Sodium 1-amino-9,10-dihydro-4-[5-[(2-hydroxyethyl)sulphamoyl]-3,4-xylidino]-9,10-dioxoanthracene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was evaluated for genotoxic potential in the standard Ames/Salmonella gene mutation test and in the in vitro mammalian cell gene mutation test (HPRT-locus) and the in vitro cytogenecity chromosome aberration study. The test substance was positive in the Ames test but negative in the in vitro mammalian cell gene mutation test and in the in vitro mammalian chromosomal aberration test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name: FAT 20033/L
Common Name: TECTILON BLUE 4R CRUDE MOIST (LAB DRIED) Acid Blue 277
Batch No.: AT-PD13-359A1
CAS No.: 25797-81-3
Physical State / Colour: solid / dark blue
Storage Conditions: room temperature, protected from light
Active Components: >95 %
Analysation Date: 07 January 2014
Expiry Date: 07 January 2019
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment:
with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL

Experiment I:
without metabolic activation: 25, 50, and 100 µg/mL
with metabolic activation: 200, 400 and 500 µg/mL

Experiment II:
without metabolic activation: 25, 50, and 100 µg/mL
with metabolic activation: 75, 150 and 350 µg/mL

Vehicle / solvent:

- Vehicle (s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 5 mg/mL. DMSO was used as solvent (1 % DMSO). Different test item stock solutions were prepared and added to the samples. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation (400 and 900 µg/mL)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation (1.11 µg/mL)

Details on test system and experimental conditions:

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)

FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SEEDED: 1 x 10E4 - 5 x 10E4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture)
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell count

Evaluation criteria:

There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4 % aberrant cells without and 4.3 % with metabolic activation).

Statistics:

A statistical evaluation was used as an aid for interpretation of the results. Statistical significance at the 5 % level (p <0.05) was evaluated by The Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative/solvent control.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

According to the guidelines the highest recommended concentration was 5000 μg/mL. The test item was suspended in an appropriate solvent (DMSO) or in cell culture medium. Precipitation of the test item was noted at concentrations of 2500 μg/mL and higher. The highest dose group evaluated in the pre-experiment was 5000 μg/mL. The relative mitotic index and cell count were used as parameters for toxicity. The concentrations evaluated in the main experiment were chosen on the basis of the results from the pre-experiment. The mitotic index and/or cell count could not be determined at concentrations of 1000 μg/mL and higher due to the high toxicity of the test item.

 

	Dose Group	Concentration [µg/mL]	Relative Mitotic Index [%]	Relative Cell Count [%]	Mean % Aberrant Cells		Historical Laboratory Negative Control Range	Precipitation^a	Statistical Significance^b
					incl. Gaps	excl. Gaps
Experiment I 4 h treatment, 20 h preparation interval	C	0	111	116	8.0	4.0	0.0 % - 4.0 % aberrant cells	-	-
	S	0	100	100	2.0	0.5		-	-
	3	25	106	80	2.5	1.0		-	-
	4	50	101	74	2.5	1.0		-	-
	5	100	107	54	1.0	0.0		-	-
	EMS	900	94	87	12.5	8.5		-	+
Experiment II 20 h treatment, 20 h preparation interval	C	0	86	96	1.5	0.0	0.0 % - 4.0 % aberrant cells	-	-
	S	0	100	100	2.0	1.5		-	-
	4	25	100	86	5.0	1.5		-	-
	5	50	90	82	4.5	2.0		-	-
	6	100	121	73	2.0	0.0		-	-
	EMS	400	54	72	18.5	16.0		-	+

 

