C.I. Acid Yellow 246

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April 2013 to 08 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Name: FAT 20306/B
Batch No.: HT 2025/50
Physical State at RT: solid powder
Colour: red
Purity: 93.6 %
Expiry Date: 28 August 2015
Storage Conditions: at room temperature

Method

Target gene:

V79 Chinese Hamster V79 cell line, HPRT (hypoxanthine-guanine-phosphoribosyl-transferase) system

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsomal

Test concentrations with justification for top dose:

experiment I:
without metabolic activation: 10, 20, 30, 40, 50, 60, 65, 70 and 75 µg/ml
with metabolic activation: 5, 10, 25, 50, 75, 100, 200, 250, 300 and 350 µg/ml
experiment II:
without metabolic activation: 5, 10, 25, 50, 100, 200, 300, 400 and 500 µg/ml
with metabolic activation: 11.25, 22.5, 45, 90, 130, 170, 210, 240, 270 and 300 µg/ml

Vehicle / solvent:

cell culture medium (MEM + 0% FBS 4h treatment; MEM+10% FBS 20h treatment) and diluted prior to treatmen. The solvent was compatible with the survival of the cells and the S9 activation.

Controls

Controls 1

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

Controls 2

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 cell population of pre-existing HPRT' mutant cells were exposed to HAT medium containing 100 hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine and 10.0 µM glycine for several cell doublings (2-3 days)

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, 11 µg/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 ceils 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 prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with Phenobarbital (80 mg/kg bw) and B-Naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
The following quality control determinations were performed:
a) Biological activity in:
-the Salmonella typhimurium assay using 2-aminoanthracene and benzo[α]pyrene -the mouse lymphoma assay using benzo[α]jpyretie -the chromosome aberration assay using cyclophosphamide.
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in ampoules of 2 and 4 mL and stored at <-75 °C.
The protein concentration in the S9 preparation (Lot: 250113B) was 33 mg/mL. The S9 mix preparation was performed according to Ames et al.

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 ceils 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 subcultured 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 [%] = mean of colonies (dose group)/200x100
The mutation rate will be calculated as follows:
Mutants per 10 cells = mean number of mutants (dose group)/400 000 x CE [%] /100 (dose group) x 10E6
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.

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 as evalutaion 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:

the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results in not regarded as necessary.

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:

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.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation:
No precipitation of the test item was noted in any of the experiments.
Toxicity:
A biologically relevant growth inhibition (reduction of relative growth below 70 %) was observed after the treatment with the test item in experiment I and II with and without metabolic activation.
In experiment I without metabolic activation the relative growth was 14.1 % for the highest concentration (75µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 350 µg/mL with a relative growth of 18.1 %. Furthermore, a slight toxic effect was observed at concentrations of 50 and 75 µg/ml with a relative growth of 60.4 and 62.8%. In experiment II without metabolic activation the relative growth was 11.5 % for the highest concentration (500 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 300 µg/mL with a relative growth of 22.7 %.

Mutagenicity:
In experiment I without metabolic activation most 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 14.56 and 17.45 mutants/10E6 cells and in the range of 4.91 to 47.39 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2,96 was found at a concentration of 50 µg/mL with a relative growth of 82.4 %. 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 18.64 and 24.16 mutants/10E6 cells and in the range of 15.28 to 43.00 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.01 was found at a concentration of 250 µg/mL with a relative growth of 108.9 %.
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 biologically relevant increase as compared to the negative controls. Mutation frequencies with the negative control were found to be 19.02 and 19.26 mutants/10E6 cells and in the range of 7.33 to 23.38 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.22 was found at a concentration of 100 µg/mL with a relative growth of 73.6 %.
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 19.62 and 7.12 mutants/10E6 cells and in the range of 9.26 to 21.78 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.63 was found at a concentration of 90 µg/mL with a relative growth of 81.0 %. The elevated number of mutant colonies and the elevated mutation factor observed in experiment I at a concentration of 50 µg/mL (without metabolic activation) could not be reproduced in experiment II (long time exposure) and is therefore to be considered as not biologically relevant. 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.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

FAT 20306/B is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

A GLP-compliant study was performed to assess the potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster according to OECD guideline 476. 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. The test item was investigated at the following concentrations:

experiment I:

without metabolic activation: 10, 20, 30, 40, 50, 60, 65, 70 and 75 µg/ml

with metabolic activation: 5, 10, 25, 50, 75, 100, 200, 250, 300 and 350 µg/ml

experiment II:

without metabolic activation: 5, 10, 25, 50, 100, 200, 300, 400 and 500 µg/ml

with metabolic activation: 11.25, 22.5, 45, 90, 130, 170, 210, 240, 270 and 300 µg/ml

No precipitation of the test item was noted in the experiments. 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 14.1 % for the highest concentration (75 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 350 µg/mL with a relative growth of 18.1 %. Furthermore, a slight toxic effect was observed at concentrations of 50 and 75 µg/ml with a relative growth of 60.4 and 62.8 %.In experiment II without metabolic activation the relative growth was 11.5 % for the highest concentration (500 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 300 µg/mL with a relative growth of 22.7 %.

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. No dose-response relationship could be observed. Based on the study results, FAT 20306/B is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.