Reaction mass of Disodium [3-{[4-chloro-2-(hydroxy-kO)-5-methoxyphenyl]diazenyl-kN2}-4-(hydroxy-kO)naphthalene-2,7-disulfonato(4-)](hydroxy)chromate(2-) and Trisodium [3-{[4-chloro-2-(hydroxy-kO)-5-methoxyphenyl]diazenyl-kN2}-4-(hydroxy-kO)naphthalene-2,7-disulfonato(4-)](dihydroxy)chromate(3-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Acid Blue 156 lead to increase in back mutants for Salmonella typhimurium TA98 strain in absence of metabolic activation at the highest concentration at 5000 µg/plate only. This increase (51 back mutants) was very close to the acceptable range (20-50) for negative controls as established for the historical control data. Hence, this finding considered to be not a definite indication of the mutation potential of Acid Blue 156. The mutation potential was further assessed in the Chinese hamster ovary cells in the HPRT assay, where Acid Blue 156 failed to lead to increased mutations. It was further found to be non-clastogenic in an in vitro chromosomal aberration assay. Hence based on the overall weight of evidence, Acid Blue 156 is considered to be not genotoxic.

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:

20 April 1994 to 12 August 1994

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:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test material: FAT 20044/B (Neolan Blau 3R roh trocken SFO)
Batch No.: 96
Purity: about 80 %
Stability: July 1999
Appearance: Blue mass
Expiry date: July 1999
Storage: Room temperature

Species / strain / cell type:

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

Metabolic activation system:

With and without metabolic activation

Test concentrations with justification for top dose:

Range finding test: ranging from 20.6 to 5000 µg/plate
From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate with and without metabolic activation.
Main study: 61.7 to 5000 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with metabolic activation: TA 98, TA100, TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation: TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation: TA100, TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation: TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation: TA 1537

Details on test system and experimental conditions:

The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 1535, TA 1537) were obtained from Prof. B. Ames, Berkeley, USA. Strain TA 100 was obtained from Dr. M. Schüpbach, Hoffmann-La Roche Limited, Basel, Switzerland.

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:

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

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Four salmonella strains were used

Range finding test

Six concentrations of FAT 20044/B (Neolan Blau 3R roh trocken SFO) ranging from 20.6 to 5000.0 ug/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. 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, original experiment

In the experiment performed without metabolic activation, treatment of strain TA 98 with FAT 20044/B (Neolan Blau 3R roh trocken SFO) led to a slight increase in the number of histidineprototrophic mutants at the concentration of 5000 µg/plate. No effect was seen in the experiment with activation conducted on this strain and in the experiments with and without activation conducted on strains TA 100, TA 1535 and TA 1537.

 

Mutagenicity test, confirmatory experiment

In the experiment performed without metabolic activation, again, treatment of strain TA 98 with FAT 20044/B (Neolan Blau 3R roh trocken SFO) led to a slight increase in the number of revertant counts at the concentration of 5000.0 µg/plate. No effects occurred in the experiment with activation conducted on this strain and in the experiments with and without activation conducted on strains TA 100, TA 1535 and TA 1537. 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 concentration. Therefore, the test substance exerted no toxic effect on the growth of the bacteria. There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the test data.

 

SUMMARY OF THE MUTAGENICITY EXPERIMENTS: ORIGINAL EXPERIMENT with metabolic activation

 

Strain	Treatment	Mean Counts	Strain	Treatment	Mean Counts
TA 100	Negative control	121.67	TA 1535	Negative control	11.33
	61.73 µg/plate	145.00		61.73 µg/plate	16.67
	185.19 µg/plate	143.67		185.19 µg/plate	8.33
	555.56 µg/plate	147.00		555.56 µg/plate	12.67
	1666.67 µg/plate	147.00		1666.67 µg/plate	10.67
	5000.00 µg/plate	158.33		5000.00 µg/plate	11.67
	Positive Control	1893.67		Positive Control	358.00
			TA 98	Negative control	27.67
				61.73 µg/plate	33.33
				185.19 µg/plate	32.0
				555.56 µg/plate	35.33
				1666.67 µg/plate	38.67
				5000.00 µg/plate	39.33
				Positive Control	1972.33
TA1537	Negative control	7.67
	61.73 µg/plate	5.33
	185.19 µg/plate	7.33
	555.56 µg/plate	9.67
	1666.67 µg/plate	9.33
	5000.00 µg/plate	9.33
	Positive Control	143.33

