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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the Ames assay, Chromosomal abberation test and HPRT test, the Reactive Orange 136 is considered to be non-genotoxic or non-mutagenic.

Link to relevant study records

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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:
Nov. 19, 1996 to Jan. 27, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
"Japanese Guidelines for Screening Toxicity Testings of Chemicals:Testing Methods for New Chemical Substances, enacted July 13, 1974, amended December 5, 1986
GLP compliance:
yes (incl. certificate)
Remarks:
German GLP
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: WP2/96
- Expiration date of the lot/batch: November 30, 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: pure: stable under storage conditions
in solvent: 48 h in H2O, saline, polyethylene glycol, and CMC
- Solubility of the test substance in the solvent/vehicle: dissolved in deionised water
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
E. coli WP2
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:
deionised water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100: Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
TA 1537, TA 98: Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA, WP2: Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA, WP2 : With metabolic activatior
Details on test system and experimental conditions:
The bacterial strains were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
Evaluation criteria:
Evaluation of Results
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a reproducible increase for at least one test concentration is induced. A test article producing neither a dose related and reproducible increase in the number of revertants nor a reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
A mutagenic response is described as follows:
A test article is considered mutagenic if in the strains TA 98, TA 100, WP2, and its uvrA derivative the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 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 article regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No statistical evaluation of the data is required.
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
FAT 45176/A is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed with FAT 45176/A according to OECD 471 and EU B.14 guideline to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strains WP2 and WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate.

No toxic effects evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test item at any dose level, neither in the presence nor 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.

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

Based on the study results, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, FAT 45176/A is considered to be non-mutagenic.

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:
November 15, 1996 to March 26, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Japanese Guidelines for Screening Toxicity Testings of Chemicals: Testing Methods for New Chemical Substances, enacted July 13, 1974, amended December 05, 1986
GLP compliance:
yes (incl. certificate)
Remarks:
German GLP
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: WP2/96
- Expiration date of the lot/batch: November 30, 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: pure: stable under storage conditions
in solvent: 48 h in H2O, Saline, PEG and CMC
- Solubility of the test substance in the solvent/vehicle: the test article was dissolved in culture medium (MEM)
Target gene:
V79 cells of the Chinese Hamster in vitro
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
s9 mix
Test concentrations with justification for top dose:
Without S9 mix:
-18h (Experiment I): 3.0, 5.0, 10.0, 30.0, 100.0, 500.0 μg/ml
-18h (Experiment II): 3.0, 5.0, 10.0, 30.0, 100.0, 300.0 μg/ml
-28h (Experiment I): 10.0, 30.0, 100.0, 500.0 μg/ml
-28h (Experiment II): 10.0, 30.0, 100.0, 300.0 μg/mll

with S9 mix:
-18h (Experiment I): 3.0, 5.0, 10.0, 30.0, 100.0, 500.0 μg/ml
-18h (Experiment II): 3.0, 5.0, 10.0, 30.0, 100.0, 500.0 μg/ml
-28h (Experiment I): 10.0, 30.0, 100.0, 500.0 μg/ml
-28h (Experiment II): 10.0, 30.0, 100.0, 500.0 μg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
no
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:
Cell Cultures
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, 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 mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because
of standardized characteristics of the cells.
Thawed stock cultures were propagated at 37° C in 80 cm plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x 105 cells per flask were seeded into 15 ml of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS, Boehringer Mannheim, D-68261 Mannheim). The cells were subcultured twice weekly. The cell cultures were incubated at 37 ° C in a humidified atmosphere with 4.5 % carbon dioxide (95.5 % air).
Evaluation criteria:
A test article is classified mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosome 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 chromosome aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
Statistics:
Statistical significance was confirmed by means of the Fischer's exact test. However, both biological and statistical significance should be considered together.
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
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
.
Conclusions:
FAT 45'176/A is considered to be non-mutagenic in this chromosome aberration test.
Executive summary:

FAT 45'176/A dissolved in culture medium (MEM), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments according to OECD 473 and EU B.10 guideline. The chromosomes were prepared 18 h and 28 h after start of treatment with the test article. 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 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test (5000 µg/ml) was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. Test article concentrations between 3 and 5000 µg/ml (with and without S9 mix) were chosen for the assessment of the cytotoxic potential. In the absence of S9 mix toxic effects indicated by reduced cell numbers of about 50 % of control and below were observed after treatment with 100 µg/ml and above. In the presence of S9 mix reduced cell numbers were observed after treatment with 300 µg/ml and above. Precipitation of the test article was observed 4 h after start of treatment at concentrations of 30 ug/ml and above.

