4-anilino-3-nitro-N-phenylbenzenesulphonamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Disperse Yellow 042 was neither mutagenic in the bacterial reverse mutation assay and/or in vitro mammalian cell gene mutation assay (HPRT) nor clastogenic in the in vitro mammalian chromosomal aberration assay, hence can be considered to be not genotoxic.

Link to relevant study records

Reference

Reference 1

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

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

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.

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):
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).

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.

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: FAT 36'014/F, Terasil Gelb GWL roh feucht (laborgetrocknet)
Batch No: 292347.26
Aggregate State at RT: Solid
Colour: yellow to brown
Purity: 90.7 % (active ingredient)
Analysis: cf. information in sponsor's file
Stability: Pure: until April, 1998. In solvent: if necassary it will be performed by the sponsor at a later date
Storage: Room temperature
Expiration Date: April, 1998

Target gene:

Histidine gene

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally, due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the uvrB"-mutation. Additionally, TA 102 contains the multicopy plasmid pAQ1, which carries the hisG428 mutation and a tetracycline resistance gene. TA 102 contains the ochre mutation in hisG gene.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate of active ingredient.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

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:

not specified

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:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

(for TA 102 without metabolic activation)

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(for all strains with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and the pre-incubation test (experiment II)

For each strain and dose level, including the controls, a minimum of three plates were used.

Experiment 1: The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL: Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),
- 500 µL: S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
- 100 µL: Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL: Overlay agar

Experiment 2: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix/S9 mix substitution buffer and 100 µL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2 mL overlay agar (45°) 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.

Number of replication: Triplicate
Number of independent experiments: Two

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are: corresponding background growth on both negative control and test plates as well as normal range of spontaneous reversion rates. Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time. A test substance is considered as positive if either a dose related or reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced. A test substance producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. A significant response is described as follows: A test substance is considered as mutagenic if in strain TA 100 and TA 102 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test substance regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

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.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(occurred in strain TA 1537 at 2500 and 5000 µg/plate and in strain TA 98 at 5000 µg/plate without metabolic activation, as well as at 2500 and 5000 µg/plate with metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In experiment II toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1537 at 2500 and 5000 µg/plate and in strain TA 98 at 5000 µg/plate without metabolic activation, as well as at 2500 and 5000 µg/plate with metabolic activation. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.

Remarks on result:

other: all strains/cell types tested

Pre-experiment for toxicity result

The plates with the test article showed normal background growth up to 5000 µg/plate in strain TA 98 and TA 100, respectively.

Conclusions:

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

Executive summary:

A bacterial reverse mutation assay was performed to investigate the potential of FAT 36014/F, Terasil Gelb GWL roh feucht (laborgetrocknet) to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. 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 article was tested at the following concentrations: 33.3; 100 ; 333.3; 1000 ; 2500 ; and 5000 µg/plate (of active ingredient). In experiment II toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1537 at 2500.0 and 5000 µg/plate and in strain TA 98 at 5000 µg/plate without metabolic activation, as well as at 2500 and 5000 µg/plate with metabolic activation. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with FAT 36014/F, Terasil Gelb GWL roh feucht (laborgetrocknet) 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 significance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, FAT 36014/F, Terasil Gelb GWL roh feucht (laborgetrocknet) is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Clastogenicity

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name: FAT 36014/Z TE
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): 94.0 % (w/w)
Date of Analysis: 07/08/2013
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

Target gene:

Not available

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

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:
with and without metabolic activation: 0.1, 1.0, 2.5, 5.0, 7.5, 10, 15, 25, 50, 75, 100 µM

Experiment I:
without metabolic activation: 10, 25 and 50 µM
with metabolic activation: 30, 60 and 90 µM
Experiment II:
without metabolic activation: 2.5, 7.5 and 15 µM
with metabolic activation: 20, 40 and 50 µM

Vehicle / solvent:

- Vehicle (s)/solvent(s) used: DMSO, cell culture medium
- Justification for choice of solvent/vehicle: Based on the results of the solubility test DMSO was used as solvent (1% DMSO final concentration). The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation (600 µg/mL)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation (1.11 µg/mL)

Details on test system and experimental conditions:

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)

FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SEEDED: 1 x 10E4 - 5 x 10E4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture), except for experiment I group 2 and the positive control without metabolic activation (100 for the 1st and 200 for the 2nd culture).
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell count

Evaluation criteria:

There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0 % - 4.0 % aberrant cells without metabolic activation and 0.0 % - 4.3 % aberrant cells with metabolic activation).

Statistics:

A statistical evaluation was used as an aid for interpretation of the results. Statistical significance at the 5 % level (p <0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative/solvent control.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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

Summary: Experiment I and II, without metabolic activation

	Dose Group	Concentration [µM]	Relative Mitotic Index [%]	Relative Cell Count [%]	Mean % Aberrant Cells		Historical Laboratory Negative Control Range	Precipi-tation^a	Statistical Signifi-cance^b
					incl. Gaps	excl. Gaps
Experiment I 4 h treatment, 20 h preparation interval	C	0	88	111	3.0	1.0	0 % - 4.0 % aberrant cells	-	-
	S	0	100	100	4.0	1.5		-	-
	2	10	123	95	5.7	3.3		-	-
	5	25	60	63	2.5	2.5		-	-
	6	50	31	55	2.0	1.0		-	-
	EMS	600 µg/mL	98	87	9.7	6.7		-	+
Experiment II 20 h treatment, 20 h preparation interval	C	2.5	65	101	1.0	0.0	0 % - 4.0 % aberrant cells	-	-
	S	7.5	100	100	1.5	1.0		-	-
	3	2.5	97	91	0.5	0.0		-	-
	5	7.5	76	83	1.0	1.0		-	-
	7	15	34	69	0.0	0.0		-	-
	EMS	600 µg/mL	39	74	15.5	15.0		-	+

The mitotic index was determined in 1000 cells per culture of each test group.

