Pentasodium 4-amino-6-[[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-sulphonatophenyl]azo]-3-[(2,5-disulphonatophenyl)azo]-5-hydroxynaphthalene-2,7-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Black 039 is considered to be not genotxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 01 October, 1992 to 05 November, 1992

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

Principles of method if other than guideline:

Guidelines followed

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

not specified

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

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Aqua bidest
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

no

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:

no

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:

no

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:

no

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. After pre-incubation 2 mL overlay agar 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 whether the highest dose induced the above described enhancement factors or not.

Statistics:

No data

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(occurred in strain TA 1537 without metabolic activation at the highest concentration in experiment I)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A slight toxic effect, evidenced by a reduction in the number of revertants, occurred in strain TA 1537 without metabolic activation at the highest concentration in experiment I. 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. No substantial increases in revertant colony numbers of any of the five tester strains were observed at any dose level, either in the presence or absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.

Remarks on result:

other: all strains/cell types tested

None

Conclusions:

The test substance was considered to be non-mutagenic in a Salmonella typhimurium reverse mutation assay.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance (of ca. 100 % 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 metabolic 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.

A slight toxic effect, evidenced by a reduction in the number of revertants, occurred in strain TA 1537 without metabolic activation at the highest concentration in Experiment I. 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. No substantial increases in revertant colony numbers of any of the five tester strains were observed at any dose level.

Under the study conditions, the test substance was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay. 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

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:

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

Deviations:

no

Principles of method if other than guideline:

Guildelines followed

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I
without S9 mix:
18 h: 100; 300; 1000 µg/ml
28 h: 1000 µg/ml

with S9 mix:
18 h: 300; 2500; 5000 µg/ml
28 h: 5000 µg/ml

Experiment II
without S9 mix:
18 h: 300; 1500; 2000 µg/ml
28 h: 300 µg/ml

with S9 mix:
18 h: 300; 2500; 5000 µg/ml
28 h: 2500 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Reasons for the Choice of the Cell Line V79
The V79 cell line has been used successfully for many years in in vitro experiments. Especially the high proliferation rate (doubling time of clone V79/T5 in stock cultures: 12 h, determined on March 17, 1994) and a high plating efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
Lacking metabolic activities of cells under in vitro conditions are a disadvantage of assays with cell cultures as many chemicals only develop mutagenic potential when they are metabolized by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with liver microsome preparations (S9 mix).

Cell Cultures
Large stocks of the V7 9 cell line (supplied by LMP, Technical University Darmstadt, D-64287 Darmstadt) were stored in liquid nitrogen in the cell bank of C C R allowing the repeated use of the same cell culture batch in experiments. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remained similar because of the reproducible characteristics of the cells. Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x 105 cells per flask were seeded in 15 ml of MEM (minimal essential medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; Boehringer Mannheim, D-68298 Mannheim). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in an atmosphere with 4.5 % carbon dioxide (95.5 % air).

Evaluation criteria:

A test article is classified as mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.
A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosomal aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

None

Conclusions:

FAT 45168/A is considered to be non-clastogenic in this chromosome aberration test.

Executive summary:

The test article FAT 45168/A was assessed for its potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments.

The chromosomes were prepared 18 h and 28 h after start of treatment with the test article, which was dissolved in culture medium. The treatment interval was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following concentrations were evaluated (18 h: 3 concentrations; 28 h: highest evaluable concentration):

Experiment I

without S9 mix: 18 h: 100; 300; 1000 µg/ml 28 h: 1000 µg/ml

with S9 mix: 18 h: 300; 2500; 5000 µg/ml 28 h: 5000 µg/ml

Experiment II

without S9 mix: 18 h: 300; 1500; 2000 µg/ml 28 h: 300 µg/ml

with S9 mix: 18 h: 300; 2500; 5000 ug/ml 28 h: 2500 µg/ml

The concentration range of the test article had been determined in a pre-test using the XTT-assay and a qualitative assessment of high density cultures as indicator for toxicity response. Toxic effects were observed at concentrations of 2500 and 5000 µg/ml without S9 mix (observation in high density cultures) whereas in the presence of S9 mix testing up to the maximum recommended concentration (5000 µg/ml) revealed no cytotoxic effects. In the cytogenetic experiments the test article was tested up to cytotoxic concentrations (without S9 mix) or up to the maximum recommended concentration (with S9 mix; 5000 µg/ml). In both independent experiments, there were no biologically relevant increases in cells with structural aberrations after treatment with the test article. In both experiments, no biologically relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the negative controls.

Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosomal aberrations.

CONCLUSION

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosomal aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, FAT 45168/A is considered to be non-clastogenic in this chromosome aberration test.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

For a detailed read across justification please refer to chapter 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (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

Principles of method if other than guideline:

Guildelines followed

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I
without S9 mix:
18 h: 100; 300; 1000 µg/ml
28 h: 1000 µg/ml

with S9 mix:
18 h: 300; 2500; 5000 µg/ml
28 h: 5000 µg/ml

Experiment II
without S9 mix:
18 h: 300; 1500; 2000 µg/ml
28 h: 300 µg/ml

with S9 mix:
18 h: 300; 2500; 5000 µg/ml
28 h: 2500 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Reasons for the Choice of the Cell Line V79
The V79 cell line has been used successfully for many years in in vitro experiments. Especially the high proliferation rate (doubling time of clone V79/T5 in stock cultures: 12 h, determined on March 17, 1994) and a high plating efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
Lacking metabolic activities of cells under in vitro conditions are a disadvantage of assays with cell cultures as many chemicals only develop mutagenic potential when they are metabolized by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with liver microsome preparations (S9 mix).