In experiment I in the absence of S9 mix and in both experiments in the presence of S9 mix, test article concentrations in a range of 3 - 500 µg/ml were applied for the investigation of the potential to induce cytogenetic damage. In absence of S9 mix in experiment II, the applied concen-tration range was 3 - 300 µg/ml. In the cytogenetic experiments, precipitation of the test article was observed 4 h after start of treatment at concentrations of 30 µg/ml and above.

In the absence and the presence of S9 mix, in both experiments no substantial reduction of the mitotic indices occurred at the evaluated experimental points, neither at concentrations with visible precipitation nor at concentrations without precipitation, except in experiment I in the presence of S9 mix at interval 18 h in cultures after treatment with 500 µg/ml.

In both experiments, in the absence and the presence of S9 mix, no biologically relevant increases in cells carrying structural chromosome aberrations were observed. Unreproduced significant increases in experiment II at interval 28 h in the absence of S9 mix were considered as biologically irrelevant. In both experiments, no relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) 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 chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Therefore, FAT 45'176/A is considered to be non-mutagenic in this chromosome aberration test.

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:
May 16 to September 12, 2013
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:
yes
Remarks:
revised archiving passage
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Remarks:
German GLP
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:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: WP 2/96
- Expiration date of the lot/batch: 23 October 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
Target gene:
HPRT-locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mixture-rat liver microsome
Test concentrations with justification for top dose:
Experiment I without metabolic activation:
5, 10, 20, 40, 60, 80, 100 and 125 µg/mL and with metabolic activation:
10, 25, 50, 100, 125, 150, 175, 200, 225 and 250 µg/mL
Experiment II without metabolic activation:
25, 50, 100, 200, 250, 300, 350, 400, 450 and 500 µg/mL and with metabolic activation:
80, 120, 160, 180, 200, 220, 240, 260, 280 and 300 µg/mL
Vehicle / solvent:
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:
not specified
Positive controls:
yes
Remarks:
Without metabolic activation
Positive control substance:
ethylmethanesulphonate
Remarks:
300 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
With metabolic activation
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
0.8 and 1.0 µg/mL
Details on test system and experimental conditions:
Cells
V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate (12 - 14 h doubling time of the BSL BIOSERVICE stock cultures) and their high cloning efficiency of untreated cells, usually more than 50%. These facts are necessary for the appropriate performance of the study.
The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE. This allows the repeated use of the same cell culture batch in experiments. Each cell batch was routinely checked for mycoplasma infections (PCR). Thawed stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM).
For purifying the ceil population of pre-existing HPRT' mutants cells were exposed to HAT medium containing 100 µM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine and 10.0 µM glycine for several cell doublings (2-3 days).
Medium
Complete Culture Medium
MEM medium supplemented with
10 % fetal bovine serum (FBS) 100 U/100 µg/mL penicillin/streptomycin 2 mM L-glutamine 25 mM HEPES
2.5 µg/mL amphotericin B
Treatment Medium
MEM medium supplemented with
0 % fetal bovine serum (FBS): short-term exposure
10 % fetal bovine serum (FBS): long-term exposure
100 U/100 µg/mL penicillin/streptomycin 2 mM L-glutamine
25 mM HEPES
2.5 µg/mL amphotericin B
Selective Medium
MEM complete culture medium supplemented with thioguanine (TG, 11µg/mL).
Mammalian Microsomal Fraction S9 Mix
An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under study. However due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary (2). Many substances only develop mutagenic potential when they are metabolized by the mammalian organism. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).
The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Giessen, Germany. Male Sprague Dawley rats were induced with phenobarbital / p-naphthoflavone.
The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresomfm-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-amino anthracene and benzo[a]pyrene)
The following additional quality control determination was performed by BSL BIOSERVICE GmbH:
Biological activity in:
-the mouse lymphoma assay using benzo[a]pyrene
-the HPRT assay using 7,12-Dimethylbenz[a]anthracene
A stock of the supernatant containing the microsomes is frozen in aliquots of 5 mL and stored at < -75 °C.
The protein concentration in the S9 preparation (Lot: 2996) was 47.7 mg/mL.
S9Mix
An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below:
8 mM MgCl2
33 mM KC1
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4, During the experiment the S9 mix was stored on ice.
Experimental Design
Pre-Test for Toxicity
The toxicity of the test item was determined in pre-experiments.
Ten concentrations [25, 50, 100, 250, 500, 1000, 1750, 2500, 3750 and 5000 µg/mL] were tested with and without metabolic activation. The experimental conditions in these pre-experiments were the same as described below for the main experiment with short-term exposure. For the 20 h long-term exposure assay (experiment II, only without metabolic activation) eight concentrations [5, 10, 25, 50, 100, 250, 500 and 1000 µg/mL] were tested. The experimental conditions in this pre-experiment were the same as described below for the long-term exposure experiment.
Exposure Concentrations
The test item was investigated at the following concentrations:
Experiment I
without metabolic activation:
5, 10, 20, 40, 60, 80, 100 and 125 µg/mL and with metabolic activation:
10, 25, 50, 100, 125, 150, 175, 200, 225 and 250 µg/mL Experiment II
without metabolic activation:
25, 50, 100, 200, 250, 300, 350, 400, 450 and 500 µg/mL and with metabolic activation:
80, 120, 160, 180, 200, 220, 240, 260, 280 and 300 µg/mL
Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 20 h long-term exposure assay.
According to OECD Guidelines at least 8 concentrations of the test item were set up in the experiments with and without metabolic activation.
Experimental Performance
Seeding of the Cultures
Prior to use, cultures have been cleansed of pre-existing cells. Two or three days old exponentially growing stock cultures (more than 50% confluent) were trypsinised at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsine concentration for all subculturing steps was 0.05%.
Approximately 1.0 x 10E6 cells per concentration, solvent/negative and positive control, were seeded in complete culture medium (MEM supplemented with 10% FBS) in a culture flask, respectively (see experimental scheme).
Treatment
Approx. 24 h after seeding the cells were exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 h (short time exposure) or 20 h (long time exposure) the treatment medium containing the test item was removed and the cells were washed twice with PBS. Subsequently complete medium (MEM supplemented with 10% FBS) was added. During the following expression period the cells of the logarithmic growing culture were subcuitured 48 to 72 h after treatment. Additionally the cell density was measured (for toxicity criteria) and adjusted to 1 x 10E6 cells/mL.
At the end of the expression period for selection the mutants, about 4 x 10E5 cells from each treatment group, were seeded in cell culture petri dishes (diameter 90 mm) with selection medium containing 11 µg/mL thioguanine (TG) for further incubation (about one week). At the end of the selection period, colonies were fixed and stained for counting.
The cloning efficiencies (CE) were determined in parallel to the selection of mutants. For each treatment group two 25 cm2 flasks were seeded with approx. 200 cells to determine cloning efficiencies. After incubation for an appropriate time colonies were fixed with methanol, stained with Giemsa and counted.
The cloning efficiency will be calculated as follows:
CE (100%)=mean of colonies (dose group)/ 200 x100

