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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
HRPT Locus (V79)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Constituent 1
Chemical structure
Reference substance name:
4-anilino-3-nitro-N-phenylbenzenesulphonamide
EC Number:
225-862-7
EC Name:
4-anilino-3-nitro-N-phenylbenzenesulphonamide
Cas Number:
5124-25-4
Molecular formula:
C18H15N3O4S
IUPAC Name:
4-anilino-3-nitro-N-phenylbenzenesulfonamide
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: FAT 36014/Z TE
Product: TERASIL YELLOW GWL CRUDE MOIST (LAB DRIED) Disperse Yellow 042
Chemical Name: 3-Nitro-N-phenyl-4-(phenylamino)benzene sulfonamide
Batch No.: Q30661AASY
Physical State: solid
Storage Conditions: room temperature, protected from light
Expiry Date: 08/08/2018
Colour: yellow
pH (specify): 9.0 (2%(w/w) aqu. Suspension), RT
Melting point: 158 °C
Active components: > 90 %
Purity (qualitative and quantitative): 94.0 % (w/w)
Molecular Weight: 369.40 g/mol
Date of Analysis: 07 August 2013
Hazards and Precautions: safety glasses with side shields
Solubility in water: 5.72 mg/l
Specific details on test material used for the study:
Name: FAT 36014/Z
Product: Terasil Yellow GWL crude moist (Lab dried)
Disperse Yellow 042
Batch No.: Q30661AASY
Physical State: solid
Storage Conditions: room temperature, protected from light
Expiry Date: 08/08/2018
Colour: Yellow
pH (specify): 9 (2 %(w/w) aqu. Suspension), RT
Melting point: 158 °C
Active components: >90 %
Purity (qualitative and quantitative): 94 % (w/w)
Molecular Weight: 369.40 g/mol
Date of Analysis: 07 August 2013
Hazards and Precautions: safety glasses with side shields
Solubility in water: 5.72 mg/l
Safety Precautions: safety gloves, mask and safety glasses were worn while handling the test item.

Method

Target gene:
hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)
Species / strain
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):
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 µM

Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
0.5, 1, 2.5, 5, 10, 20, 35, 50, 75, 100 µM
Experiment I
without metabolic activation: 0.5, 1, 2.5, 5.0, 10, 11, 12.5, 14, 15.5, 17 µM
and with metabolic activation: 10, 20, 30, 40, 45, 50, 55, 60, 65, 70 µM

Experiment II
without metabolic activation: 5, 10, 20, 30, 40, 50, 55, 60, 65, 70 µM
and with metabolic activation: 8, 16, 22, 30, 38, 46, 52, 58, 60 µM
Vehicle / solvent:
Vehicle (Solvent) used: DMSO (1% v/v).
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
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:
not specified
True negative controls:
not specified
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 DMSO
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION AGENT (mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I + II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth
Evaluation criteria:
A test is considered to be negative if there is no biologically relevant increase in the number of mutants. There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
Statistics:
A statistical analysis of the test data was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.

Results and discussion

Test results
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: ≥ 11.0 µM; experiment I with S9: ≥ 50 µM; Experiment II without S9: ≥ 20 µM; Experiment II with S9:≥ 38 µM
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
None
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Precipitation:

Precipitation of the test item was noted in the pre-experiment at a concentration of 200 μM (with and without metabolic activation). No precipitation of the test item was noted in the main experiments.

Cytotoxicity:

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 23.6 % for the highest concentration (17 μM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 70 μM with a relative growth of 12.1 %.

In experiment II without metabolic activation the relative growth was 12.6 % for the highest concentration (70 μM) evaluated. The highest concentration evaluated with metabolic activation was 60 μM with a relative growth of 11.7 %.

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 34.95 and 21.95, of the solvent control 25.27 and 19.66 mutants/106 cells and in the range of 18.99 to 35.74 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.59 was found at a concentration of 17 μM with a relative growth of 23.6 %. 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 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 solvent controls. Mutation frequencies with the negative control were found to be 26.59 and 24.79, of the solvent control 33.53 and 24.71 mutants/106 cells and in the range of 11.18 to 46.63 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.60 was found at a concentration of 70 μM with a relative growth of 12.1 %. 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 couldbe 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 21.50 and 24.26, of the solvent control 38.06 and 40.96 mutants/106 cells and in the range of 19.44 to 64.86 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.64 was found at a concentration of 65 μM with a relative growth of 16.6 %. In experiment II 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 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 solvent controls. Mutation frequencies with the negative control were found to be 41.21 and 24.93, of the solvent control 17.37 and 22.05 mutants/106 cells and in the range of 12.56 to 40.00 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 2.03 was found at a concentration of 52 μM with a relative growth of 15.7 %. DMBA (0.8 and 1 μg/mL) and EMS (300 μg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Applicant's summary and conclusion

Conclusions:
FAT 36014/Z is considered to be non-mutagenic in the HPRT assay using V79 cells of the Chinese Hamster.
Executive summary:

The study was conducted according to OECD guideline no. 476 and in accordance with GLP. In a mammalian cell gene mutation assay (HPRT locus), V79 cells cultured in vitro were exposed to FAT 36014/Z TE dissolved in DMSO at concentrations of

- 0.5, 1.0, 2.5, 5.0, 10.0, 11.0, 12.5, 14.0, 15.5, 17.0 µM (without metabolic activation, Experiment I)

- 10, 20, 30, 40, 45, 50, 55, 60, 65, 70 µM (with metabolic activation, Experiment I)

- 5, 10, 20, 30, 40, 50, 55, 60, 65, 70 µM (without metabolic activation, Experiment II)

- 8, 16, 22, 30, 38, 46, 52, 58, 60 µM (with metabolic activation, Experiment II).

FAT 36014/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 23.6 % for the highest concentration (17 µM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 70 µM with a relative growth of 12.1 %. In experiment II without metabolic activation the relative growth was 12.6 % for the highest concentration (70 µM) evaluated. The highest concentration evaluated with metabolic activation was 60 µM with a relative growth of 11.7 %.

In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.59 was found at a concentration of 17 µM with a relative growth of 23.6 %.

In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.60 was found at a concentration of 70 µM with a relative growth of 12.1 %.

In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.64 was found at a concentration of 65 µM with a relative growth of 16.6 %.

In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.03 was found at a concentration of 52 µM with a relative growth of 15.7 %.

In experiment I without metabolic activation the requirements of testing one concentration with relative growth between 10 % and 20 % could not be fulfilled.

The highest concentration of 17 µM resulted in a relative growth of 23.6 %. Due to the fact that in this experiment the gradient of toxicity is very steep and that all stages of toxicity have been detected without any hint at mutagenicity, this deficiency is considered to be not biologically relevant. The positive controls did induce the appropriate response.  There was no evidence of a concentration related positive response of induced mutant colonies over background. This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data. In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item FAT 36014/Z is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.