	Dose Group	Concentration [µg/mL]	Relative Mitotic Index  [%]	Relative Cell Count [%]	Mean % Aberrant Cells		Historical Laboratory Negative Control Range	Precipitation^a	Statistical Significance^b
					incl. Gaps	excl. Gaps
Experiment I 4 h treatment, 20 h preparation interval	C	0	91	108	2.5	1.0	0.0 % - 4.3 % aberrant cells	-	-
	S	0	100	100	7.0	3.0		-	-
	3	200	84	102	3.0	1.5		-	-
	5	400	75	68	2.0	1.5		-	-
	6	500	61	38	5.0	2.0		-	-
	CPA	1.1	85	94	10.0	7.0		-	-
Experiment II 4 h treatment, 20 h preparation interval	C	0	100	97	2.5	1.0	0.0 % - 4.3 % aberrant cells	-	-
	S	0	100	100	3.0	2.5		-	-
	1	75	94	81	5.0	2.5		-	-
	2	150	93	78	6.0	3.0		-	-
	4	350	88	46	7.0	3.5		-	-
	CPA	1.11	80	73	12.0	8.5		-	+

Conclusions:

FAT 20033/L is considered to be non-clastogenic in this chromosome aberration test.

Executive summary:

To investigate the potential of FAT 20033/L to induce structural chromosome aberrations in Chinese hamster V79 cells,an in vitro chromosome aberration assay was carried out according to OECD TG 473. The metaphases were prepared around 20 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation in experiment I. In experiment II, the treatment interval was around 20 h without and 4 h with metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations. DMSO was used as solvent (1 % DMSO). Different test item stock solutions were prepared and added to the samples. The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 25, 50 and 100 µg/mL

with metabolic activation: 200, 400 and 500 µg/mL

 

Experiment II:

without metabolic activation: 25, 50 and 100 µg/mL

with metabolic activation: 75, 150 and 350 µg/mL

 

No precipitation of the test item was observed without and with metabolic activation in all dose groups evaluated in experiment I and II. In experiment I (short-term treatment) without metabolic activation, cytotoxicity of the test item was determined at the highest concentration of 100 µg/mL considering the relative cell count. However, considering the relative mitotic index no cytotoxic effects were observed. With metabolic activation cytotoxic effects of the test item were determined at a concentration of 400 µg/mL and higher considering the relative cell count. However, considering the relative mitotic index cytotoxicity was observed at the highest concentration of 500 µg/mL. In experiment II (long-term treatment) without metabolic activation, cytotoxic effects of the test item were not observed up to a concentration of 100 µg/mL considering the relative mitotic index and cell count. Above this concentration of the test item a very high and steep toxicity was observed considering the relative mitotic index. With metabolic activation, no cytotoxic effects of the test item considering the relative mitotic index were observed up to the highest evaluated concentration. However, considering the relative cell count cytotoxicity was observed at the highest concentration of 350 µg/mL. In both experiments, no biologically relevant increase of the aberration rates was determined after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the negative / solvent controls. The distinct increase in the number of structural chromosome aberrations induced by the positive controls (EMS, CPA) clearly demonstrated the sensitivity of the test system. There was no evidence of chromosome aberration induced over background. Based on the above findings, it can be concluded that the test item FAT 20033/L did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, FAT 20033/L  is considered to be non-clastogenic in this chromosome aberration test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: FAT 20033/H, (Tectilon blau 4R-01) EN-No.: 672001.29
Aggregate State at RT: solid
Colour: blue
Purity: 72 % active ingredient
Stability: Pure: see expiration date
Storage: room temperature
Expiration Date: January 1994

Target gene:

Histidine gene

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the uvrB"-mutation. Additionally TA 102 contains the multicopy plasmid pAQ1, which carries the hisG428 mutation and a tetracycline resistance gene. TA 102 contains the ochre mutation in hisG gene.

Additional strain / cell type characteristics:

other: see above

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate of active ingredient.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity for the bacteria.