In case of experiment I without metabolic activation the cloning efficiency will be calculated as follow
CE (%)=mean of colonies (dose group)/438 x 100
The mutation rate will be calculated as follows:
Mutants per 10E6 cells =mean number of mutants (dose group)/400 000 x CE[%] /100 (dose group) x 106





Evaluation criteria:
A test is considered to be negative if there is no biological 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.
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Statistics:
The data generated are recorded in the raw data. The results are presented in tables, including experimental groups with the test item, negative and positive controls. Individual colony counts for the treated and control groups are presented for both mutation induction and survival.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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 18.6% for the highest concentration (125 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 250 µg/mL with a relative growth of 14.2%.
In experiment II without metabolic activation the relative growth was 19.2% for the highest concentration (500 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 300 µg/mL with a relative growth of 19.8%.

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 10E6 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 10.76 and 22.30 mutants/10E6 cells and in the range of 5.25 to 36.99 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.24 was found at a concentration of 125 µg/mL with a relative growth of 18.6%.
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 10E6 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 14.39 and 9.97 mutants/10E6 cells and in the range of 4.56 to 26.06 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.14 was found at a concentration of 100 µg/mL with a relative growth of 83.0%.
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 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologicaiiy relevant increase as compared to the negative controls.
Mutation frequencies with the negative control were found to be 10.46 and 19.02 mutants/10E6 cells and in the range of 3.95 to 26.58 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.80 was found at a concentration of 250 µg/mL with a relative growth of 86.0%.
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 10E6 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 5.79 and 7.07 mutants/10E6 cells and in the range oif 3.95 to 18.84 mutants/10E6 cells with the test item, respectively. The highest mutation, rate (compared to the negative control values) of 2.93 was found at a concentration of 200 µg/mL with a relative growth of 72.2%.
Conclusions:
FAT 45176/A is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

FAT 45176/A was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster. The study was carried out according to OECD 476 and EU B.17 guideline.

