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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Yellow 039 did not induce reverse mutation when tested with Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. However, the substance was found to increase mutant frequency in absence of metabolic activation when tested for forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells. Hence, the mutagenic potential of Reactive Yellow 039 could not be denied. Taking the above results into account, a higher tier study, an in vivo UDS assay according to OECD Guideline 486, is being proposed. Reactive Yellow 39 can be considered to be not clastogenic as it returned negative reult in the in vitro chromosomal aberration assay.

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MC-20/2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
Target gene:
The test substance, FAT 40061/F, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO-K1-BH4 cell line is a proline auxotroph with a modal chromosome number of 20, a population doubling time of 12-14 hours, and a cloning efficiency generally greater than 80% (Li et al., 1987). The CHO-K1-BH4 cells used in this study were obtained from A.W. Hsie, Oak Ridge National Laboratories (Oak Ridge, TN).
Frozen stock cultures were tested to confirm the absence of mycoplasma contamination and for karyotpye stability.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.
Test concentrations with justification for top dose:
- Preliminary toxicity assay: 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL with and without S9.
- Definitive mutagenicity: 17.8, 35.6, 71.2, 142, 190, 253, 338 and 450 μg/mL with S9 and 235.6, 71.2, 142, 190, 253, 338, 450 and 600 μg/mL without S9.
Test substance dilutions were prepared immediately before use and delivered to the test system.
Vehicle / solvent:
SOLUBILITY TEST:
Water was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in sterile water at a concentration of ~50 mg/mL, the maximum tested.
The vehicle used to prepare the test substance dose formulations, and also used as the vehicle control was distilled water, as indicated below:
- Vehicle: distilled water
- CAS number: 7732-18-5
- Supplier: Gibco
- Lot number: 1662088
- Purity/Grade: Sterile, distilled
- Expiration date: 30 December 2016
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Remarks:
EMS is the positive control for the mutagenicity assay without S9. Benzo(a)pyrene is the positive control for themutagenicity assay with S9.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Cells were treated for 5 ± 0.5 hours in the presence and absence of S9, by addition of the test and control substance formulations to the treatment medium (with or without S9, as appropriate). This technique has been shown to be an effective method for detecting various chemical mutagens in this test system (Hsie et al., 1981; Li et al., 1987).

Preliminary Toxicity Test for Selection of Dose Levels
Cells were treated with 10 test substance concentrations, as well as the vehicle control, in the presence and absence of S9 using single cultures. The maximum concentration evaluated (5000 μg/mL). Lower concentrations were prepared by 2-fold dilutions. The pH of the cultures was within pH 7 ± 0.5. Therefore, no pH adjustment was necessary to maintain neutral pH in the treatment medium. Osmolality of the vehicle control and the highest concentration also was measured at the beginning of treatment. Concentrations evaluated in the definitive mutation assay were based on adjusted relative survival.

NUMBER OF REPLICATIONS: Duplicate
Evaluation criteria:
See any other information on materials and methods section
Statistics:
See any other information on materials and methods section
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the pH or osmolality of the cultures
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the pH or osmolality of the cultures
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Results:

Definitive mutagenicity assay:

Based on the results of the preliminary toxicity assay, FAT 40061/F was evaluated in the definitive mutagenicity assay at concentrations of 17.8, 35.6, 71.2, 142, 190, 253, 338 and 450 μg/mL with S9, and 35.6, 71.2, 142, 190, 253, 338, 450 and 600 μg/mL without S9. No visible precipitate was observed at the beginning or end of treatment, and the test substance again had no adverse impact on the pH of the cultures. However, due to unacceptable vehicle cloning efficiency values at the time of selection this trial was not scored for mutagenicity (not shown), and the assay was repeated.

