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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 93450 was found to have no mutagenic potential in both Ames assay and in vitro mammalian cell gene mutation assay, while FAT 41034/A did not have clastogenic action when tested in the in vitro mammalian chromosomal aberration assay. Hence, using the principles of read across, both substances can be considered to be neither mutagenic nor clastogenic, and hence not genotoxic.

Link to relevant study records

Referenceopen allclose all

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:
1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only four strains were tested)
GLP compliance:
no
Remarks:
(quality assurance statement was provided)
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: EN 63279.32
Target gene:
Histidine gene

Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
20, 80, 320, 1280 and 5120 µg/0.1 mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: The test substance was soluble in DMF.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: daunorubicin-HCl
Remarks:
(for strain TA 98 without metabolic activation)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
(for strain TA 100 without metabolic activation)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
(for strain TA 1535 without metabolic activation)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 9(5)aminoacridine hydrochloride
Remarks:
(for strain TA 1537 without metabolic activation)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
(for strain TA 1535 with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

Each Petri dish contained:
1) approx. 20 mL of minimum agar (Vogel-Bonner Medium E) and glucose,
2) 0.1 mL of the solution of the test substance or the vehicle and 0.1 mL of a bacterial culture in 2.0 mL of soft agar.

The soft agar was composed of: 100 mL of 0.6 % agar solution with 0.6% NaCl and 10 mL of a solution of 1-histidine, 0.5 mM and +biotin 0.5 mM. In the experiments in which the substance was metabolically activated, 0.5 mL of an activation mixture was added also. 1 mL activation mixture contained: 0.3 mL S9 fraction of liver from rats (Tif:RAIf(SPF)) induced with Aroclor 1254 and 0.7 mL of a solution of cofactors. In the experiments without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). The plates were incubated for about 48 h at 37±1.5⁰C in darkness.
Evaluation criteria:
When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Statistics:
no data
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the experiments performed without and with microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with the test substance revealed no marked differences. At the concentrations of 320 µg/0.1 mL and above the substance precipitated in soft agar.
Conclusions:
The test substance was not considered to be mutagenic in the Salmonella typhimurium reverse mutation assay.
Executive summary:

The test substance (of commercial grade purity) was tested for mutagenic effects on histidine auxotrophic mutants of Salmonella typhimurium in compliance/equivalent to OECD 471 with deviation, i.e. only four strains were tested. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 with and without microsomal activation at 20, 80, 320, 1280 and 5120 µg/0.1 mL. In order to confirm the results, the experiments were repeated. In the experiments performed with and without microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test substance revealed no marked deviations. At 320 µg/0.1 mL and above, the substance precipitated in soft agar. Under the study conditions, the test substance was not considered to be mutagenic in the Salmonella typhimurium reverse mutation assay.

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:
2014-03-11 to 2014-05-22
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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Q30420AAYB
- Expiration date of the lot/batch: 08/08/2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
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
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):
0.001, 0.002, 0.005, 0.010, 0.025, 0.050, 0.100, 0.150, 0.175, 0.20 mM

Experiment I
without and with metabolic activation: 0.005, 0.010, 0.025, 0.050, 0.075, 0.100, 0.125, 0.150, 0.175 and 0.20 mM

Experiment II
without metabolic activation: 0.0010, 0.0025, 0.0050, 0.0075, 0.010, 0.025, 0.05, 0.10, 0.15 and 0.20 mM and
with metabolic activation: 0.006, 0.008, 0.01, 0.02, 0.03, 0.04, 0.12, 0.16 and 0.20 mM
Vehicle / solvent:
Vehicle (Solvent) used: acetone
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: suspended in acetone
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: ≥ 0.075 mM; Experiment II without S9: 0.20 mM)
Vehicle controls validity:
valid
Positive controls validity:
valid

Precipitation:

In the main experiments precipitation of the test item was noted at concentrations of 0.150 mM and higher at the beginning of the treatment and at concentrations of 0.100 mM and higher at the end of the treatment period (with and without metabolic activation).