 

 

SUMMARY OF THE MUTAGENICITY EXPERIMENTS: ORIGINAL EXPERIMENT without metabolic activation

 

Strain	Treatment	Mean Counts	Strain	Treatment	Mean Counts
TA 100	Negative control	126.33	TA 1535	Negative control	14.00
	61.73 µg/plate	126.67		61.73 µg/plate	8.33
	185.19 µg/plate	136.67		185.19 µg/plate	12.00
	555.56 µg/plate	121.67		555.56 µg/plate	12.00
	1666.67 µg/plate	142.00		1666.67 µg/plate	11.33
	5000.00 µg/plate	158.67		5000.00 µg/plate	10.00
	Positive Control	1292.67		Positive Control	1015.00
			TA 98	Negative control	16.00
				61.73 µg/plate	17.33
				185.19 µg/plate	16.00
				555.56 µg/plate	21.33
				1666.67 µg/plate	29.33
				5000.00 µg/plate	51.00
				Positive Control	1750.33
TA1537	Negative control	9.67
	61.73 µg/plate	5.00
	185.19 µg/plate	8.00
	555.56 µg/plate	9.33
	1666.67 µg/plate	12.00
	5000.00 µg/plate	11.00
	Positive Control	2050.33

 

 

SUMMARY OF THE MUTAGENICITY EXPERIMENTS: CONFIRMATORY EXPERIMENT with metabolic activation

 

Strain	Treatment	Mean Counts	Strain	Treatment	Mean Counts
TA 100	Negative control	115.67	TA 1535	Negative control	8.00
	61.73 µg/plate	121.00		61.73 µg/plate	10.33
	185.19 µg/plate	111.67		185.19 µg/plate	10.33
	555.56 µg/plate	128.33		555.56 µg/plate	10.67
	1666.67 µg/plate	138.67		1666.67 µg/plate	10.67
	5000.00 µg/plate	158.67		5000.00 µg/plate	9.00
	Positive Control	1758.00		Positive Control	357.00
			TA 98	Negative control	33.00
				61.73 µg/plate	30.00
				185.19 µg/plate	39.33
				555.56 µg/plate	36.33
				1666.67 µg/plate	34.00
				5000.00 µg/plate	41.00
				Positive Control	1743.33
TA1537	Negative control	9.33
	61.73 µg/plate	5.33
	185.19 µg/plate	7.00
	555.56 µg/plate	10.33
	1666.67 µg/plate	8.67
	5000.00 µg/plate	16.33
	Positive Control	176.33

 

 

SUMMARY OF THE MUTAGENICITY EXPERIMENTS: CONFIRMATORY EXPERIMENT without metabolic activation

 

Strain	Treatment	Mean Counts	Strain	Treatment	Mean Counts
TA 100	Negative control	120.33	TA 1535	Negative control	8.67
	61.73 µg/plate	108.33		61.73 µg/plate	11.33
	185.19 µg/plate	113.00		185.19 µg/plate	11.33
	555.56 µg/plate	124.00		555.56 µg/plate	9.33
	1666.67 µg/plate	139.00		1666.67 µg/plate	9.67
	5000.00 µg/plate	172.00		5000.00 µg/plate	11.67
	Positive Control	1260.33		Positive Control	1137.00
			TA 98	Negative control	18.33
				61.73 µg/plate	18.67
				185.19 µg/plate	15.67
				555.56 µg/plate	16.00
				1666.67 µg/plate	24.67
				5000.00 µg/plate	51.67
				Positive Control	1792.67
TA1537	Negative control	8.33
	61.73 µg/plate	9.33
	185.19 µg/plate	7.00
	555.56 µg/plate	9.33
	1666.67 µg/plate	12.67
	5000.00 µg/plate	13.67
	Positive Control	2029.67