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(for TA 1535 and TA 100 without metabolic activation)

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

(for TA 1537 and TA 98 without metabolic activation)

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

(for TA 102 without metabolic activation)

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(for all strains with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and the pre-incubation test (experiment II)

For each strain and dose level, including the controls, a minimum of three plates were used.
Experiment 1: The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL: Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),
- 500 µL: S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
- 100 µL: Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL: Overlay agar

Experiment 2: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix /S9 mix substitution buffer and 100 µL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 h at 37 °C in the dark.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are: corresponding background growth on both negative control and test plates as well as normal range of spontaneous reversion rates. Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time. A test substance is considered as positive if either a dose related or reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced. A test substance producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. A significant response is described as follows: A test substance is considered as mutagenic if in strain TA 100 and TA 102 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test substance regardless of whether the highest dose induced the above-described enhancement factors or not.

Statistics:

no data

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(only with preincubation test)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(only with preincubation test)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(only with preincubation test)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(with both plate incorporation and preincubation test)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(with both plate incorporation and preincubation test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In experiment I toxic effects, evidenced by a reduction in the number of revertants, occurred in strain 1537 at 5000 µg/plate with metabolic activation. Additionally, the effects in the strains TA 98 and TA 100 at 2500 and 5000 µg/plate with metabolic activation are considered as toxic effects with regard to the results obtained in experiment II.

In experiment II toxic effects evidenced by the reduction of induced revertants were obtained in the strains TA 1537 (from 100 µg/plate), TA 98 (from 100 µg/plate), and TA 100 (from 1000 µg/plate) in the presence of metabolic activation. Additionally, toxicity evidenced by a reduction of the number of revertants was observed in TA 102 at 5000 µg/plate in the presence of S9 mix in experiment II.

The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

In experiment I a non-significant dose-dependent increase in revertant colony numbers was observed in the strains TA 98 and TA 100 in the presence of extrinsic metabolic activation up to 1000 µg/plate. The factor of 3.0 and 2.0, respectively which are recommended for a mutagenic response in the above-mentioned strains could not be reached. Additionally slight increases in revertant colony numbers were found in strain TA 102 in the presence of metabolic activation at 333.3, 1000, 2500 and 5000 µg/plate in experiment I. The results of experiment I indicate a possibly existing mutagenic effect in strain TA 98 and 100 in the presence of metabolic activation. Due to the higher sensitivity of the pre-incubation method (especially in the presence of S9 mix) in comparison to the plate incorporation method, the second experiment was carried out as a pre-incubation test.

In the pre-incubation test (experiment II) a significant dose dependent effect was observed in the strains TA 1537 and TA 98 up to 100 µg/plate and in the strain TA 100 up to 1000 µg/plate in the presence of metabolic activation. At higher concentrations the number of induced revertants decreased due to overlapping toxic effects of the test substance. As in experiment I slight increases in revertant colony numbers were observed in strain TA 102 at 333.3 and 1000 µg/plate in the presence of S9 mix. The results of both experiments indicate a mutagenic potential of the test substance in the strains TA 98 and TA 100. The results obtained in strain TA 1537 could not be reproduced in the independent experiment. However, the normally more sensitive preincubation method (especially in the presence of S9 mix) showed a significant dose-dependent increase of revertant colony numbers in this strain. The results obtained with strain TA 102 are considered not to be biologically relevant, since the factor of 2.0 which is recommended for a mutagenic response in this strain could not be reached.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies

Conclusions:

The test substance was considered to be mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance (of ca. 72 % purity) to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP. The assay was performed in two independent experiments both with and without liver microsomal activation. Experiment I was performed as a plate incorporation assay and experiment II as a pre-incubation assay using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each concentration, including the controls, was tested in triplicate at following concentrations; 33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate of active ingredient.

 

In Experiment I, toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1537 at 5000 µg/plate with metablolic activation. Additionally, the effects in the strains TA 98 and TA 100 at 2500.0 and 5000.0 µg/plate with metabolic activation are considered as toxic effects with regard to the results obtained in experiment II.