Cell Cultures
Large stocks of the V7 9 cell line (supplied by LMP, Technical University Darmstadt, D-64287 Darmstadt) were stored in liquid nitrogen in the cell bank of CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remained similar because of the reproducible characteristics of the cells. Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x 105 cells per flask were seeded in 15 ml of MEM (minimal essential medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; Boehringer Mannheim, D-68298 Mannheim). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in an atmosphere with 4.5 % carbon dioxide (95.5 % air).

Evaluation criteria:

A test article is classified as mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.
A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosomal aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

Conclusions:

Source chemical FAT 45168/A is considered to be non-clastogenic in this chromosome aberration test.

Executive summary:

Currently, no study evaluating potential of FAT 40171/Y to induce chromosomal aberration is available. However, source chemical FAT 45'168/A was assessed for its potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments.

The chromosomes were prepared 18 h and 28 h after start of treatment with the test article, which was dissolved in culture medium. The treatment interval was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following concentrations were evaluated (18 h: 3 concentrations; 28 h: highest evaluable concentration):

Experiment I

without S9 mix: 18 h: 100; 300; 1000 ug/ml 28 h: 1000 µg/ml

with S9 mix: 18 h: 300; 2500; 5000 ug/ml 28 h: 5000 µg/ml

Experiment II

without S9 mix: 18 h: 300; 1500; 2000 ug/ml 28 h: 300 µg/ml

with S9 mix: 18 h: 300; 2500; 5000 ug/ml 28 h: 2500 µg/ml

The concentration range of the test article had been determined in a pre-test using the XTT-assay and a qualitative assessment of high density cultures as indicator for toxicity response. Toxic effects were observed at concentrations of 2500 and 5000 µg/ml without S9 mix (observation in high density cultures) whereas in the presence of S9 mix testing up to the maximum recommended concentration (5000 µg/ml) revealed no cytotoxic effects. In the cytogenetic experiments the test article was tested up to cytotoxic concentrations (without S9 mix) or up to the maximum recommended concentration (with S9 mix; 5000 µg/ml). In both independent experiments, there were no biologically relevant increases in cells with structural aberrations after treatment with the test article. In both experiments, no biologically relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the negative controls.

Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosomal aberrations.

CONCLUSION

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosomal aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, FAT 45168/A is considered to be non-clastogenic in this chromosome aberration test.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-02-05 to 2014-03-13

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:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Principles of method if other than guideline:

Guidelines followed

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

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
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Experiment I
without and with metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL
Experiment II
without metabolic activation: 100, 250, 500, 750, 1000, 2000, 3000, 4000 and 5000 µg/mL
and with metabolic activation: 45, 90, 180, 375, 750, 1500, 3000, 4000 and 5000 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4 h treatment; MEM + 10 % FBS 20 h treatment)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; 300 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; 0.8 and 1.0 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Statistics:

None

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: ≥ 1000 μg/mL; experiment I with S9: ≥ 2500 μg/mL; Experiment II with S9:≥ 3000 μg/mL

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

None

Conclusions:

FAT 40171/Y TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In a mammalian cell gene mutation assay (HPRT locus), V79 cells cultured in vitro were exposed to FAT 40171/Y TE at concentrations of

- 5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL (with and without metabolic activation, Experiment I)

- 100, 250, 500, 750, 1000, 2000, 3000, 4000 and 5000 µg/mL (without metabolic activation, Experiment II)

- 45, 90, 180, 375, 750, 1500, 3000, 4000 and 5000 µg/mL (with metabolic activation, Experiment II).

FAT 40171/Y TE was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I with and without metabolic activation and in experiment II with metabolic activation. No biologically relevant growth inhibition was observed in experiment II without metabolic activation

In experiment I without metabolic activation the relative growth was 27.1 % for the highest concentration (5000 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 21.1 %.

In experiment II without metabolic activation the relative growth was 72.5 % for the highest concentration (5000 µg/mL) evaluated. In experiment II with metabolic activation the highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 34.9 %.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.37 was found at a concentration of 500 µg/mL with a relative growth of 76.2 %.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 2.04 was found at a concentration of 2500 µg/mL with a relative growth of 54.1 %. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 0.74 was found at a concentration of 1000 µg/mL with a relative growth of 77.5%. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 2.88 was found at a concentration of 4000 µg/mL with a relative growth of 52.4 %.

The positive controls did induce the appropriate response. 

There was no evidence of a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item FAT 40171/Y TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

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:

An in vitro study key study was performed to investigate the potential of the test substance (of ca. 100 % 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 metabolic 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. Under the study conditions, the test substance was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay. 

 

Mammalian cell gene mutation assay (in vitro)

A second key study was conducted according to OECD guideline 476 and in accordance with GLP. In a mammalian cell gene mutation assay (HPRT locus), V79 cells cultured in vitro were exposed to FAT 40171/Y TE dissolved in culture medium at various concentrations. In the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item FAT 40171/Y TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

In vitro mammalian chromosomal aberration assay with source chemical

A third key study was conducted with source chemical FAT45168/A according to OECD guideline no. 473 and in accordance with GLP. The test article FAT 45168/A was assessed for its potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. FAT 45'168/A is considered to be non-mutagenic in this chromosome aberration test.

Results of these three studies indicate that reactive Black 039 should be considered as not genotoxic.

Justification for classification or non-classification

Reactive Black 039 is considered to be not genotoxic, hence it does not warrant classification according to Regulation (EC) No. 1272/2008 (CLP) criteria.