C.I. Acid Yellow 246

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on three in-vitro studies Ames, chromosomal aberration and hprt assay the test substance is found non-genotoxic.

Link to relevant study records

Reference

Reference 1

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

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

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

Target gene:

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

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.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

300 µg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

0.8 and 1.0 µg/ml

Details on test system and experimental conditions:

Cells:
V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate (12 - 14 h doubling time of the BSL BIOSERVICE stock cultures) and their high cloning efficiency of untreated cells, usually more than 50 %. These facts are necessary for the appropriate performance of the study. The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE. This allows the repeated use of the same cell culture batch in experiments. Each cell batch was routinely checked for mycoplasma infections (PCR), Thawed stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM). For purifying the 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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 July 1987- 06 June 1991

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:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name: FAT 20'306/B
Batch No.: HT 2025/G
Stability: Pure: 60 months; In solvent: >8 hours in H20, ethanol, methanol and DMSO
Storage: room temperature
Expiration Date: June 1989

Target gene:

V79 cells of the Chinese Hamster in vitro

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

s9 mix

Test concentrations with justification for top dose:

Without S9 mix:
-7h: 40.0, 50.0, 60.0, 72.0 μg/ml
-18h: 0.4, 20.0, 40.0, 50.0, 60.0, 72.0 μg/ml
-28h: 40.0, 50.0, 60.0, 72.0 μg/ml

with S9 mix:
-7h: 70.0, 75.0, 80.0, 85.0 μg/ml
-18h: 0.7, 35.0, 70.0, 75.0, 80.0, 85.0 μg/ml
- 28h: 70.0, 75.0, 80.0, 85.0 μg/ml

Vehicle / solvent:

saline G:
NaCl: 8000 mg
KCl: 400 mg
Glucose: 1100 mg
Na2HPO4 7H2O 290 mg
KH2PO4 150 mg

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

witho S9 mix

Details on test system and experimental conditions:

S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.3 mg/ml in the cultures. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix:
8 mM MgCl
33 mM KCl
5 mM glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

Evaluation criteria:

The test article will be classified as mutagenic if it induces a significantly increased aberration rate with at least one of the concentrations tested as compared with the negative control. This can be confirmed by means of the nonparametric Mann-Whitney test.
A test article which produce no significant positive response at any test point will be regarded as non-mutagenic in this assay.

Statistics:

No data

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

partial inhibition of mitosis

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: all strains/cell types tested

Amendment to Report:

CONCERNING: SUMMARY- page 7

PRESENT:

The test article FAT 20306/B was assessed for its potential to induce chromosomal aberrations in V7 9 cells of the Chinese Hamster in vitro.Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following dose level were evaluated:

without S9 mix: with ,S9 mix:

7 h: 72.0 µg/ml 7 h: 85.0 µg/ml

18 h: 0.4; 20.0; 72.0 µg/ml 18 h: 0.7; 35.0; 85.0 µg/ml

28 h: 72.0 µg/ml , 28 h: 85.0 µg/ml

 

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response.

Treatment of the cells with 72.0 and 85.0 µg/ml, respectively, reduced clearly the plating efficiency of the V79 cells. The mitotic index was slightly reduced with the highest concentrations at fixation interval 18 hours in the presence and absence of S9 mix.

There was no relevant enhancement of cells with structural aberrations after treatment with the test article at any fixation interval neither without nor with metabolic activation by S9 mix.

Appropriate reference mutagens were used as positive controls and showed distinct increases of cells with structural chromosome aberrations.

 

CONCLUSION:

Inconclusion, it can be stated that in the described study and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, FAT 20'306/B is considered to be non-mutagenic in this chromosomal aberration test.

 

NEW:

The test article FAT 20'306/B was assessed for its potential to induce chromosomal aberrations in V79 cells of the Chinese Hamster in vitro in two independent experiments.

Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h.

In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following dose level were evaluated:

Experiment I

without S9 mix:

7 h: 72.0 µg/ml

18 h: 0.4; 20.0; 72.0 µg/ml

28 h: 72.0 µg/ml

with S9 mix:

7 h: 85.0 µg/ml

18 h: 0.7; 35.0; 85.0 µg/ml

28 h: 85.0 µg/ml

Experiment II

without S9 mix:

7 h: 80.0 µg/ml

18 h: 100.0; 150.0 µg/ml

28 h: 100.0 µg/ml

with S9 mix:

7 h: 80.0 µg/ml

18 h: 150.0 µg/ml

28 h: 150.0 µg/ml

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response.