 

In Experiment II, toxic effects evidenced by the reduction of induced revertants were obtained in the strains TA 1537 (from 100 µg/plate), TA 98 (from 100 µg/plate) and TA 100 (from 1000 µg/plate) in the presence of metabolic activation. Additionally, toxicity evidenced by a reduction in the number of revertants was observed in TA 102 at 5000 µg/plate in the presence of S9 mix in experiment II.

 

The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. 

Significant dose-dependent increases were observed in Experiment II in the presence of S9 mix in the strains TA 1537, TA 98, and TA 100. 

Based on the findings of the study, the test substance was concluded to have induced point mutations by base pair changes and frameshifts in the genome of the strains TA 1537, TA 98 and TA 100. Therefore, the test substance was considered to be mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: FAT 20'033/I Tectilon Blau 4R-01 (Roh)
Batch No.: Partie 158 (1992)
Aggregate State at RT: solid
Purity: >97.3 %
Analysis : HPLC
Stability: Pure: stable until January 01, 1998
Storage: room temperature
Expiration Date: January 01, 1998

Target gene:

Histidine gene

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the uvrB"-mutation. Additionally TA 102 contains the multicopy plasmid pAQ1, which carries the hisG428 mutation and a tetracycline resistance gene. TA 102 contains the ochre mutation in hisG gene.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate of active ingredient.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(for TA 1535 and TA 100 without metabolic activation)

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

(for TA 1537 and TA 98 without metabolic activation)

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

(for TA 102 without metabolic activation)

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(for all strains with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and the pre-incubation test (experiment II)

For each strain and dose level, including the controls, a minimum of three plates were used.

Experiment 1: The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL: Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),
- 500 µL: S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
- 100 µL: Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL: Overlay agar

Experiment 2: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix /S9 mix substitution buffer and 100 µL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 h at 37 °C in the dark.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are: corresponding background growth on both negative control and test plates as well as normal range of spontaneous reversion rates. Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time. A test substance is considered as positive if either a dose related or reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced. A test substance producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. A significant response is described as follows: A test substance is considered as mutagenic if in strain TA 100 and TA 102 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test substance regardless of whether the highest dose induced the above-described enhancement factors or not.

Statistics:

no data

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(only with preincubation test)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(only with preincubation test)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(only with preincubation test)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(with both plate incorporation and preincubation test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(although positive results were obtained in plate incorporation test, it could not be reproduced in the normally more sensitive pre-incubation test)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In experiment I, toxic effects evidenced by a reduction of revertant colony numbers occurred in strain TA 1535 (with S9 mix) at 2500 and 5000 µg/plate, in strain TA 1537 (with and without S9), in strain TA 98 (with and without S9 mix) and in strain TA 100 (without S9 mix) at 5000 µg/plate. Additionally in experiment II in strain TA 98 at 2500 and 5000 µg/plate without metabolic activation. The induced revertant rate (mutagenic effect) decreased at higher concentrations in the strains TA 1537, TA 98 and TA 100 in the presence of metabolic activation, which is also an indication for a toxic effect. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. A clear dose-dependent increase in revertant colony numbers was obtained in the strains TA 1537 and TA 98 from 33.3 up to 100 µg/plate, as well as in strain TA 100 from 33.3 up to 2500 µg/plate in the presence of metabolic activation in the preincubation assay (experiment II.). At higher concentrations the number of induced revertant rates decreased due to overlapping toxic effects of the test substance at higher concentrations. In experiment I (plate incorporation assay) there was no tendency for a mutagenic response in the strains TA 1537 and TA 100 with metabolic activation, whereas in strain TA 98 in the presence of S9 mix a slight dose-dependent effect could be obtained from 33.3 up to 333.3 µg/plate. This effect may support the result obtained in experiment II in the strain TA 98 (with S9 mix). Nevertheless, the effects observed in experiment II are considered as biologically relevant since the pre-incubation assay is more sensitive than the plate-incorporation assay. Due to the selected test design (exp. I as a plate test; exp. II as a pre-incubation test) the results obtained in experiment II could not be reproduced in the strains TA 1537 and TA 100. In strain TA 102 in the presence of S9 mix a dose-dependent increase in revertant colony numbers was obtained in experiment I from 100 to 1000 µg/plate. This result is considered not to be biologically relevant since it could not be reproduced in the normally more sensitive pre-incubation test (experiment II). Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