A re-test was conducted under identical conditions. No visible precipitate was observed at the beginning or end of treatment, and the test substance again had no adverse impact on the pH of the cultures. The average adjusted relative survival was 18.11 and 38.84 % at concentrations of 338 μg/mL with S9 and 600 μg/mL without S9, respectively. Cultures treated at concentrations of 71.2, 142, 190, 253 and 338 μg/mL with S9, and 142, 253, 338, 450 and 600 μg/mL without S9, were chosen for mutant selection (cultures treated at concentrations of 17.8 and 35.6 μg/mL with S9 and 35.6, 71.2 and 190 μg/mL without S9 were discarded prior to selection because a sufficient number of other concentrations was available; cultures treated at a concentration of 450 μg/mL with S9 were discarded prior to selection due to excessive cytotoxicity). Statistically significant* increases in mutant frequency were observed with and without S9 (*: p <0.05 and **: p <0.01, respectively). With S9, the increases in mutant frequency observed were 5.30, 7.82*, 5.90, 7.36* and 7.35* at 71.2, 142, 190, 253 and 338 μg/mL concentrations, respectively. Thus, it was evident that the increases observed with S9 were not dose dependent (p >0.05) and were within the historical negative control 95 % confidence limit. In contrast, the increases observed (i.e. 8.96*, 10.33*, 14.89**, 15.44** and 19.80** at the test concentrations of 142, 253, 338, 450 and 600 μg/mL, respectively) without S9 were dose dependent (p <0.01) and exceeded this limit. The positive controls also induced significant increases in mutant frequency (p< 0.01).

All criteria for a valid assay ultimately were met.

CHO/HPRT Assay Historical Control Data (2011 -2013)

      Non-activated     S9 -activated
   Solvent control  0.2 microL/mL EMS  Solvent control  4.0 microL/mL B(a)P
 Mean MF  4.2 239.2  4.4  143.1 
 SD  3.6 144  3.9  76.2 
 Maximum  15.7 764.2  16.9  314.5 
 Minimum  0.0 12  0.0  5.8 

Solvent control (culture medium, distilled water, saline, DMSO, ethanol, acetone or vehicle supplied by Sponsor). It has been demonstrated that all of the above solvents exhibit the same mutant frequency range.

EMS Ethyl methanesulfonate

B(a)P Benzo(a)pyrene MF Mutant frequency per 106 clonable cells

SD Standard deviation

Conclusions:
FAT 40061/F TE was positive in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures in the absence of S9. The results observed in the presence of S9 were considered to be equivocal.
Executive summary:

The test substance, FAT 40061/F, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). FAT 40061/F was prepared in distilled water and evaluated in a preliminary toxicity assay at concentrations of 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL with and without S9. No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the pH or osmolality of the cultures. Adjusted relative survival was 8.49 and 25.47 % at a concentration of 313 μg/mL with and without S9, respectively. Adjusted relative survival approximated 0 % at all higher concentrations. Based on these results, FAT 40061/F was evaluated in the definitive mutagenicity assay at concentrations of 17.8, 35.6, 71.2, 142, 190, 253, 338 and 450 μg/mL with S9, and 35.6, 71.2, 142, 190, 253, 338, 450 and 600 μg/mL without S9 (an earlier trial, performed under identical conditions, was not scored for mutagenicity due to unacceptable vehicle cloning efficiency values at the time of selection; not shown). No visible precipitate was observed at the beginning or end of treatment, and the test substance again had no adverse impact on the pH of the cultures. The average adjusted relative survival was 18.11 and 38.84 % at concentrations of 338 μg/mL with S9 and 600 μg/mL without S9, respectively. Cultures treated at concentrations of 71.2, 142, 190, 253 and 338 μg/mL with S9, and 142, 253, 338, 450 and 600 μg/mL without S9, were chosen for mutant selection (cultures treated at concentrations of 17.8 and 35.6 μg/mL with S9, and 35.6, 71.2 and 190 μg/mL without S9, were discarded prior to selection because a sufficient number of other concentrations was available; cultures treated at a concentration of 450 μg/mL with S9 were discarded prior to selection due to excessive cytotoxicity). Statistically significant increases in mutant frequency were observed with and without S9 (*: p <0.05 and **: p <0.01, respectively). With S9, the increases in mutant frequency observed were 5.30, 7.82*, 5.90, 7.36* and 7.35* at 71.2, 142, 190, 253 and 338 μg/mL concentrations, respectively. Thus, it was evident that the increases observed with S9 were not dose dependent (p >0.05) and were within the historical negative control 95 % confidence limit. In contrast, the increases observed (i.e. 8.96*, 10.33*, 14.89**, 15.44** and 19.80** at the test concentrations of 142, 253, 338, 450 and 600 μg/mL, respectively) without S9 were dose dependent (p <0.01) and exceeded this limit. The positive controls also induced significant increases in mutant frequency (p <0.01). Thus, based on the above findings, FAT 40061/F was considered to be positive in the CHO/HPRT Assay with Duplicate Cultures in the absence of S9. The results observed in the presence of S9 were considered to be equivocal.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Pre-test on cell growth inhibition with 4h and 24h treatment was performed in order to determine the toxicity of the test article and also in the evaluations of results. These deviations had no detrimental impact on the outcome of the study.
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DUW 1410/21
- Expiration date of the lot/batch: June 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: 100 g/l (at 20 °C)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimal essential Medium; SEROMED; D-12247 Berlin)
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix liver microsomal fraction
Test concentrations with justification for top dose:
- Test concentrations in the pre-test on toxicity (with and without S9 mix, exposure time 4 hours and 24 hours):
39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0, 5000.0 µg/ml.