 

 

Conclusions:

In the experiments performed without metabolic activation, treatment of strain TA 98 with FAT 20044/B led to a slight increase in the number of back-mutant colonies at the highest concentration only. No effect occurred in the experiments with activation conducted on this strain and on the other strains.

Executive summary:

In a GLP-compliant study, FAT 20044/B, was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium according to OECD guideline 471. The strains of Salmonella typhimurium used were 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 dissolved in bi-distilled water and tested at five concentrations in the range of 61.7 to 5000.0 µg/plate. In order to confirm the results, the experiments were repeated with and without metabolic activation in the same concentration range. 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 the experiments performed without metabolic activation, treatment of strain TA 98 with FAT 20044/B led to a slight increase in the number of back-mutant colonies at the highest concentration only. No effect occurred in the experiments with activation conducted on this strain and on the other strains.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 December 2020 to 04 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Physical Appearance: Solid
Purity by HPLC (Area %): 70.5 %
Batch No.: SI/May 2020/CR/001 (India)
Manufactured Date: 08 June 2020
Expiry Date: 07 June 2025

Target gene:

HPBL

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human Peripheral Blood Lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver metabolic activation system

Test concentrations with justification for top dose:

28, 83, and 250 µg/mL

Vehicle / solvent:

Sterile water

Untreated negative controls:

yes

Remarks:

Sterile water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Presence of metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

In absence of Metabolic Activation

Details on test system and experimental conditions:

Whole blood cultures incubated in a tissue culture rotator inside a CO2 incubator with humidified atmosphere of 84 to 85 %, 5 ± 0.2 % CO2 at 37 ± 1 °C for 48 hours were used.

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:

1. A test chemical is considered to be clearly positive and considered to induce chromosomal aberrations 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 and considered unable to induce chromosomal aberration 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. Additional experimental work may be required by the Study Director to clarify such results. In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.
4. An increase in the number of polyploid cells may indicate that the test substances have the potential to inhibit mitotic processes and incidence of polyploid cells and cells with endoreduplicated chromosomes should be recorded separately.

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:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tested up to highest concentration exhibiting 50 % cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: All strains have been used

Preliminary Solubility Test

Test item was soluble in SW at 200 mg/mL.

 

Preliminary Cytotoxicity Test and Justification for the Selection of Test

Concentrations

At the end of the 3-hour exposure period, FAT 20044/C did not cause precipitation in the test solution at any of the test doses, both in the presence and absence of metabolic activation. 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 7.20 and 7.25 with a pH value of 7.24 in the SW control. In the absence of metabolic activation, the pH ranged between 7.21 and 7.24 with a pH value of 7.24 in the SW control. At the end of 3-hour exposure, osmolality of the test medium at the highest test item treatment condition (2000 μg/mL) was 0.292 and 0.304 OSMOL/kg in the presence and absence of metabolic activation, respectively. The corresponding osmolality in the SW control was 0.294 and 0.289 OSMOL/kg in the presence and absence of metabolic activation, respectively. FAT 20044/C exhibited the required level of cytotoxicity (reduction in the mitotic index by 45±5 % of the concurrent vehicle control) at 250 μg/mL both in the presence and absence of metabolic activation with 3-hour and 22-hour exposure. Based on these observations, in the chromosomal aberration assay, a top concentration of 250 μg/mL was tested in the 3-hour and 22-hour exposure, in the presence and absence of metabolic activation. Test item showed, 54 and 54 % at 500 μg/mL toxicity, 62 and 63 % toxicity at 1000 μg/mL and 71 % and 68 % toxicity at 2000 μg/mL, respectively, in the presence and absence of metabolic activation at 3-hour exposure, compared to the concurrent vehicle control. Test item exhibited 56, 62 and 75 % toxicity at 500, 1000 and 2000 μg/mL, respectively, at 22-hour exposure, compared to the vehicle control.