Conclusions:

The test substance was considered to be mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance (of ca. >97.3 % purity) to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP. The assay was performed in two independent experiments both with and without liver microsomal activation. Experiment I was performed as a plate incorporation assay and Experiment II as a pre-incubation assay using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. Each concentration, including the controls, was tested in triplicate. The substance was tested up to 5000 µg/plate. Toxic effects evidenced by a reduction of spontaneously occurring revertant colony numbers and induced revertant colony numbers were observed in the strains TA 1535, TA 1537, TA 98, and TA 100 in Experiment I and II. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. A clear dose-dependent increase in revertant colony numbers was obtained in the strains TA 1537, TA 98, and TA 100 in the presence of metabolic activation in Experiment II. In conclusion, it can be stated that the test substance induced point mutations in strains TA 1537, TA 98 and TA 100. Therefore, the test substance was considered to be mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

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:

Test substance: FAT 20033/K [TECTILON B LAU 4R-01 FEUCHT (Laborgetrocknet)]
Batch No: 280-284
Purity: ca 72 %
Storage conditions: Room temperature
Expiry date: August 31, 1999
Material submitted by: CIBA-GEIGY Limited, Dyestuffs Division Basle, Switzerland.

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F10 medium supplemented with 10% pre-tested foetal calf serum, 100 U/mL penicillin and 100 µg/mL streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

Cytotoxicity test: 0.49 to 1000 µg/mL (for both with and without metabolic activation)
Mutagenicity test: 27.78 to 750 µg/mL (with metabolic activation); 4.63 to 125 µg/mL (without metabolic activation)
Confirmatory experiment: 22.22 to 600 µg/mL (with metabolic activation); 5.56 to 150 µg/mL (without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The highest concentration of the test substance was determined in a preliminary solubilisation test to be 100 mg/mL soluble in DMSO.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: N-Nitrosodimethylamine

Remarks:

(with metabolic activation)

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

(without metabolic activation)

Details on test system and experimental conditions:

Depending on the toxicity of the test substance 2.5-5.0x10E6 cells of passage 27 (original experiment) and passage 31 (confirmatory experiment) were plated in 30 mL growth medium into 175 sq cm flasks and incubated overnight. The growth medium was replaced for 5 h by 27 mL treatment medium and 3.0 mL S9 activation mixture or for 21 h by 30 mL treatment medium alone. In each assay, cultures were treated in duplicate with four test substance concentrations, a positive and a negative (DMSO) control. In the non-activated part of the experiment, the positive control was the ultimate mutagen Ethylmethansulphonate (EMS) at a concentration of 0.3 µl/ml. In the part with metabolic activation the positive control was the promutagen N-Nitrosodimethylamine (DMN) at a concentration of 1.0 µl/ml. The treatment was terminated by washing the cell layer extensively with PBS. After washing, the cells were suspended by trypsinisation, pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter, diluted with fresh growth medium and replated into flasks at 2E+6 cells. The cultures were incubated at 37 °C for seven to eight days during which the cells could recover and divide to express the mutant phenotype. The cultures were subcultered after the second or third day transferring 2E6 cells to a fresh flask to maintain exponential growth during the expression phase. In parallel cytotoxicity of the test substance was estimated from the cloning efficiency immediately after treatment. The counted cell suspension of each concentration level was further diluted so that 100 cells were seeded per 9.6 sq cm in 2.5 mL of growth medium and incubated at 37 °C. The number of colonies which developed within seven to eight days in these cultures reflected the viability at the end of the treatment (survival values). At the end of the expression period the cultures were trypsinised pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter. The cell suspension of each culture was diluted with fresh growth medium and an aliquot replated into four flasks (75 cm² growth area) each containing 2E6 cells for the mutant selection. The high-density cultures were subjected to the mutant selection procedure by supplementing the growth medium with 8 µg/mL 6-thioguanine (6-TG). Only cells mutated at the hprt locus could survive the 6-thioguanine treatment. The number of colonies formed in these flasks during the following days reflected the overall number of mutations induced by the treatment with the test substance or the mutagen (positive control). After seven to eight days incubation at 37 °C, the cultures were fixed and stained with Giemsa. The mutant clones were counted with the naked eye. In parallel the viability at the end of the expression period was estimated from the cloning efficiency. The remaining cell suspensions from the various expression cultures were further diluted such that 100 cells were seeded per 9.6 cm² in 2.5 mL of growth medium and were incubated at 37 °C. The number of colonies which developed within these low-density cultures reflected the viability at the end of the expression period (viability values).