- Main experiment (18h, Without S9-mix):
40, 80, 120, 160 µg/ml.

- Main experiment (With S9-mix, 4h):
30, 60, 90, 120 µg/ml.
Vehicle / solvent:
On the day of the experiment (immediately before treatment), the test article was dissolved in deionised water.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
MES (Without metabolic activation); CPA (with metabolic activation)
Details on test system and experimental conditions:
DURATION
- Exposure duration: 18 hrs without S9 mix; 4 hrs with S9 mix.

NUMBER OF CELLS EVALUATED: 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis.

DETERMINATION OF TOTOXICITY
- In the pre-test the toxicity of the test article was examined using the determination of the cell number. Cell numbers of two cultures (10 coordinate defined fields per culture) were determined for each experimental group.



Evaluation criteria:
A test article is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- no significant increase of the number of structural chromosome aberrations are observed.

A test article is classified as mutagenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cellsexclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the Fisher's-exact-test. Evaluation was performed only for cells carrying aberrations exclusive gaps.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
-In the pre-test, no precipitation of the test article in culture medium was observed neither in the presence nor in the absence of S9 mix. No influence of the test article on the pH value or osmolarity was observed (solvent control: 280 mOsm, pH 7.3 versus 284 mOsm and pH 7.3 at 5000 ug/ml).

- In the main experiment in the presence and absence of S9 mix no reduced mitotic indices (81.1 % and 95.4 % of control) were observed. In the absence of S9 mix the cell number was not reduced after treatment with 120 ug/ml (85 % of control) whereas the cell number in the presence of S9 mix was reduced to 55 % of control. However, at the next higher concentration in the absence of S9 mix (160 ug/ml) cytogenetic evaluation was not possible, since no mitotic cells were observed.

- In the absence and presence of S9 mix, neither a significant nor a statistically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In the absence and in the presence of S9 mix, the aberration rates of the cells after treatment with the test article (1.0 % and 3.5 %) were near the range of the solvent control values (1.0% - 2.0%, see table 7) and within the range of our historical control data:
0.0 % - 4.0 %.

EMS (600 ug/ml) and CPA (0.71 ug/ml) were used as positive controls and showed distinct increases in cells with structural chromosome
aberrations.