Treatment of the cells with 72.0 and 85.0 µg/ml, respectively, reduced clearly the plating efficiency of the V79 cells. In experiment I, the mitotic index was slightly reduced with the highest concentration at fixation interval 18 hours in the presence and absence of S9 mix.

In experiment II, the mitotic index was reduced at each fixation interval with and without S9 mix except at interval 28 h without S9 mix after treatment with the highest scorable concentration. In both experiments, there was no biologically relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval neither without nor with metabolic activation by S9 mix.

Appropriate reference mutagens were used as positive controls and showed distinct increases of cells with structural chromosome aberrations.

 

CONCLUSION:

In conclusion, it can be stated that in the described study and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, FAT 20'306/B is considered to be non-mutagenic in this chromosomal aberration test.

 

REASON FOR THE ALTERATION: a second experiment was requested by the sponsor

 

 

CONCERNING: OBJECTIVE

PRESENT:

AIMS OF THE STUDY

This in vitro experiment was performed to assess the mutagenic potential of the test article to induce structural chromosome aberrations by means of the chromosome aberration assay in the Chinese Hamster cell line V79.

NEW:

AIMS OF THE STUDY:

This in vitro experiment was performed to assess the mutagenic potential of the test article to induce structural chromosome aberrations by means of two independent chromosome aberration assays in the Chinese Hamster cell line V79.

REASON FOR THE ALTERATION: a second experiment was by the sponsor

 

CONCERNING: DOSE SELECTION- page 13

PRESENT:

DOSE SELECTION:

According to the results from this pre-experiment at least six concentrations to be applied in the chromosomal aberration assay were chosen. The highest concentration should reduce the survival

rate to approximately 20 - 30 %. In addition, this concentration should suppress if possible mitotic activity (% cells in mitosis) by approximately 50 %, but not so great a reduction that insufficient scorable mitotic cells can be found.

Treatment was performed with the following concentrations : *

without S9 mix:

7 h: 40.0; 50.0; 60.0; 72.0 ng/ml

18 h: 0.4; 20.0; 40.0; 50.0; 60.0; 72.0 µg/ml

28 h: 40.0; 50.0; 60.0; 72.0 µg/ml

with S9 mix:

7 h: 70.0; 75.0; 80.0; 85.0 µg/ml

18 h: 0.7; 35.0; 70.0; 75.0; 80.0; 85.0 µg/ml

28 h: 70.0; 75.0; 80.0; 85.0 µg/ml

According to the criteria mentioned above one (7 h and 28 h) and three concentrations (18 h) were selected to evaluate metaphases for cytogenetic damage.

In the pre-experiment for toxicity the colony forming ability of the V79 cells was reduced to 10 - 20 % after treatment with 72.0 and 85.0 µg/ml, respectively, in the absence and presence of S9 mix.

In the main experiment cultures after treatment with the maxima concentrations 7 2.0 and 85.0 µg/ml were selected to evaluate metaphases for cytogenetic damage. With this concentrations applied in the absence and presence of S9 mix the mitotic index was slightly suppressed.

 

NEW:

DOSE SELECTION

According to the results from this pre-experiment at least six concentrations to be applied in the chromosomal aberration assay were chosen. The highest concentration should reduce the survival rate to approximately 20 - 30 %. In addition, this concentration should suppress if possible mitotic activity (% cells in mitosis) by approximately 50 %, but not so great a reduction that insufficient scorable mitotic cells can be found.

