Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Acid Yellow 219 showed negative results in the key Salmonella typhimurium reverse mutation assay (OECD 471) and in the in vitro chromosome aberrations test (OECD 473) as well as in vitro mammalian cell gene mutation assay (OECD 476). The substance is therefore considered to be not genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experiment start date - 16 October 1984; Experiment completion date - 12 November 1984; Study completion date - 29 November 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:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Name: FAT 20004/G
Purity: 98.4 %
Batch no: DM 4694/13/1
Target gene:
Histidine gene

Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
The bacteria on which the tests were performed were the following histidine-auxotrophic strains of Salmonella typhimurium: TA 98, TA 100, TA 102, TA 1535 and TA 1537 (origin: Prof.B. Ames, Berkeley, U.S.A.).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
With and without metabolic activation: 20, 78, 313, 1250 and 5000 µg/0.1 ml.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunorubicin- HCl
Remarks:
for TA 98 without -S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
for TA 100 without -S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
for TA 102: without -S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
for TA 1535 without -S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: aminoacridine hydrochloride monohydrate
Remarks:
TA 1537 without -S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
strains TA 98, TA 100, TA 1537, TA 102 with activation mixture
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with strain TA 1535 with activation mixture
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
Positive controls validity:
valid
Additional information on results:
In the experiments performed without and with microsomal activation none of the tested concentrations of FAT 20004/G led to an increase in the incidence of histidine-prototrophic mutants in comparison with the controls. After exposure to FAT 20004/G at the highest concentration in the experiments without and with microsomal activation, the number of histidine-prototrophic mutants was reduced, as a result of the inhibitory effect of the substance on the growth of the bacteria.

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST EXPERIMENTS WITHOUT MICROSOMAL ACTIVATION NUMBER OF BACK-MUTANT COLONIES PER PLATE (ARITHMETIC MEAN)

Strain

TA 98

TA 100

TA 102

TA 1535

TA 1537

Control

25

160

285

12

4

20 µg/0.1 ml

23

200

299

9

7

78 µg/0.1 ml

25

181

285

11

6

313 µg/0.1 ml

21

168

301

11

6

1250 µg/0.1 ml

18

164

288

6

7

5000 µg/0.1 ml

14

45

166

8

4

Positive control (Vehicle)

26

168

269

13

9

Positive control

(Concentration 1)

309

916

916

790

43

Positive control

(Concentration 2)

576

1464

2431

1236

835

 

 SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST EXPERIMENTS WITH MICROSOMAL ACTIVATION NUMBER OF BACK-MUTANT COLONIES PER PLATE (ARITHMETIC MEAN)

Strain

TA 98

TA 100

TA 102

TA 1535

TA 1537

Control

53

162

406

12

13

20 µg/0.1 ml

66

162

380

12

8

78 µg/0.1 ml

53

155

407

12

9

313 µg/0.1 ml

54

167

393

13

9

1250 µg/0.1 ml

47

154

416

9

8

5000 µg/0.1 ml

36

81

216

13

6

Positive control (Vehicle)

43

143

345

9

7

Positive control

(Concentration 1)

1657

2041

1188

602

249

  

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST EXPERIMENTS WITHOUT MICROSOMAL ACTIVATION NUMBER OF BACK-MUTANT COLONIES PER PLATE (ARITHMETIC MEAN)

Strain

TA 98

TA 100

TA 102

TA 1535

TA 1537

Control

28

131

328

18

7

20 µg/0.1 ml

28

138

291

14

8

78 µg/0.1 ml

26

143

310

18

11

313 µg/0.1 ml

29

165

324

15

8

1250 µg/0.1 ml

27

131

298

14

8

5000 µg/0.1 ml

16

52

140

10

7

Positive control (Vehicle)

26

129

305

16

9

Positive control

(Concentration 1)

572

775

1008

582

66

Positive control

(Concentration 2)

856

1275

1368

813

982

 

 SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST EXPERIMENTS WITH MICROSOMAL ACTIVATION NUMBER OF BACK-MUTANT COLONIES PER PLATE (ARITHMETIC MEAN)

Strain

TA 98

TA 100

TA 102

TA 1535

TA 1537

Control

52

141

326

17

19

20 µg/0.1 ml

46

132

309

17

24

78 µg/0.1 ml

60

126

353

13

21

313 µg/0.1 ml

70

123

350

11

25

1250 µg/0.1 ml

55

136

486

12

17

5000 µg/0.1 ml

24

67

133

4

5

Positive control (Vehicle)

50

119

278

22

14

Positive control

(Concentration 1)