In the main experiment in the presence and absence of S9 mix no reduced mitotic indices (81.1 % and 95.4 % of control) were observed. In the absence of S9 mix the cell number was not reduced after treatment with 120 ug/ml (85 % of control) whereas the cell number in the presence of S9 mix was reduced to 55 % of control. However, at the next higher concentration in the absence of S9 mix (160 ug/ml) cytogenetic evaluation was not possible, since no mitotic cells were observed.

In the absence and presence of S9 mix, neither a significant nor a statistically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In the absence and in the presence of S9 mix, the aberration rates of the cells after treatment with the test article (1.0 % and 3.5 %) were near the range of the solvent control values (1.0 % - 2.0 %) and within the range of our historical control data: 0.0 % - 4.0 %.

Conclusions:
FAT 40061/E did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) and considered not clastogenic.
Executive summary:

An in vitro screening assay was performed to assess the potential of FAT 40061/E to induce structural chromosome aberrations. Evaluation of cytogenetic damage induced in V79 cells (cell line from the lung of the Chinese Hamster) was performed in the absence and the presence of metabolic activation. Cultures of the highest evaluable concentration were investigated after 18 hours. The experiment was performed according OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) and follows GLP methodology. In the absence and the presence of S9 mix the highest possible test article concentrations were evaluated for cytogenetic damage. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

Based on the results of the pre-test on toxicity, following test concentrations were used for the main study:

- Main experiment (18h, Without S9-mix): 40, 80, 120, 160 µg/ml.

- Main experiment (4h, With S9-mix): 30, 60, 90, 120 µg/ml.

In the main experiment, reduced cell numbers were observed in the presence of 120 ug/ml S9 mix after treatment, however no reduced cell numbers or mitotic indices were seen at other test concentrations. In the absence of S9 mix, at the evaluated concentrations, neither reduced mitotic indices nor reduced cell numbers were observed. Thus, no relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls.Therefore, FAT 40061/E is considered to be not clastogenic in this chromosome aberration test.

 

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

 

Therefore, FAT 40061/E is considered to be not clastogenic in this chromosome aberration test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Directive 84/449, L 251, B 14, p. 143-145.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 28073626
- Expiration date of the lot/batch: May, 1998

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: sponsor at a later date
- Stability under test conditions: Pure: until May, 1998
Target gene:
Salmonella typhimurium histidine (his) reversion system.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
None
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix (Rat liver)
Test concentrations with justification for top dose:
Pre-experiment for toxicity: 3.3, 10, 33.3, 100, 333.3, 1000, 2500, 5000 µg/plate
Main study ( in two independent experiments):33.3, 100, 333.3, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
FAT 40061/D was dissolved in aqua bidest. The solvent was chosen because of its solubility properties.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, (4-NOPD); 2-aminoanthracene (2-AA)
Remarks:
Without metabolic activation (Sodium azide, 4-NOPD, MMS); Without metabolic activation (2-AA).
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiemnt I: Plate incorporation test.
Experiment II: Pre-incubation test.
On the day of the experiment, the test article FAT 4 0'061/D was dissolved in aqua bidest. The solvent was chosen because of its solubility
properties.

DURATION
- Precultures: 10 hours at 37 °C
- Experimental culture: 48 hours at 37 °C

STRAINS (pre-experiment for toxicity): TA 98 and TA 100

NUMBER OF REPLICATIONS: Each concentration, including the controls, was tested in triplicate.

DETERMINATION OF TOTOXICITY
- To evaluate the toxicity of the test article a prestudy was performed with strains TA 98 and TA 100.

-Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.
Evaluation criteria:
The generally accepted conditions for the evaluation of the results are:
- Corresponding background growth on both negative control and test plates
- Normal range of spontaneous reversion rates.
Range of spontaneous reversion frequencies (5.9)*
TA 1535: 3-37
TA 1537: 4-31
TA 1538: 12-37
TA 98: 15-60
TA 100: 75-200
TA 102 (+): 120-300
* These values refer to the negative control without metabolic activation.
(+) The range of strain TA 102 was determined from our historical control datas.

