results of the preliminary cytotoxicity test, the following test concentrations were selected for testing in the gene mutation test:

Experiments 1 and 2 (Presence and Absence of Metabolic Activation, respectively)

a) 25; b) 100; c) 400; and d) 1600 µg/mL (factor of 4)

Vehicle / solvent:

DMSO

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

Cells were grown in tissue culture flasks at 37 ± 1 °C in a humidified carbon dioxide incubator (5 ± 0.2 % CO2 in air)

Rationale for test conditions:

Test approaches currently accepted under the OECD for the assessment of mammalian cell gene
12 mutation involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals. Established CHO cell line is useful in in vitro gene mutation testing because it is easily cultured in
standard medium, has a small number of large chromosomes each with a more or less distinctive morphology and a relatively short cycle time.

Evaluation criteria:

CRITERIA FOR ACCEPTABILITY OF THE TEST
The assay will be considered valid if the following criteria are met:
a) The concurrent vehicle control data is within the range of the laboratory historical control data.
b) The concurrent positive control substances should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent vehicle control.
c) Two experimental conditions are tested unless one results in positive response.
d) Adequate number of cells and analyzable concentrations are tested under each of the experimental conditions.
e) The criteria for the selection of top concentration are consistent with those described in the guideline.

EVALUATION AND INTERPRETATION OF RESULTS
When all the validity criteria are fulfilled:
1. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• The increase is concentration-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
2. A test chemical is considered to be clearly negative if, in all experimental conditions examined:
• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Preliminary Solubility Test:
FAT 21036/H was insoluble in sterile water at 200 mg/mL.
FAT 21036/H formed a free flowing susension in DMSO at 160 mg/mL.
DMSO is one of the vehicles compatible with this test system. Hence, based on the results of solubility test, DMSO was selected as the vehicle in the mammalian cell gene mutation assay.

Preliminary Cytotoxicity Test and Justification for the Selection of Target Top Concentration:

At the end of 3-hour exposure, there was no precipitation of the test item in the test medium at any of the tested concentrations, both, in the presence and absence of metabolic activation. At the end of 3-hour exposure, the pH of the test medium in the presence of metabolic activation ranged from 7.11 to 7.22 with 7.22 in the DMSO control while in the absence of metabolic activation it was between 7.12 and 7.22 with 7.25 in the DMSO control. At the end of the 3-hour exposure period, the osmolality of the test medium, at the highest test item concentration level (1600 μg/mL) was 445 and 442 mOSMOL/kg in the presence and absence of metabolic activation, respectively. The corresponding osmolality in the DMSO was 460 mOSMOL/kg in the presence and absence of metabolic activation. The Relative Survival at 1600 μg/mL was 27 and 34 %, in the presence and absence of metabolic activation, respectively. The test item did not show evidence of significant cell growth inhibition as Relative survival (10 to 20 % RS compared to vehicle control) at any of the tested concentrations, both, in the presence and absence of metabolic activation. Based on the observations of precipitation and preliminary cytotoxicity test, it was decided to test up to the highest soluble test concentration of 1600 μg/mL in the gene mutation assay both in the presence and absence of metabolic activation.

Gene Mutation Assay:
There was no evidence of excessive cytotoxicity (i.e., <10 % RS) at any of the tested concentrations either in the presence or absence of metabolic activation. The RS values in the presence of metabolic activation, ranged from 30 to 76 % while in the absence of metabolic activation, ranged from 31 to 79 % compared to the vehicle control. The frequency of mutants in the vehicle control was within the range of the in-house historical control data. The test item did not cause a significant increase in the frequencies of mutants compared to the vehicle control in the presence or absence of metabolic activation at any of the tested concentrations. Under similar conditions the positive control 3-methylcholanthrene (3-MCA) induced statistically significant increases in the mutant frequency as compared with the vehicle control.

DISCUSSION:
The assay conditions fulfilled all the specifications as per the OECD 476 guideline. All the acceptance criteria for a valid test were met. The Adjusted Cloning Efficiency of the vehicle controls was ≥1.463. The mean mutant frequency of the vehicle controls was 10.41 and 10.89 mutants per 106 clonable cells in the presence and in the absence of metabolic activation, respectively. In experiment in the presence of metabolic activation, the positive control chemical produced a statistically significant increase in the mutant frequencies, demonstrating both the activity of the metabolic activation system and the responsiveness of the test system. No evidence for the induction of gene mutation was observed in any of the concentrations of the test item either in the presence or in the absence of metabolic activation. Taken together, the results indicated that FAT 21036/H does not have the potential to induce gene mutation either in the presence or in the absence of metabolic activation at the doses tested.