The selection of the concentrations was based on data from the pre-experiments. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay.

The test item was investigated at the following concentrations:

Experiment I without metabolic activation:

5, 10, 20, 40, 60, 80, 100 and 125 µg/mL and with metabolic activation:

10, 25, 50, 100, 125, 150, 175, 200, 225 and 250 µg/mL

Experiment II without metabolic activation:

25, 50, 100, 200, 250, 300, 350, 400, 450 and 500 µg/mL and with metabolic activation:

80, 120, 160, 180, 200, 220, 240, 260, 280 and 300 µg/mL

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

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 18.6% for the highest concentration (125 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 250 µg/mL with a relative growth of 14.2%. In experiment II without metabolic activation the relative growth was 19.2% for the highest concentration (500 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 300 µg/mL with a relative growth of 19.8%.

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.

In conclusion, in the described mutagenicity test under the experimental conditions reported, FAT 45176/A 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

Additional information from genetic toxicity in vitro:

Three in vitro tests were performed on FAT 45176/A, including Ames test, chromosome aberration test and gene mutation.

Ames Assay:

This study was performed with FAT 45176/A according to OECD 471 and EU B.14 guideline to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strains WP2 and WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate.

No toxic effects evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test item at any dose level, neither in the presence nor 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.

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

Based on the study results, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, FAT 45176/A is considered to be non-mutagenic.

Chromosomal Abberation:

FAT 45'176/A dissolved in culture medium (MEM), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments according to OECD 473 and EU B.10 guideline. The chromosomes were prepared 18 h and 28 h after start of treatment with the test article. 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 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test (5000 µg/ml) was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. Test article concentrations between 3 and 5000 µg/ml (with and without S9 mix) were chosen for the assessment of the cytotoxic potential. In the absence of S9 mix toxic effects indicated by reduced cell numbers of about 50 % of control and below were observed after treatment with 100 µg/ml and above. In the presence of S9 mix reduced cell numbers were observed after treatment with 300 µg/ml and above. Precipitation of the test article was observed 4 h after start of treatment at concentrations of 30 ug/ml and above.

In experiment I in the absence of S9 mix and in both experiments in the presence of S9 mix, test article concentrations in a range of 3 - 500 µg/ml were applied for the investigation of the potential to induce cytogenetic damage. In absence of S9 mix in experiment II, the applied concen-tration range was 3 - 300 µg/ml. In the cytogenetic experiments, precipitation of the test article was observed 4 h after start of treatment at concentrations of 30 µg/ml and above.

In the absence and the presence of S9 mix, in both experiments no substantial reduction of the mitotic indices occurred at the evaluated experimental points, neither at concentrations with visible precipitation nor at concentrations without precipitation, except in experiment I in the presence of S9 mix at interval 18 h in cultures after treatment with 500 µg/ml.

In both experiments, in the absence and the presence of S9 mix, no biologically relevant increases in cells carrying structural chromosome aberrations were observed. Unreproduced significant increases in experiment II at interval 28 h in the absence of S9 mix were considered as biologically irrelevant. In both experiments, no relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) 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 chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, FAT 45'176/A is considered to be non-mutagenic in this chromosome aberration test.

HPRT:Gene mutation test in vitro

FAT 45176/A was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster. The study was carried out according to OECD 476 and EU B.17 guideline.

The selection of the concentrations was based on data from the pre-experiments. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay.

The test item was investigated at the following concentrations:

Experiment I without metabolic activation:

5, 10, 20, 40, 60, 80, 100 and 125 µg/mL and with metabolic activation:

10, 25, 50, 100, 125, 150, 175, 200, 225 and 250 µg/mL

Experiment II without metabolic activation:

25, 50, 100, 200, 250, 300, 350, 400, 450 and 500 µg/mL and with metabolic activation:

80, 120, 160, 180, 200, 220, 240, 260, 280 and 300 µg/mL

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

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 18.6% for the highest concentration (125 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 250 µg/mL with a relative growth of 14.2%. In experiment II without metabolic activation the relative growth was 19.2% for the highest concentration (500 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 300 µg/mL with a relative growth of 19.8%.

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.

In conclusion, in the described mutagenicity test under the experimental conditions reported, FAT 45176/A is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Justification for classification or non-classification

Based on the available genotoxicity studies, Reactive Orange 136 does not considered to be classified for genotoxicity according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.