Treatment was performed with the following concentrations*

Experiment I

without S9 mix:

7 h: 40.0; 50.0; 60.0; 72.0 ng/ml

18 h: 0.4; 20.0; 40.0; 50.0; 60.0; 72.0 µg/ml

28 h: 40.0; 50.0; 60.0; 72.0 µg/ml

with S9 mix:

7 h: 70.0; 75.0; 80.0; 85.0 µg/ml

18 h: 0.7; 35.0; 70.0; 75.0; 80.0; 85.0 µg/ml

28 h: 70.0; 75.0; 80.0; 85.0 µg/ml

* concentrations were chosen from the plotted dose-response curve

 

Experiment II

without S9 mix:

7h: 80.0; 100.0; 150.0; 200.0 µg/ml

18h: 10.0; 30.0; 80.0; 100.0; 150.0; 300.0 µg/ml

28h: 80.0; 100.0; 150.0; 200.0 µg/ml

with S9 mix:

7h: 80.0; 100.0; 150.0; 200.0 µg/ml

18h: 10.0; 30.0; 80.0; 150.0; 300.0; 600.0 µg/ml

28h: 80.0; 100.0; 150.0; 200.0 µg/ml

According to the criteria mentioned above one (7 h and 28 h) and three concentrations (18 h) were selected to evaluate metaphases for cytogenetic damage.

In the pre-experiment for toxicity the colony forming ability of the V79 cells was reduced to 10 - 20 % after treatment with 72.0 and 85.0 µg/ml, respectively, in the absence and presence of S9 mix.

In the main experiment I cultures after treatment with the maxima concentrations 72.0 and 85.0 µg/ml were selected to evaluate metaphases for cytogenetic damage. With this concentrations applied in the absence and presence of S9 mix the mitotic index was slightly suppressed.

In experiment II, cultures after treatment with 80.0 µg/ml (7 h with and without S9 mix), 150 µg/ml (18 h with and without S9 mix) 100 and 150 µg/ml (28 h without and with S9 mix, respectively) were evaluated as top dose levels. At each interval with the next higher dose level no or not enough scorable cells could be found.

REASON FOR THE ALTERATION: a second experiment was requested by the sponsor

Conclusions:

FAT 20306/B did not induce structural chromosome aberrations in the V79 Chinese Hamster cell line.

Executive summary:

FAT 20306/B was assessed for its potential to induce chromosomal aberrations in V79 cells of the Chinese Hamster in-vitro in two independent experiments according to OECD guideline 473 and EU method B.10. Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following dose level were evaluated:

Experiment I

without S9 mix:

7 h: 72.0 µg/ml

18 h: 0.4; 20.0; 72.0 µg/ml

28 h: 72.0 µg/ml

with S9 mix:

7 h: 85.0 µg/ml

18 h: 0.7; 35.0; 85.0 µg/ml

28 h: 85.0 µg/ml

Experiment II

without S9 mix:

7 h: 80.0 µg/ml

18 h: 100.0; 150.0 µg/ml

28 h: 100.0 µg/ml

with S9 mix:

7 h: 80.0 µg/ml

18 h: 150.0 µg/ml

28 h: 150.0 µg/ml

 

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response. Treatment of the cells with 72.0 and 85.0 µg/ml, respectively reduced clearly the plating efficiency of the V79 cells. In experiment I, the mitotic index was slightly reduced with the highest concentration at fixation interval 18 hours in the presence and absence of S9 mix. In experiment II, the mitotic index was reduced at each fixation interval with and without S9 mix except at interval 28 h without S9 mix after treatment with the highest scorable concentration. In both experiments, there was no biologically relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval neither without nor with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases of cells with structural chromosome aberrations. In conclusion, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, FAT 20306/B is considered to be non-mutagenic in this chromosomal aberration test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August 1984 to 05 December 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Principles of method if other than guideline:

The tests were carried out in accordance with the method proposed by AMES et al. (lit.1,2,3) and with the modifications described by the Ministry of Labour of Japan.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test material: FAT 20 306/B
Batch No.: HT 2025/50
Purity: >98 %
Soluble in: water
Solubility: >50 g /l
Validity: 30 years (2014)

Target gene:

histidine-auxotrophic strains of Salmonella typhimurium and on a tryptophanauxotrophic strain of E. coli.

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

5, 10, 50, 100, 500, 1000 and 5000 ug/0.1 ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without microsomal activation: TA 98 (1 ug/0.1ml DMSO) and TA 1538 (2 ug/0.1 ml DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without microsomal activation: TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without microsomal activation: TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 9(5)aminoacridine hydrochloride mondhydrate

Remarks:

without microsomal activation: TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without microsomal activation: E. coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with microsomal activation: TA 98, TA 100, TA 1537 and TA 1538

Positive control substance:

other: 2-aminoanthracene

Remarks:

with microsomal activation: E.. coli WP2 uvrA

Evaluation criteria:

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.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the experiments performed without and with microsomal activation, treatment of the cultures with the various concentrations of FAT 20306/B did not increase the incidence of back-mutant colonies by comparison with the negative control. Owing to a growth-inhibiting effect of the substance a slight reduction in the colony count was observed at the highest concentration.