Conclusions:

It is concluded that the test item, FAT 21036/H does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed

Executive summary:

The genotoxic potential of the test item FAT 21036/H to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells. The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver). FAT 21036/H was insoluble in sterile water and formed a free flowing suspension in DMSO at 160 mg/mL. In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 27 and 34 % at the highest tested concentration of 1600 µg/mL, in the presence and absence of metabolic activation, respectively. At the end of the three hours exposure period, there was no precipitation of the test item in the test medium at any of the tested concentrations, both in the presence and absence of metabolic activation. There was no appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 1600 µg/mL was tested in the gene mutation assay. In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 25, 100, 400 and 1600 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate. There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions. The results of the forward gene mutation test at the hprt locus with FAT 21036/H indicated that the test item was non-mutagenic under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The read-across substance FAT 20297 was found to be not clastogenic in the micronucleus assay. Hence, based on the principles of read across, FAT 21036 was also considered to be not clastogenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Both, Acid Blue 225 and Acid Blue 344, are blue dyes originating from the bromamine acid. Both these possess sulphonic acid groups. Hence, the two dyes are claimed to be analogues.
For further details, please refer read across justification document.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

FAT 20297/C was considered to be non clastogenic in the micronucleus assay.

Executive summary:

Currently no invivo genotoxicity study is available for Acid Blue 225. However, a structurally similar substance, Acid Blue 344 was investigated fin a study conducted according to EEC Directive 79/831, Annex V, Part B, Paragraph 4.3.1.

The compound was administered orally to mice at a concentration of 1500 mg/kg body weight. No increase in the number of micronucleated polychromatic erythrocytes was observed in the bone marrow smears taken 20, 44 and 68 hours after administration of the test substance. A positive control (Thio-TEPA) administered at a concentration of 80 mg/kg body weight showed pronounced evidence of mutagenicity 20 hours after administration, proving the sensitivity of the testing protocol.

Hence, FAT 20297/C was considered to be non clastogenic in this micronucleus assay.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 March 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EEC Directive 79/831, Annex V, Part B, Paragraph 4.3.1

Deviations:

no

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Name: FAT 20297/C
Purity: 98 %

Species:

mouse

Strain:

other: OF-1 albino

Details on species / strain selection:

Recommended by the Guideline

Sex:

male/female

Details on test animals or test system and environmental conditions:

The experiment was performed using non-consanguinous albino mice originating from an SPF colony (IFFA-CREDO, L'Arbresle, France). Mice of both sexes (25 g) were used after a quarantine period of 2 weeks at Battelle; during the quarantine the animals were allowed ad libitum access to food (Aliment Rats-Souris Charles River, produced by U.A.R., Villemoisson, France) and drinking water. Animals are housed 5 of the same sex by cage in Makrolon type III cages.

Route of administration:

oral: gavage

Vehicle:

The test substance was dissolved in distilled water.

Duration of treatment / exposure:

1. Control (distilled water): 0.5 mL - sacrificed at 20 hours
2. Positive control (Thio-TEPA): 80 mg/kg bw - sacrificed at 20 hours
3. FAT 20297/C: 1500 mg/kg bw - sacrificed at 20 hours
4. FAT 20297/C: 1500 mg/kg bw - sacrificed at 44 hours
5. FAT 20297/C: 1500 mg/kg bw - sacrificed at 68 hours.

Frequency of treatment:

Once

Dose / conc.:

1 500 other: mg/kg bw

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

Thio-TEPA (N,N', N"-triethylenethiophosphoramide)
- Route of administration: oral
- Doses: 80 mg/kg bw

Tissues and cell types examined:

Bone marrow and micronucleated polychromatic erythrocytes

Details of tissue and slide preparation:

After sacrifice of the animals the femurs were taken and broken open at one end. Bone marrow cells were suspended in foetal calf serum using a small syringe and the cells were centrifuged at 120 x g for 5 minutes. The supernatant was removed with a Pasteur pipette, cells were spread on a microscope slide and the smears allowed to dry in air. The following day smears were stained with Giemsa (1:6 in water) and mounted with a coverslip after drying.