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 only without metabolic activation. In experiment I without metabolic activation the relative growth was 44.4 % for the highest concentration (0.20 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 0.20 mM with a relative growth of 81.7 %. In experiment II without metabolic activation the relative growth was 66.7 % for the highest concentration (0.20 mM) evaluated. The highest concentration evaluated with metabolic activation was 0.20 mM with a relative growth of 107.3 %.

Mutagenicity:

In experiment I without metabolic activation all mutant values of the negative controls, the solvent 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 solvent controls. Mutation frequencies with the negative control were found to be 16.93 and 29.07, of the solvent control 17.35 and 22.71 mutants/106 cells and in the range of 6.25 to 42.49 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 2.12 was found at a concentration of 0.025 mM with a relative growth of 80.0 %. With metabolic activation all mutant values of the negative controls, the solvent 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 solvent controls. Mutation frequencies with the negative control were found to be 20.56 and 26.35, of the solvent control 22.67 and 29.56 mutants/106 cells and in the range of 15.74 to 36.87 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to solvent control values) of 1.41 was found at a concentration of 0.150 mM with a relative growth of 88.7 %. In experiment II without metabolic activation most mutant values of the negative controls, the solvent 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 solvent controls. Mutation frequencies with the negative control were found to be 22.61 and 30.06, of the solvent control 34.62 and 34.12 mutants/106 cells and in the range of 17.61 to 51.02 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.48 was found at a concentration of 0.10 mM with a relative growth of 83.2 %. In experiment II with metabolic activation most mutant values of the negative controls, the solvent 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 solvent controls. Mutation frequencies with the negative control were found to be 34.58 and 19.88, of the solvent control 32.86 and 26.23 mutants/106 cells and in the range of 20.00 to 52.53 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.78 was found at a concentration of 0.02 mM with a relative growth of 108.3 %. 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 93450/Z 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 93450/Z at concentrations of 0.005, 0.010, 0.025, 0.050, 0.075, 0.100, 0.125, 0.150, 0.175 and 0.20 mM (with and without metabolic activation, Experiment I), 0.0010, 0.0025, 0.0050, 0.0075, 0.010, 0.025, 0.05, 0.10, 0.15 and 0.20 mM (without metabolic activation, Experiment II), 0.006, 0.008, 0.01, 0.02, 0.03, 0.04, 0.12, 0.16 and 0.20 mM (with metabolic activation, Experiment II). The test was carried as as per OECD 476, Commission Regulation (EC) No 440/2008, L142, Annex Part B, B 17, and EPA Health Effects Test Guidelines, OPPTS 870.5300. FAT 93450/Z 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 44.4 % for the highest concentration (0.20 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 0.20 mM with a relative growth of 81.7 %. In experiment II without metabolic activation the relative growth was 66.7 % for the highest concentration (0.20 mM) evaluated. The highest concentration evaluated with metabolic activation was 0.20 mM with a relative growth of 107.3 %.

In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.12 was found at a concentration of 0.025 mM with a relative growth of 80.0 %. In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values)of 1.41 was found at a concentration of 0.150 mM with a relative growth of 88.7 %. In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.48 was found at a concentration of 0.10 mM with a relative growth of 83.2 %. In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.78 was found at a concentration of 0.02 mM with a relative growth of 108.3 %. 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 asacceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The target chemical, Solvent Yellow 163has been tested for mutagenic effects in the Ames assay, and in the in vitro mammalian cell gene mutation assay. However, study assessing the clastogenic potential of it in the mammalian cells is not available. Further, the Ames assay with Solvent Yellow 163 was conducted with four Salmonella strains i.e. TA 98, TA 100, TA 1535 and TA 1537, and testing on E. coli WP2 uvrA was not done in this study. Hence to complete the data requirement and assessment of its genotoxicity potential, additional study investigating mutagenic potential in E. coli WP2 uvrA and a study investigating clastogenicity in mammalian cells is required. The source chemical, Disperse Red 381was found to be not mutagenic in an Ames assay when tested with Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 as well as E. coli WP2 uvrA, and not clastogenic in the in vitro mammalian chromosomal aberration assay. Hence, the data available with the source chemical Disperse Red 381 was used for fulfilment of data requirement as well as to arrive at the conclusion regarding the genotoxicity potential of Solvent Yellow 163.