Remarks on result:

other: all strains used

Conclusions:

FAT 20306/B is considered as non-mutagenic in Ames assay.

Executive summary:

In an Ames assay, FAT 20306/B was tested according to equivalent to OECD guideline 471 for mutagenic effects on histidine-auxotrophic strains of Salmonella typhimurium and on a tryptophanauxotrophic strain of E. coli. The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 5, 10, 50, 100, 500, 1000 and 5000 ng/0.1 ml. In order to confirm the results the experiments were repeated. These tests permit, the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone backmutation to histidine- or tryptophan-prototrophism. To ensure that mutagenic effects of metabolites of the test substance formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors).

In the experiments performed without and with microsomal activation, treatment of the cultures with the various concentrations of FAT 20306/B did not increase the incidence of back-mutant colonies by comparison with the negative control. Owing to a growth-inhibiting effect of the substance a slight reduction in the colony count was observed at the highest concentration.

No evidence of the induction of point mutations by FAT 20306/B or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and E. coli used in these experiments. Based on the test results, FAT 20306/B is considered as non-mutagenic in Ames assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance is not considered as mutagenic in micronucleus assay in mouse.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

ion

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 September 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: OF-1 albino mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA-CREDO, L'Arbresle, France
- Weight at study initiation: 25 g
- Assigned to test groups randomly: yes
- Housing: Animals were housed 5 of the same sex per cage in Makrolon type III cages
- Diet: Aliment Rats-Souris Charles River, produced by U.A.R., Villemoisson/Orge, France ad libitum
- Water: ad libitum
- Acclimation period: 1 week

Route of administration:

oral: gavage

Vehicle:

distilled water

Details on exposure:

A preliminary range finding test by treating three groups of three male mice at concentrations of 3000, 2000 and 1000 mg/kg were conducted. For the micronucleus test, each treatment group was comprised of five male and five female mice receiving one intragastric intubation (2500 mg/kg) using 0.5 ml of a solution at 125 mg/ml per 25 g body weight.

Duration of treatment / exposure:

20, 44 and 68 h

Frequency of treatment:

single application

Post exposure period:

20, 44 and 68 h for main test

Remarks:

Doses / Concentrations:
2500 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

Thio-TEPA

Tissues and cell types examined:

Two types of erythrocytes were observed in the bone marrow smears: normochromatic (mature red blood cells about to pass into the blood stream) and polychromatic (immature red blood cells) . The latter are stained blue by Giemsa for around 24 h after the expulsion of the erythroblast nucleus: the colouration is probably due to traces of RNA remaining in these cells.

Details of tissue and slide preparation:

After sacrifice of the animals the femurs were taken and broken open at one end. Bone marrow cells were suspended in foetal calf serum using a small syringe, and the cells were centrifuged at 120 x g for 5 minutes. The supernatant was removed with a Pasteur pipette, cells were spread on a microscope slide and the smears allowed to dry in air. The following day smears were stained with Giemsa (1:6 in water) and mounted after drying with a coverslip.

Statistics:

A complete statistical analysis, using BMPD computer programme 7D is performed.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

At low magnification of the microscope no noticeable differences in bone marrow nucleated cells were observed between animals treated with FAT 20'306/B and negative controls.
In the positive control group (Thio-TEPA) decreased numbers of bone marrow nucleated cells were noted.

There was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in animals exposed to 2500 mg/kg of FAT 20306/B compared to negative control animals. In animals treated with Thio-TEPA there was a statistically significant increased number of micronucleated cells. The ratio of polychromatic to normochromatic erythrocytes was markedly decreased in mice treated with Thio-TEPA. There was no difference between animals treated with FAT 20306/B and the negative control for this ratio.

Conclusions:

FAT 20306/B did not present a mutagenic effect in the micronucleus test using mice treated by oral administration at 2500 mg/kg.