The cell count was determined by a cell counter per culture for each test group.

The relative values of the mitotic index and cell count are related to the solvent controls.

 

C:       Negative Control (Culture Medium)

S:        Solvent Control (DMSO)

EMS: Ethylmethanesulfonate

a:        - without precipitation, + with precipitation

b:        statistical significant increase compared to negative controls (Fisher’s exact test, p< 0.05),
         +:       significant; -not significant

Summary: Experiment I and II, with metabolic activation

	Dose Group	Concentration [µM]	Relative Mitotic Index  [%]	Relative Cell Count [%]	Mean % Aberrant Cells		Historical Laboratory Negative Control Range	Precipi-tation^a	Statistical Signifi-cance^b
					incl. Gaps	excl. Gaps
Experiment I 4 h treatment, 20 h preparation interval	C	0	100	107	2.5	1.5	0.0% - 4.3% aberrant cells	-	-
	S	0	100	100	3.0	1.5		-	-
	2	30	87	92	5.5	0.5		-	-
	4	60	72	80	6.5	4.0		-	-
	6	90	14	48	5.0	2.0		-	-
	CPA	1.11 µg/mL	88	97	11.0	8.5		-	+
Experiment II 4 h treatment, 20 h preparation interval	C	0	95	83	1.5	1.0	0.0% - 4.3% aberrant cells	-	-
	S	0	100	100	3.5	2.0		-	-
	2	20	99	84	3.0	2.0		-	-
	4	40	124	67	3.0	1.0		-	-
	5	50	91	59	4.0	1.5		-	-
	CPA	1.11 µg/mL	93	81	10.5	10.0		-	+

The mitotic index was determined in 1000 cells per culture of each test group.

The cell count was determined by a cell counter per culture for each test group.

The relative values of the mitotic index and cell count are related to the solvent controls.

 

C:       Negative Control (Culture Medium)

S:        Solvent Control (DMSO)

CPA:   Cyclophosphamide

a:        - without precipitation, + with precipitation

b:        statistical significant increase compared to negative controls (Fishers exact test, p< 0.05),
         +: significant; -not significant

Conclusions:

FAT 36014/Z did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.

Executive summary:

To investigate the potential of FAT 36014/Z to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was conducted according to OECD TG 473, EC No 440/2008 B10 and OPPTS 870.5375. The metaphases were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation in experiment I. In experiment II, the treatment interval was 20 h without and 4 h with metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations. Based on the results of the solubility test DMSO was used as solvent (1 % DMSO final concentration). The solvent was compatible with the survival of the cells and the S9 activity.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 10, 25 and 50 µM

with metabolic activation: 30, 60 and 90 µM

Experiment II:

without metabolic activation: 2.5, 7.5 and 15 µM

with metabolic activation: 20, 40 and 50 µM

No precipitation effects of the test item were noted without and with metabolic activation in all dose groups evaluated in experiment I and II. In experiment I without metabolic activation, cytotoxic effects of the test item were noted at concentrations of 25 µM and higher considering the relative mitotic index and the relative cell count. With metabolic activation cytotoxic effects of the test item were noted at concentrations of 90 µM and higher considering the relative mitotic index and the relative cell count. In experiment II without metabolic activation, cytotoxic effects of the test item were observed at concentrations of 15 µM and higher considering the relative mitotic index the relative cell count. With metabolic activation no cytotoxic effects of the test item were noted considering the relative mitotic index. However, considering the relative cell count cytotoxic effects were observed not until 40 µM. In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the solvent controls. EMS (600 µg/mL) and CPA (1.11 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects. In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item FAT 36014/Z did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test item FAT 36014/Z is considered to be non-clastogenic in this chromosome aberration test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay:

An in vitro study was performed to investigate the potential of the test substance (of ca. >90.7 % purity) to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP. The assay was performed in two independent experiments both with and without liver metabolic activation. Experiment I was performed as a plate incorporation assay and Experiment II as a pre-incubation assay using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each concentration, including the controls, was tested in triplicate at up to 5000 µg/plate. Under the study conditions, the test substance was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

In a supporting study, FAT 36014/C was found to be not mutagenic when tested in the bacterial reverse mutation assay.

In vitro mammalian chromosomal aberration assay:

FAT 36014/Z was assessed for potential to induce structural chromosome aberrations in Chinese hamster V79 cells, in an in vitro chromosome aberration assay. The findings of the study suggest that, FAT 36014/Z did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, FAT 36014/Z is considered to be non-clastogenic.

In vitro mammalian cell gene mutation assay:

In a mammalian cell gene mutation assay (HPRT locus), V79 cells cultured in vitro were exposed to FAT 36014/Z dissolved in DMSO at various concentrations. In the described in vitro cell gene mutagenicity test under the experimental conditions reported, FAT 36014/Z is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Disperse Yellow 042 was neither mutagenic in the bacterial reverse mutation assay and/or in vitro mammalian cell gene mutation assay (HPRT) nor clastogenic in the in vitro mammalian chromosomal aberration assay, hence can be considered to be not genotoxic.

Justification for classification or non-classification

Disperse Yellow 042 was found to be not genotoxic, hence it does not warrant classification for mutagenicity accordingto Regulation (EC) No. 1272/2008.