Evaluation criteria:

Statistically significant increase in the number of micronucleated polychromatic erythrocytes

Statistics:

using BMPD computer programme 7D

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

GENERAL APPEARANCE OF THE SMEARS:

At low magnification of the microscope no noticeable differences in bone marrow nucleated cells were observed between animals treated with FAT 20297/C and negative control.

In the positive control group (Thio-TEPA) decreased numbers of bone marrow nucleated cells were noted.

NUMBERS OF OBSERVED MICRONUCLEI AND RATIO OF POLYCHROMATIC TO NORMOCHROMATIC ERYTHROCYTES

There was no statistical difference or sex effect between animals exposed to FAT 20297/C and negative control animals. There was no increase in the number of micronucleated polychromatic erythrocytes in animals exposed to 1500 mg/kg bw of FAT 20297/C. In animals treated with Thio-TEPA there is a statistically significant increase number of micronucleated cells.

The ratio of polychromatic to normochromatic erythrocytes in both control and FAT 20297/C treated animals is the same. This ratio is decreased in mice treated with Thio-TEPA.

Conclusions:

FAT 20297/C was considered to be non-clastogenic in the micronucleus assay.

Executive summary:

FAT 20297/C was assayed for mutagenicity using the micronucleus test. This test was conducted in accordance with EEC Directive 79/831, Annex V, Part B, Paragraph 4.3.1.

The compound was administered orally to mice at a concentration of 1500 mg/kg body weight. No increase in the number of micronucleated polychromatic erythrocytes was observed in the bone marrow smears taken 20, 44 and 68 hours after administration of the test substance. A positive control (Thio-TEPA) administered at a concentration of 80 mg/kg body weight showed pronounced evidence of mutagenicity 20 hours after administration, proving the sensitivity of the testing protocol.

Hence, FAT 20297/C was considered to be non clastogenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The genetic toxicity database of Acid Blue 225 consists of a bacterial reverse mutation assay, an in vitro mammalian cell gene mutation assay and an in vitro mammalian cell chromomsomal aberration assay.

Bacterial reverse mutation assay

A study was performed to investigate the potential of FAT 21036/C to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. This test was conducted in accordance with OECD test guideline 471. 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: 61.7, 185.1, 555.5, 1666.6, 5000 µg/plate. A marginal increase in backmutants was reported with strain TA 102 with metabolic activation at 555.6 µg/plate only in the original experiment and at 1666.7 µg/plate only in the confirmatory experiment. This marginal effect seen was regarded as not significant as the increase was seen at two different concentrations during original and confirmatory experiments, without being seen at higher concentrations in these experiments. Thus, the effect seen cannot be considered as dose-dependent or concentration driven. No effects were observed with the other strains with or without metabolic activation in either experiments. Hence, based on the results of these experiments and on standard evaluation criteria, it is concluded that FAT 21036/C was not mutagenic in this bacterial reverse mutation assay.

