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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Blue 72 was assessed for mutagenic potential in a bacterial reverse mutation assay and anin vitromammalian cell gene mutation assay (HPRT). However, currently no study for assessing clastogenic potential of it is available. Hence, a study with structurally similar substance, FAT 40336, is used to fulfil the data requirement.

Bacterial reverse mutation assay

FAT 40045/D (of 74.36 % purity) was assessed for the potential to induce gene mutations according to OECD Guideline 471, in two independent experiments both with and without metabolic activation, usingSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98 and TA 100. Each concentration, including the controls, was tested in triplicate at up to 5000 µg/plate. No relevant increases in revertant colony numbers of any of the four tester strains were observed at any dose level, either in the presence or absence of metabolic activation. Hence, the test substance was not considered to be mutagenic inSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, and TA 100.

In vitromammalian cell gene mutation assay

FAT 40045/Z TE was assessed for mutagenic action in mammalian cells in a mammalian cell gene mutation assay (HPRT locus) conducted as two independent experiments, using V79 cells cultured in vitro with and without metabolic activation. In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed. Hence, FAT 40045/Z TE is considered to be not mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

In vitro mammalian chromosomal aberration assay

In an in vitro chromosomal aberration assay with and without metabolic activation conducted with read-across substance FAT 40336, no relevant increase in cells with structural aberrations was seen. Therefore, FAT 40'336/B is considered to be not clastogenic in this chromosomal aberration test.

Conclusion:

Reactive Blue 72 was not mutagenic in bacterial as well as mammalian cells. Though no study assessing its clastogenic potential is available, therefore, read across to substance FAT 40336 was performed which demonstrated to be not clastogenic in an in vitro mammalian chromosomal aberration assay.
Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-02-05 to 2014-03-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
Pre-experiment for experiment I (with and without metabolic activation):
5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
5, 10, 25, 50, 100, 200, 350, 500, 750, 1000 µg/mL
Experiment I
without metabolic activation: 25, 50, 75, 100, 125, 150, 200, 225 and 250 µg/mL
and with metabolic activation: 5, 10, 25, 50, 100, 250, 500 and 1000 µg/mL

Experiment II
without metabolic activation: 10, 20, 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL
and with metabolic activation: 70, 150, 300, 400, 500, 600, 700, 800 and 900 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:
yes
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:
yes
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.
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: ≥ 50 μg/mL; experiment I with S9: ≥ 500 μg/mL; Experiment II without S9: ≥ 250 μg/mL; Experiment II with S9:≥ 400 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Precipitation:

No precipitation of the test item was noted in any of the experiments.

Toxicity:

A biologically relevant growth inhibition (reduction of relative growth below 70%) was observed after the treatment with the test item in experiment I and II with and without metabolic activation.

In experiment I without metabolic activation the relative growth was 11.5% for the highest concentration (250 μg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 1000 μg/mL with a relative growth of 10.6%.

In experiment II without metabolic activation the relative growth was 12.6% for the highest concentration (1500 μg/mL) evaluated. The highest concentration evaluated with metabolic activation was 900 μg/mL with a relative growth of 11.5%.

Mutagenicity:

In experiment I without metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 18.35 and 20.81 mutants/106 cells and in the range of 4.71 to 29.49 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.51 was found at a concentration of 225 μg/mL with a relative growth of 11.1%.

With metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 16.30 and 11.84 mutants/106 cells and in the range of 6.29 to 18.71 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.33 was found at a concentration of 10 μg/mL with a relative growth of 113.8%.

In experiment II without metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 26.82 and 29.28 mutants/106 cells and in the range of 12.74 to 40.26 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.44 was found at a concentration of 50 μg/mL with a relative growth of 90.4%.

In experiment II with metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 23.10 and 13.15 mutants/106 cells and in the range of 13.89 to 42.28 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.33 was found at a concentration of 300 μg/mL with a relative growth of 77.4%.

DMBA (0.8 and 1.0 μg/mL) and EMS (300 μg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Conclusions:
FAT 40045/Z TE is considered to be not mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

In this mammalian cell gene mutation assay (HPRT locus), V79 cells cultured in vitro were exposed to FAT 40045/Z TE at concentrations of

- 25, 50, 75, 100, 125, 150, 200, 225 and 250 µg/mL (without metabolic activation, Experiment I)

- 5, 10, 25, 50, 100, 250, 500 and 1000 µg/mL (with metabolic activation, Experiment I)

- 10, 20, 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL (without metabolic activation, Experiment II)

- 70, 150, 300, 400, 500, 600, 700, 800 and 900 µg/mL (with metabolic activation, Experiment II).

FAT 40045/Z TE was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 11.5 % for the highest concentration (250 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 1000 µg/mL with a relative growth of 10.6 %. In experiment II without metabolic activation the relative growth was 12.6 % for the highest concentration (1500 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 900 µg/mL with a relative growth of 11.5 %.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.51 was found at a concentration of 225 µg/mL with a relative growth of 11.1 %.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.33 was found at a concentration of 10 µg/mL with a relative growth of 113.8 %. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 1.44 was found at a concentration of 50 µg/mL with a relative growth of 90.4 %. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 2.33 was found at concentrations of 300 µg/mL with a relative growth of 77.4 %.

The positive controls did induce the appropriate response. 