 

Bacterial reverse mutation assays

In order to detect the mutagenic potential of Solvent Yellow 163, a bacterial cell gene mutation study (Ames Test) was performed on four strains of Salmonella (S. typhimuriumTA 98, TA 100, TA 1535 and TA 1537) without and with microsomal activation at 20, 80, 320, 1280 and 5120 µg/0.1 mL (Deparade, 1984). At 320 µg/0.1 mL and above, the substance precipitated in soft agar. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. Hence, the substance was considered to be non-mutagenic under the conditions applied in this test.

Disperse Red 381 (FAT 41034/A) was assessed for mutagenic potential in a bacterial reverse mutation assay. The assay was conducted in two steps, 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 strain WP2 uvrA (Wollny, 2002). The assay was performed in two independent experiments both with and without liver microsomal activation using test concentrations of 33, 100, 333, 1000, 2500, and 5000 µg/plate. 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 toxic effects (below the factor of 0.5), evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 41034/A at any dose level, neither in the -/+ 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. Hence, under the study conditions, the test substance was not considered to be mutagenic in the bacterial reverse mutation assay.

In vitro mammalian cell gene mutation assay

Solvent Yellow 163 (FAT 93450/Z) was tested in the HPRT locus using V79 cells of the Chinese Hamster according to OECD Guideline 476 (Wallner, 2014). In this mammalian cell gene mutation assay (HPRT locus), V79 cells culturedin vitrowere exposed to FAT 93450/Z at concentrations of 0.005, 0.010, 0.025, 0.050, 0.075, 0.100, 0.125, 0.150, 0.175 and 0.20 mM (+/- S9 -mix, Experiment I), 0.0010, 0.0025, 0.0050, 0.0075, 0.010, 0.025, 0.05, 0.10, 0.15 and 0.20 mM (+/- S9-mix, Experiment II), 0.006, 0.008, 0.01, 0.02, 0.03, 0.04, 0.12, 0.16 and 0.20 mM (+/- S9-mix, Experiment II). FAT 93450/Z was tested up to cytotoxic concentrations. In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.12 was found at a concentration of 0.025 mM with a relative growth of 80.0 %. In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.41 was found at a concentration of 0.150 mM with a relative growth of 88.7 %. In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.48 was found at a concentration of 0.10 mM with a relative growth of 83.2 %. In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.78 was found at a concentration of 0.02 mM with a relative growth of 108.3 %. The positive controls did induce the appropriate response. There was no evidence of a concentration related positive response of induced mutant colonies over background. In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item FAT 93450/Z is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

In vitro mammalian chromosomal aberration assay with the source chemical

Disperse Red 381 (FAT 41'034/A), dissolved in minimal essential medium (MEM), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamsterin vitroin two independent experiments. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentrations in the pre-test on toxicity (2000 ug/ml without S9 mix and 1800 ug/ml with S9 mix) were chosen with regard to the ability to formulate a homogeneous suspension of the test item in an appropriate solvent. Since no relevant toxicity was observed in the pre-test on toxicity, the test item was tested up to a concentration exhibiting clear test item precipitation as recommended in the OECD Guideline 473. No toxic effects indicated by clearly reduced mitotic indices or cell numbers were observed after treatment with the test item. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No biologically increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. In conclusion, under the experimental conditions reported, the test item FAT 41'034/A did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) and hence considered to be non-clastogenic in the absence and the presence of S9 mix.

Conclusion on genotoxicity

The Ames assay and Mammalian Cell Gene Mutation (HPRT) assay conducted with Solvent Yellow 163 and the Ames assay and the in Vitro Mammalian Chromosome Aberration assay with Disperse Red 381 were assessed to investigate the potential of Solvent Yellow 163 to induce gene mutations. All the assays had negative outcome. Hence, using the principles of read across, both substances can be considered to be neither mutagenic nor clastogenic and hence not genotoxic.

Justification for classification or non-classification

Solvent Yellow 163 is considered to be not genotoxic, hence does not warrant classification for mutagenicity as per the criteria of Regulation (EC) No. 1272/2008.