- A test article is considered as positive if either a dose related and reproducible increase in the number of revertants or a significant
and reproducible increase for at least one test concentration is induced.
- A test article 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 article 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 article regardless whether the highest dose induced the above described enhancement factors or not.
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100.
The plates with the test article showed normal background growth up to 5000.0 ug/plate in strain TA 98 and TA 100, respectively. According to the dose selection criteria, the test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 ug/plate
Conclusions:
FAT 40061/D is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay with and without metabolic activation.
Executive summary:

FAT 40061 was assessed for the potential to induce gene mutations in bacterial cells 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. This test was performed following GLP methodology and according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay). 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, 100.0, 333.3, 1000, 2500 and 5000 µg/plate. No toxic effects occurred in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000.0 ug/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 40061/D 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 (Without metabolic activation (Sodium azide, 4-NOPD, MMS); without metabolic activation (2-AA) 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 40061/D is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay with and without metabolic activation.

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

The genetic toxicity potential of Reactive Yellow 039 was assessed in a bacterial reverse mutation assay, an in vitro mammalian cell gene mutation assay and an in vitro mammalian chromosomal aberration assay.

Bacterial reverse mutation assay:

FAT 40061 was assessed for the potential to induce gene mutations in bacterial cells 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 test article was tested with and without metabolic activation at the following concentrations: 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate. No toxic effects occurred in the test groups with and without 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 40061/D 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.

In vitro mammalian cell gene mutation assay:

FAT 40061/F, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). In this assay, statistically significant increases in mutant frequency were observed with and without S9 (*: p< 0.05 and **: p< 0.01, respectively). With S9, the increases in mutant frequency observed were 5.30, 7.82*, 5.90, 7.36* and 7.35* at 71.2, 142, 190, 253 and 338 μg/mL concentrations, respectively. Thus, it was evident that the increases observed with S9 were not dose dependent (p> 0.05) and were within the historical negative control 95 % confidence limit. In contrast, the increases observed (i.e. 8.96*, 10.33*, 14.89**, 15.44** and 19.80** at the test concentrations of 142, 253, 338, 450 and 600 μg/mL, respectively) without S9 were dose dependent (p< 0.01) and exceeded this limit. The positive controls also induced significant increases in mutant frequency (p< 0.01). Thus, based on the above findings, FAT 40061/F was considered to be positive in the CHO/HPRT Assay with Duplicate Cultures in the absence of S9. The results observed in the presence of S9 were considered to be equivocal.

In vitro mammalian chromosomal aberration assay:

In an in vitro screening assay, performed to assess the potential of FAT 40061/E to induce structural chromosome aberrations in V79 cells (cell line from the lung of the Chinese Hamster) in the absence and the presence of metabolic activation. Based on the results of the pre-test on toxicity, following test concentrations were used for the main study:

- Main experiment (18 h, without S9-mix): 40, 80, 120, 160 µg/ml.

- Main experiment (4 h, with S9-mix): 30, 60, 90, 120 µg/ml.

In the main experiment, reduced cell numbers were observed in the presence of 120 µg/ml S9 mix after treatment, however no reduced cell numbers or mitotic indices were seen at other test concentrations. In the absence of S9 mix, at the evaluated concentrations, neither reduced mitotic indices nor reduced cell numbers were observed. Thus, no relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. Therefore, FAT 40061/E is considered to be not clastogenic in this chromosome aberration test.

Conclusion:

Reactive Yellow 039 did not induce reverse mutation when tested with Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. However, found to increase mutant frequency in absence of metabolic activation when tested for forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells. Hence, the mutagenic potential of Reactive Yellow 039 could not be denied. Taking into consideration the above results, a higher tier study, an in vivo UDS assay according to OECD Guideline 486, is being proposed. Reactive Yellow 039 can be considered to be not clastogenic as it returned negative result in the in vitro chromosomal aberration assay.


Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No.1272/2008).