There was no evidence of a concentration related positive response of induced mutant colonies over background. Hence FAT 40045/Z TE is considered to be not mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer chapter 13 for detailed read across justification.
Reason / purpose:
read-across source
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
Conclusions:
The test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, FAT 40'336/B (and consequently Reactive Blue 72) is considered to be non-mutagenic in this chromosomal aberration test.
Executive summary:

Currently no study for assessment of clastogenic potential of Reactive blue 72 is available. However, a structurally similar substance, FAT 40'336/B was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro. Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following dose levels were evaluated:

The following dose levels were evaluated:

without S9 mix:

7 h : 100.0 µg/ml

18 h : 5.0; 25.0; 50.0 µg/ml

28 h: 50.0 µg/ml

with S9 mix:

7 h: 100.0 µg/ml

18 h: 1.0; 5.0; 25.0 µg/ml

28 h: 100.0 µg/ml

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response. Treatment with 300.0 and 1000.0 µg/ml clearly reduced the plating efficiency of the V79 cells. However, at these concentrations the test article precipitated during incubation time. Therefore, lower dose levels had to be chosen in the main experiment. With these concentrations the mitotic index was reduced only at fixation intervals 7 and 28 hours in the presence of S9 mix. There was no relevant increase in cells with structural aberrations after treatment with the test article at each fixation interval either without or with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, FAT 40'336/B (and consequently Reactive Blue 72) is considered to be non-mutagenic in this chromosomal aberration test.

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:
18.10.1995 - 10.01.1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only four strains were tested) no tester strain to detect crosslinking mutagens was included
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene

Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. Regular checking of the properties of the strains with regard to membrane permeability, ampicillin and tetracycline-resistance as well as spontaneous mutation rates is performed in the laboratory of CCR according to Ames et al. In this way, it was ensured that the experimental conditions set down by Ames were fulfilled.
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:
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 substances 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 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 the number of reversions is at least twice the spontaneous rate in strain TA 100 and thrice on TA 1535, TA 1537, and TA 98. 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 criteria described above or not.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(in strains TA 1535 and TA98)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxic effects evident as a reduction in the number of revertants occurred in experiment I in strain TA 1535 at 5000 µg/plate without metabolic activation and at 2500 µg/plate and above with metabolic activation. Strain TA 98 showed these toxic effects at 5000 µg/plate with and without metabolic activation. The plates incubated with the test substance showed reduced background growth at 5000 µg/plate in experiment I and at 1000 µg/plate and above in experiment II. No relevant increases in revertant colony numbers of any of the four tester strains were observed following treatment with the test substance at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Conclusions:
The test substance was not 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 74.36 % purity) to induce gene mutations in bacterial cells according to OECD Guideline 471 with deviations (i.e., only four tester strains were tested), in compliance with GLP. The assay was carried 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 and TA 100. Each concentration, including the controls, was tested in triplicate at up to 5000 µg/plate. Toxic effects evident as a reduction in the number of revertants occurred in Experiment I in strain TA 1535 at 5000 µg/plate without metabolic activation and at 2500 µg/plate and above with metabolic activation. Strain TA 98 showed toxic effects at 5000 µg/plate with and without metabolic activation. The plates incubated with the test substance showed reduced background growth at 5000 µg/plate in Experiment I and at 1000 µg/plate and above in Experiment II. No relevant increases in revertant colony numbers of any of the four tester strains were observed at any dose level, either in the presence or absence of metabolic activation. Hence, the test substance was not considered to be mutagenic in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Reactive Blue 72 was assessed for mutagenic potential in a bacterial reverse mutation assay and an in vitro mammalian cell gene mutation assay (HPRT). However, currently no study for assessing clastogenic potential of it is available. Hence, a study with structurally similar substance, FAT 40336, is used to fulfil the data requirement.

Bacterial reverse mutation assay

FAT 40045/D (of 74.36 % purity) was assessed for the potential to induce gene mutations according to OECD Guideline 471, in two independent experiments both with and without metabolic activation, using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. Each concentration, including the controls, was tested in triplicate at up to 5000 µg/plate. No relevant increases in revertant colony numbers of any of the four tester strains were observed at any dose level, either in the presence or absence of metabolic activation. Hence, the test substance was not considered to be mutagenic in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100.

In vitro mammalian cell gene mutation assay

FAT 40045/Z TE was assessed for mutagenic action in mammalian cells in a mammalian cell gene mutation assay (HPRT locus) conducted as two independent experiments, using V79 cells cultured in vitro with and without metabolic activation. In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed. Hence, FAT 40045/Z TE is considered to be not mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

In vitro mammalian chromosomal aberration assay

In an in vitro chromosomal aberration assay with and without metabolic activation with source substance, FAT 40336, no relevant increase in cells with structural aberrations was seen. Therefore, FAT 40'336/B is considered to be not clastogenic in this chromosomal aberration test.

Conclusion:

Reactive Blue 72 was not mutagenic in bacterial as well as mammalian cells. Though no study assessing its clastogenic potential is available, source substance FAT 40336 was not clastogenic in an in vitro mammalian chromosomal aberration assay. Taking into account these findings, Reactive Blue 72 is considered to be neither mutagenic nor clastogenic, and hence not genotoxic.

Justification for classification or non-classification

Reactive Blue 72 is considered to be not genotoxic, hence it does not warrant classification for mutagenicity as per the criteria of Regulation (EC) No. 1272/2008 (CLP).