Executive summary:

The micronucleus test was performed to assess mutagenicity toxicity of the test article based on OECD 474. The test article was administered orally to mice at concentration of 2500 mg/kg body weight. There was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in animals exposed to 2500 mg/kg of FAT 20306/B compared to negative control animals. In animals treated with Thio-TEPA there was a statistically significant increased number of micronucleated cells (pronounced evidence of mutagenicity 44 h after administration). The ratio of polychromatic to normochromatic erythrocytes was markedly decreased in mice treated with Thio-TEPA. There was no difference between animals treated with FAT 20306/B and the negative control for this ratio. Thus, we can conclude the test article dose not present a mutagenic effect to mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Three in vitro tests and one in vivo test were performed of the test substance, including Ames test, chromosome aberration test and micronucleus assay.

In-vitro: Ames assay:

In a key study, FAT 20 306/B was tested according to equivalent to OECD guideline 471 for mutagenic effects on histidine-auxotrophic strains of Salmonella typhimurium and on a tryptophanauxotrophic strain of E. coli. The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 5, 10, 50, 100, 500, 1000 and 5000 ug/0.1 ml. In order to confirm the results the experiments were repeated. These tests permit, the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone backmutation to histidine- or tryptophan-prototrophism. To ensure that mutagenic effects of metabolites of the test substance formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors).

In the experiments performed without and with microsomal activation, treatment of the cultures with the various concentrations of FAT 20306/B did not increase the incidence of back-mutant colonies by comparison with the negative control. Owing to a growth-inhibiting effect of the substance a slight reduction in the colony count was observed at the highest concentration.

No evidence of the induction of point mutations by FAT 20 306/B or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and E. coli used in these experiments. Based on the test results, FAT 20306/B is considered as non-mutagenic in Ames assay.

 

In another supporting study, FAT 20306/A was tested with the Salmonella typhimurium (TA 98,TA 100, TA 1535, TA 1537) at five dose levels of 20, 80, 320, 1280 and 5120 μg/0.1 ml, both in the presence and absence of metabolic activation to check the potential of mutagenicity. In this study too test substance causes no mutagenicity.

 

In-vitro: CA

FAT 20306/B was assessed for its potential to induce chromosomal aberrations in V79 cells of the Chinese Hamster in-vitro in two independent experiments according to OECD guideline 473 and EU method B.10. Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following dose level were evaluated:

Experiment I

without S9 mix:

7 h: 72.0 µg/ml

18 h: 0.4; 20.0; 72.0 µg/ml

28 h: 72.0 µg/ml

with S9 mix:

7 h: 85.0 µg/ml

18 h: 0.7; 35.0; 85.0 µg/ml

28 h: 85.0 µg/ml

Experiment II

without S9 mix:

7 h: 80.0 µg/ml

18 h: 100.0; 150.0 µg/ml

28 h: 100.0 µg/ml

with S9 mix:

7 h: 80.0 µg/ml

18 h: 150.0 µg/ml

28 h: 150.0 µg/ml

 

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response. Treatment of the cells with 72.0 and 85.0 µg/ml, respectively reduced clearly the plating efficiency of the V79 cells. In experiment I, the mitotic index was slightly reduced with the highest concentration at fixation interval 18 hours in the presence and absence of S9 mix. In experiment II, the mitotic index was reduced at each fixation interval with and without S9 mix except at interval 28 h without S9 mix after treatment with the highest scorable concentration. In both experiments, there was no biologically relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval neither without nor with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases of cells with structural chromosome aberrations.

 

In conclusion, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, FAT 20306/B is considered to be non-mutagenic in this chromosomal aberration test.

 

In-vitro: HPRT assay

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.

 

In-vivo: MNT

The micronucleus test was performed to assess mutagenicity toxicity of the test article based on OECD 474. The test article was administered orally to mice at concentration of 2500 mg/kg body weight. There was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in animals exposed to 2500 mg/kg of FAT 20306/B compared to negative control animals. In animals treated with Thio-TEPA there was a statistically significant increased number of micronucleated cells (pronounced evidence of mutagenicity 44 h after administration). The ratio of polychromatic to normochromatic erythrocytes was markedly decreased in mice treated with Thio-TEPA. There was no difference between animals treated with FAT 20306/B and the negative control for this ratio. Thus, we can conclude the test article dose not present a mutagenic effect to mice.

Justification for classification or non-classification

Based on the findings of the genetic toxicity studies, the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.