 

Chromosomal Aberration Assay

Experiment 1

At the highest concentration tested (250 μg/mL), the mitotic inhibition was 47 % 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 (250 μg/mL), the mitotic inhibition was 47% 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 (250 μg/mL), the mitotic inhibition was 47 % 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.

Conclusions:

FAT 20044/C is not considered as clastogenic under the conditions of testing employed.

Executive summary:

In a GLP-compliant study, the clastogenic potential of the test item, FAT 20044/C to induce chromosomal aberrations in mammalian cells was evaluated using cultured human peripheral blood lymphocytes was carried out according to OECD guideline 473. Human lymphocytes in whole blood culture, stimulated to divide by addition of Phytohaemagglutinin (PHA) approximately 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. The test item was soluble in Sterile water (SW) at 200 mg/mL. SW is one of the organic vehicles compatible with this test system.In a preliminary cytotoxicity test, blood cells were exposed to FAT 20044/C at the concentrations of 62.5, 125, 250, 500, 1000 and 2000 µg/mL along with a Sterile Water (SW) control. At the end of the 3-hour exposure period, FAT 20044/C did not cause precipitation in the test solution at any of the test doses, both in the presence and absence of metabolic activation. FAT 20044/C TE exhibited the required level of cytotoxicity (reduction in the mitotic index by 45±5 % of the concurrent vehicle control) at 250µg/mL both in the presence and absence of metabolic activation with 3-hour and 22-hour exposure. Test item showed, 54 and 54 % at 500 µg/mL toxicity, 62 and 63 % toxicity at 1000 µg/mL and 71 % and 68 % toxicity at 2000 µg/mL, respectively, in the presence and absence of metabolic activation at 3-hour exposure, compared to the concurrent vehicle control. Test item exhibited 56, 62 and 75 % toxicity at 500, 1000 and 2000 µg/mL, respectively, at 22-hour exposure, compared to the vehicle control. Based on these observations, in the chromosomal aberration assay, a top concentration of 250 µg/mL was tested in the 3-hour and 22-hour exposure, in the presence and absence of metabolic activation, respectively. In the chromosomal aberration assay, blood cultures were exposed to the test item induplicate at the concentrations of 28, 83 and 250mg/mL in Experiments 1 & 2 (presence and absence of metabolic activation with 3-hour test item exposure) and in Experiment 3 (absence of metabolic activation with 22-hour test item exposure). In a similar way, concurrent vehicle control (SW) and the positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation) 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 47, 47 and 47 % 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) increases in aberrant metaphases. The study indicated that, under the conditions of this study, FAT 20044/C was not clastogenic in human peripheral blood lymphocytes up to the respective highest concentration of 250 µg/mL in the presence and absence of S9 with 3-hour exposure and in the absence of S9 with 22-hour exposure. In conclusion, FAT 20044/C is not considered as clastogenic under the conditions of testing employed.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

hprt

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 December 2020 to 23 April 2021

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

Purity: 70.5 % (w/w)
Batch No.: SI/May 2020/CR/001 (India)
Manufactured Date: 08 June 2020
Expiry Date: 07 June 2025

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9. The S9 homogenate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 (0.7 mL/rat ready to use solution), 5 days prior to sacrifice.