In vitro mammalian chromosomal aberration assay

The clastogenic potential of the test item, FAT 21036/H, to induce chromosomal aberrations in mammalian cells was evaluated using cultured human peripheral blood lymphocytes. Human lymphocytes in whole blood culture, stimulated to divide by addition of Phytohaemagglutinin (PHA) 48 hours prior to treatment, were exposed to the test item in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver). The study consisted of a preliminary cytotoxicity test and a chromosomal aberration assay. Chromosomal aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 22-hour exposure. In a preliminary cytotoxicity test, blood cells were exposed to FAT 21036/H at the concentrations of 50, 100, 200, 400, 800 and 1600 µg/mL along with a DMSO control. At the end of the 3-hour exposure period, FAT 21036/H did not cause precipitation in the test media at any of the tested concentrations till 1600 µg/mL both, in presence and absence of metabolic activation, at the beginning and end of treatment. FAT 21036/H exhibited the required level of cytotoxicity (reduction in the mitotic index by 45±5 % of the concurrent vehicle control) at 200 µg/mL both in the presence and absence of metabolic activation with 3-hour exposure and absence of metabolic activation with 22-hour exposure. Test item showed 42 %, 44 % and 48 % cytotoxicity at 200 µg/mL in the presence and absence of metabolic activation at 3-hour exposure and absence of metabolic activation at 22-hour exposure respectively, compared to the concurrent vehicle control. Based on these observations, in the chromosomal aberration assay, a top concentration of 200 µg/mL was tested in the 3-hour exposure, in the presence and absence of metabolic activation and in the absence of metabolic activation with 22-hour exposure. In the chromosomal aberration assay, blood cultures were exposed to the test item in duplicate at the concentrations of  50, 100 and 200 mg/mL in Experiments 1 and 2 (presence and absence of metabolic activation with 3-hour test item exposure) and Experiment 3 (absence of metabolic activation with 22-hour test item exposure). In a similar way, concurrent vehicle control (DMSO) and the positive controls (cyclophosphamide monohydrate in the presence of metabolic activation in Experiment 1 and ethyl methanesulfonate in the absence of metabolic activation in Experiment 3) were tested in duplicate cultures. In each case, the cells in the C-metaphase were harvested at approximately 22 hours after the start of the treatment from the vehicle control, test item concentrations and the positive control groups. At the highest concentration tested, the reduction in mitotic index was 43, 45 and 49 % in Experiments 1, 2 and 3, respectively, compared to the vehicle control. A total of 300 metaphases from duplicate cultures from each of the controls and three treatment levels were evaluated for chromosomal aberrations. The data from the treatment groups and the positive controls were statistically compared with the vehicle control. There were no statistically significant increase in the incidence of structurally aberrant metaphases, either at 3 hours in the presence and absence of metabolic activation and 22 hours exposure, in the absence of metabolic activation in any of the tested concentrations. Under identical conditions, the respective positive control substances produced statistically significant (p <0.05) increase in aberrant metaphases confirming the sensitivity of the test system and the activity of the S9 mix. The study indicated that, under the conditions of this study, FAT 21036/H was not clastogenic in human peripheral blood lymphocytes up to the respective highest concentration of 200 µg/mL in the presence and absence of metabolic activation with 3-hour exposure and absence of metabolic activation with 22-hour exposure.

In vitro mammalian cell gene mutation assay:

The genotoxic potential of the test item FAT 21036/H to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells. The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver). FAT 21036/H was insoluble in sterile water and formed a free flowing suspension in DMSO at 160 mg/mL. In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 27 and 34 % at the highest tested concentration of 1600 µg/mL, in the presence and absence of metabolic activation, respectively. At the end of the three hours exposure period, there was no precipitation of the test item in the test medium at any of the tested concentrations, both in the presence and absence of metabolic activation. There was no appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 1600 µg/mL was tested in the gene mutation assay. 

In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 25, 100, 400 and 1600 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate. There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions. The results of the forward gene mutation test at the hprt locus with FAT 21036/H indicated that the test item was non-mutagenic under the conditions of this study.

Micronucleus assay with Acid Blue 344 (read-across):

The source substance, Acid Blue 344, was assayed for mutagenicity using the micronucleus test (EEC protocol). The compound was administered orally to mice at a concentration of 1500 mg/kg bw. No increase in the number of micronucleated polychromatic erythrocytes was observed in the bone marrow smears taken 20, 44 and 68 hours after administration of the test substance. A positive control (Thio-TEPA) administered at a concentration of 80 mg/kg bw showed pronounced evidence of mutagenicity 20 hours after administration, proving the sensitivity of the testing protocol. Hence, Acid Blue 344 was considered to be non clastogenic in this micronucleus assay.

Conclusion:

Acid Blue 225 did not cause mutations in the bacterial reverse mutation assay, as well as in the in vitro mutagenicity test and the in vitro chromosomal aberration test. In addition, data generated with a comparable substance, Acid Blue 344, demonstrated also to be not genotoxic in vivo. Therefore, Acid Blue 225 is considered to be not genotoxic in bacteria and mammalian cells.

Justification for classification or non-classification

Based on the above information, Acid Blue 225 was considered to be not genotoxic, and hence does not warrant classification according to the CLP (Regulation 1272/2008) criteria.