Evaluation criteria:

All mutant frequencies are normalized to a virtual cloning efficiency of 100 % at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15 %, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the result. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures, will be calculated.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Cytotoxicity test:
A preliminary range finding test was run assessing cytotoxicity. The test substance was tested at concentrations up to 1000 µg/mL. Higher concentration could not be applied due to solubility limitations in the vehicle. In the part with metabolic activation, at the highest concentration of 1000 µg/mL an acute growth inhibiting effect of 97.47 % could be seen, while the next lower concentration inhibited 70.88 %. Without metabolic activation treatment with test substance proved growth inhibiting by more than 99 % down to the concentration of 250 µg/mL. The next lower concentration revealed an acute inhibition of growth of 91.51 %. Accordingly, 750 µg/mL with and 125 µg/mL without metabolic activation were chosen as highest concentrations for the first mutagenicity assay.

Mutagenicity test with metabolic activation:
The original experiment was performed at the following concentrations: 27.78, 83.33, 250 and 750 µg/mL. The mean growth inhibiting values found at the highest concentration after treatment and expression were 97.71 % and 9.62 % respectively. In the confirmatory experiment the concentrations applied were 22.22, 66.67, 200 and 600 µg/mL. The highest concentration revealed a mean acute growth inhibition of 91.34 %. After the expression period growth was inhibited by 6.7 %. N-Nitrosodimethylamine (DMN, 1.0 µL/mL) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

Mutagenicity test without metabolic activation:
The original experiment was performed at the following concentrations: 4.63, 13.89, 41.67 and 125 µg/mL. The highest concentration revealed a mean acute growth inhibition of 80.20 %. After the expression period no toxicity was noted. In the confirmatory experiment the concentrations applied were 5.56, 16.67, 50 and 150 µg/mL. The highest concentration revealed a mean acute growth inhibitory effect of 78.94 %. The mean growth inhibition after the expression period was 14.61 %. Ethylmethansulfonate (EMS, 0.3 µL/mL) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

Conclusions:

The test substance did not show mutagenic activity in Chinese hamster V79 cells.

Executive summary:

An in vitro mammalian cell gene mutation assay was conducted to determine the mutagenic potential of the test substance (of ca. 72 % purity) in Chinese Hamster cell line V79 according to OECD Guideline 476, EPA OTS 798.5300 and EU Method B.17 in compliance with GLP. The test substance was dissolved in DMSO. The cells were treated in the experiments with metabolic activation for 5 h and in the experiments without metabolic activation for 21 h. Based on the result from a preliminary range finding test which was run to assess cytotoxicity, 750 µg/mL with and 125 µg/mL without metabolic activation were chosen as highest concentrations for the first mutagenicity assay. In the original experiment with metabolic activation, the growth inhibition determined after treatment and expression at the highest concentration of 750 µg/mL were 97.71 and 9.62 %. In the original experiment in absence of metabolic activation, the mean growth inhibitory effect determined after treatment was 80.2 % at the highest concentration. After the expression period no cytotoxicity could be seen. Due to the pronounced growth inhibitory response in the part with metabolic activation, the concentration range was decreased in the confirmatory experiment to 22.22 - 600 µg/mL. In the confirmatory experiment with metabolic activation, the concentrations applied were 22.22, 66.67, 200.0 and 600.0 µg/ml. The highest concentration revealed a mean acute growth inhibition of 91.34 %. After the expression period growth was inhibited by 6.70 %. In the confirmatory experiment without metabolic activation, a concentration range of 5.56 to 150 µg/mL was selected in order to reach a more complete toxicity at the highest concentration. The highest concentration revealed a mean acute growth inhibitory effect of 78.94 %, while growth inhibition after the expression period was 14.61 %. No relevant increase in mutant frequency was observed at any concentration level of the test substance in the presence and absence of metabolic activation tested in the original or the confirmatory experiment. Hence, it was concluded that the test substance did not show mutagenic activity in Chinese hamster V79 cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay:

A bacterial reverse mutation assay with FAT 20033/H was performed in two independent experiments both with and without liver microsomal activation. Experiment I was performed as a plate incorporation assay and Experiment II as a pre-incubation assay using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. A clear dose-dependent increase in revertant colony numbers was obtained in the strains TA 1537, TA 98, and TA 100 in the presence of metabolic activation in Experiment II. In conclusion, it can be stated that the test substance induced point mutations in strains TA 1537, TA 98 and TA 100. Therefore, the test substance was considered to be mutagenic in the Salmonella typhimurium reverse mutation assay. In the bacterial reverse mutation assay performed with FAT 20033/I using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102, significant dose-dependent increases were observed in Experiment II in the presence of S9 mix in the strains TA 1537, TA 98, and TA 100. Based on the findings of the study, the test substance was concluded to have induced point mutations by base pair changes and frameshifts in the genome of the strains TA 1537, TA 98 and TA 100. Therefore, the test substance was considered to be mutagenic in the Salmonella typhimurium reverse mutation assay.

 

In vitro mammalian cell gene mutation assay:

In the HPRT assay with FAT 20033/K, V79 cells from chinese hamster cell line were treated in the experiments with metabolic activation for 5 h and in the experiments without metabolic activation for 21 h. No relevant increase in mutant frequency was observed at any concentration level of the test substance in the presence and absence of metabolic activation tested in the original or the confirmatory experiment. Hence, it was concluded that the test substance did not show mutagenic activity in Chinese hamster V79 cells.

 

In vitro mammalian chromosomal aberration assay:

To investigate the potential of FAT 20033/L to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out according to OECD TG 473. The metaphases were prepared around 20 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation in experiment I. In experiment II, the treatment interval was around 20 h without and 4 h with metabolic activation. In both experiments, no biologically relevant increase of the aberration rates was determined after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the negative / solvent controls. Based on above findings, it can be concluded that the test item FAT 20033/L did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, FAT 20033/L is considered to be non-clastogenic in this chromosome aberration test.

 

Conclusion:

Acid Blue 277 was found to have mutagenic potential in bacterial cells in the presence of metabolic activation only. However, this response could not be reproduced in the mammalian cells in vitro. Further, Acid Blue 277 was not clastogenic in the mammalian cell gene mutation assay in vitro. Based on above results, Acid Blue 277 can be considered to be neither mutagenic nor clastogenic to mammalian cells, and hence not genotoxic.

Justification for classification or non-classification

Based on the above stated assessment of the genotoxic potential of 1-amino-4-({3-[(2-hydroxyethyl)sulfamoyl]-4,5-dimethylphenyl}amino)-9,10- dioxo-9,10-dihydroanthracene-2-sulfonic acid, sodium salt, it is deemed not genotoxic and accordingly does not need to be classified according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament and of The Council) as implementation of UN-GHS in the EU.