Test concentrations with justification for top dose:

7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL

Vehicle / solvent:

Sterile Water (SW)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

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 mutation involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonst rated 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

Remarks on result:

other: All strains used

Preliminary Solubility Test

FAT 20044/C formed a dark blue colored solution in sterile water at 200 mg/mL. Sterile water is one of the vehicles compatible with this test system. Hence, based on the results of solubility test, sterile water is 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 7.04 to 7.19 with 7.01 in the sterile water control while in the absence of metabolic activation it was between 7.09 and 7.23 with 7.06 in the sterile water control. At the end of the 3-hour exposure period, the osmolality of the test medium, at the highest test item concentration level (2000 μg/mL) was 0.401 and 0.380 OSMOL/kg in the presence and absence of metabolic activation, respectively.

The corresponding osmolality in the sterile water was 0.381 and 0.366 OSMOL/kg in the presence and absence of metabolic activation, respectively. The Relative Survival at 2000 μg/mL was 28 and 33 %, in the presence and absence of metabolic activation, respectively. The test item did not show evidence of significant cell growth inhibition as Relative Cloning Efficiency (10 to 20 % RCE 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 2000 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 31 to 80 % while in the absence of metabolic activation, ranged from 37 to 83 % 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. 

Conclusions:

FAT 20044/C does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations.

Executive summary:

In a GLP-compliant study, the genotoxic potential of FAT 20044/C to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells was assessed according to OECD guideline 476. 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 20044/C was soluble in sterile water at 200 mg/mL. In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 28 and 33 % at the highest tested concentration of 2000 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 2000 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 16, 80, 400 and 2000 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 20044/C 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

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In-vitro Bacterial Reverse Mutation Assay:

In a GLP-compliant study, FAT 20044/B, was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium according to OECD guideline 471. The strains of Salmonella typhimurium used were 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 dissolved in bi-distilled water and tested at five concentrations in the range of 61.7 to 5000 µg/plate. In order to confirm the results, the experiments were repeated with and without metabolic activation in the same concentration range. 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 the experiments performed without metabolic activation, treatment of strain TA 98 with FAT 20044/B led to a slight increase in the number of back-mutant colonies at the highest concentration only. No effect occurred in the experiments with activation conducted on this strain and on the other strains.

 

In-vitro chromosomal aberration test:

In the chromosomal aberration assay, blood cultures were exposed to the test item induplicate at the concentrations of 28, 83 and 250 mg/mL in Experiments 1 & 2 (presence and absence of metabolic activation with 3-hour test item exposure) and in Experiment 3 (absence of metabolic activation with 22-hour test item exposure). At the highest concentration tested, the reduction in mitotic index was 47, 47 and 47 % 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) increases in aberrant metaphases. The study indicated that, under the conditions of this study, FAT 20044/C  was not clastogenic in human peripheral blood lymphocytes up to the respective highest concentration of 250 µg/mL in the presence and absence of S9 with 3-hour exposure and in the absence of S9 with 22-hour exposure. In conclusion, FAT 2004/C is not considered as clastogenic under the conditions of testing employed.

 

In-vitro gene mutation in mammalian cells- hprt:

In a GL-P compliant study, the genotoxic potential of FAT 20044/C to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells was assessed according to OECD guideline 476. In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 28 and 33 % at the highest tested concentration of 2000 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 2000 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 16, 80, 400 and 2000 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 20044/C indicated that the test item was non-mutagenic under the conditions of this study.

 

Conclusion on genotoxic potential of Acid Blue 156:

Acid Blue 156 lead to increase in back mutants for Salmonella typhimurium TA98 strain in absence of metabolic activation at the highest concentration at 5000 µg/plate only. This increase (51 back mutants) was very close to the acceptable range (20 -50) for negative controls as established for the historical control data. Hence, this finding can be considered to be not a definite indication of the mutation potential of Acid BLue 156. The mutation potential was further assessed in the Chinese hamster ovary cells in the HPRT assay, where Acid Blue 156 failed to lead to increased mutations. It was further found to be non-clastogenic in an in vitro chromosomal aberration assay. Hence, based on the overall weight of evidence, Acid Blue 156 is considered to be not genotoxic.

Justification for classification or non-classification

Based on the findings of the genetic toxicity studies, the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.