1853

1600

1296

614

106

 

Conclusions:
No evidence of the induction of point mutations by FAT 20004/G or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.
Executive summary:

FAT 20004/G was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. This study was conducted according to method equivalent or similar to OECD test guideline 471. The investigations were performed on strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 20, 78, 313, 1250 and 5000 µg/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 back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances 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 20004/G 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 reduction in the colony count was observed at the highest concentration. No evidence of the induction of point mutations by FAT 20004/G or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation date - 29 June 2015; Experiment start date: 02 July 2015; Experiment end date - 11 September 2015; Study completion date - 10 November 2015.
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
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Identification: FAT 20004/J
Commercial Name: Tectilon Yellow 4R crude Moist
EC No.: 264-129-6
CAS No.: 63405-85-6
Batch/Lot No.: AT-0032421400
Purity: 91.3 % (provided by Sponsor)
Expiration Date: 05 January 2020
Molecular Weight: 448.43 g/mol
Description by BioReliance: Red-orange powder
Storage Conditions: Room temperature, protected from light
Receipt Date: 29 May 2015
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
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 %. The CHO-K1-BH4 cells used in this study were obtained from A.W. Hsie, Oak Ridge National Laboratories (Oak Ridge, TN).

CHO cells were maintained in Ham's F12 medium supplemented with 3 mM L-glutamine and 5 % (v/v) heat-inactivated and dialyzed fetal bovine serum (F12FCM5) under standard conditions (37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air). All media contained antimycotics and antibiotics.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenate
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3408, Expiration Date: 03 February 2017) was purchased commercially from Moltox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -70 °C or colder until used. The lot of S9 was assayed for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.

S9 Mix
The S9 mix was prepared on the day of use. The final concentrations of the components in the mix were as indicated below.
Component Final Concentration in Cultures
NADP (sodium salt) 0.8 mM
Glucose-6-phosphate 1 mM
Calcium chloride 2 mM
Potassium chloride 6 mM
Magnesium chloride 2 mM
Sodium Phosphate 10 mM
S9 homogenate 20 μL/mL
Test concentrations with justification for top dose:
FAT 20004/J was evaluated in the definitive mutagenicity assay at concentrations below;
With S9: 25.0, 35.0, 45.0, 58.0 and 78.0 μg/mL
Without S9: 10.0, 20.0, 25.0, 30.0, 35.0, 37.5, 40.0, 45.0 and 50.0 μg/mL.
Vehicle / solvent:
Solvent control (culture medium, distilled water, saline, DMSO, ethanol, acetone or vehicle supplied by Sponsor).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
Cells were plated (on Day -1) in 25 cm² cultures at a density of ~1 x 10E6 in 5 mL F12FCM5.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
: Significant increase in mutant frequency compared to vehicle controls.
Evaluation criteria:
The test substance was considered to have produced a positive response if it induced a statistically significant and dose-dependent increase in mutant frequency (p ≤0.05) that exceeded the 95 % confidence limit of the historical vehicle control data from this laboratory. If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95 % confidence interval), the results were considered equivocal. If none of these criteria were met, the results were considered to be negative. Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the study director used sound scientific judgment and clearly reported and described any such considerations.
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
Positive controls validity:
valid
Conclusions:
FAT 20004/J was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) assay.
Executive summary:

The test substance, FAT 20004/J, 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). This study was conducted according to OECD test guideline 476 in a GLP-certified laboratory. FAT 20004/J was prepared in DMSO and evaluated in a preliminary toxicity assay at concentrations of 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250 and 2500 μg/mL with and without S9. The maximum dose evaluated was based on the solubility limitations of the test substance in the vehicle. Visible precipitate was observed at concentrations ≥625 μg/mL at the beginning of treatment and at concentrations ≥78.1 μg/mL by the end of treatment. The pH of the cultures was adjusted at concentrations ≥1250 μg/mL to maintain neutral pH, and the test substance had no adverse impact on the osmolality of the cultures. Adjusted relative survival was 115.87 and 9.10 % at concentrations of 2500 μg/mL with S9 and 39.1 μg/mL without S9, respectively. Adjusted relative survival approximated 0 % at a concentration of 78.1 μg/mL without S9 (cultures treated at higher concentrations without S9 were discarded prior to plating due to excessive toxicity). Based on these results, FAT 20004/J was evaluated in the definitive mutagenicity assay at concentrations of 25.0, 35.0, 45.0, 58.0 and 78.0 μg/mL with S9 and 10.0, 20.0, 25.0, 30.0, 35.0, 37.5, 40.0, 45.0 and 50.0 μg/mL without S9. No visible precipitate was observed at the beginning of treatment; however, visible precipitate was observed at a concentration of 78.0 μg/mL with S9 by the end of treatment, and the test substance had no adverse impact on the pH of the cultures. The average adjusted relative survival was 97.03 % at a concentration of 78.0 μg/mL with S9. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with S9 (p >0.05). In contrast, the positive controls induced a significant increase in mutant frequency (p <0.01). However, the selection plates for the vehicle control cultures without S9 were contaminated (not shown), and this portion of the assay was retested under identical conditions. In the mutagenicity assay retest, 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 11.08 % at a concentration of 45.0 μg/mL without S9. Cultures treated at concentrations of 20.0, 25.0, 30.0, 35.0, 40.0 and 45.0 μg/mL without S9 were chosen for mutant selection (cultures treated at other concentrations were discarded prior to selection because a sufficient number of higher concentrations was available, or due to excessive toxicity). No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated without S9 (p >0.05). In contrast, the positive controls induced a significant increase in mutant frequency (p <0.01). In the mutagenicity assay retest, the cloning efficiency of one vehicle control culture without S9 was below the range of 60 % at initial survival. The second vehicle control culture without S9 met all acceptance criteria, and the average absolute vehicle control cloning efficiency at initial survival was 60.5 %.  Therefore, all criteria for a valid study ultimately were met. These results indicate FAT 20004/J was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures, under the conditions and according to the criteria of the test protocol.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation date - 26 October 2015; Experiment start date - 28 October 2016; Experiment completion date - 20 December 2015; Study completion date - 15 January 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Identification: FAT 20004/J TE
Commercial Name: Tectilon Yellow 4R crude Moist
EC No.: 264-129-6
CAS No. 63405-85-6
Batch/Lot No.: AT-0032421400
Purity: 91.3% (provided by Sponsor)
Expiration Date: 05 January 2020
Molecular Weight: 448.43 g/mol
Description by BioReliance: Red-orange powder
Storage Conditions: Room temperature, protected from light
Receipt Date: 29 May 2015
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

CELLS USED
Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20. The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix liver microsomal fraction:
Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3330, Exp. Date: 09 Sep 2016) was purchased commercially from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100. The S9 mix was prepared on the day of use and added to the test system at 20 % (v/v). The final concentrations of the components in the test system are indicated below:
Component Final Concentration in Cultures
NADP (sodium salt) 1 mM
Glucose-6-phosphate 1 mM
Potassium chloride 6 mM
Magnesium chloride 2 mM
S9 homogenate 20 μL/mL
Test concentrations with justification for top dose:
Non activated condition: 10, 25, 50, 60, 70, 80, 90, 100, 125, 150, 175 µg/mL.
S9 activated: 25, 50, 100, 150, 200, 250, 400 µg/mL.
Seven to fourteen dose levels were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose level evaluated for chromosome aberrations was either based on cytotoxicity (cell growth inhibition relative to the vehicle control) or visible precipitate at the conclusion of the treatment period. Two additional dose levels were included in the evaluation.
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:
mitomycin C
Remarks:
Non activated system
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9 activated system.
Details on test system and experimental conditions:
DURATION
- Exposure duration: 4 hours with S9 mix.
- 4 and 24 hours without 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 CYTOTOXICITY
- 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.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: Two

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
4 hours with S9 mix; 4 and 24 hours without S9 mix.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays):For the definitive assays only, two hours prior to cell harvest, Colcemid® was added to all cultures at a final concentration of 0.1 μg/mL.
- Methods of slide preparation:
To prepare slides, the cells were collected by centrifugation and the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with Giemsa, permanently mounted, and identified by the BioReliance study number, dose level, treatment condition, harvest date, activation system, test phase, and replicate tube design.
- Criteria for scoring chromosome aberrations: The percentage of cells in mitosis per 500 cells scored (mitotic index) was determined and recorded for each coded treatment group selected for scoring chromosomal aberrations. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture) were examined and scored for chromatid-type and chromosome-type aberrations.
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative increase in cell count (RICC).

METHODS FOR MEASUREMENTS OF GENOTOXICITY: The percentage of cells in mitosis per 500 cells scored (mitotic index) was determined and recorded for each coded treatment group selected for scoring chromosomal aberrations. Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture) were examined and scored for chromatid-type and chromosome-type aberrations. The number of metaphase spreads that were examined and scored per duplicate culture were reduced if the percentage of aberrant cells reaches a significant level (at least 10 % determined based on historical positive control data) before 150 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure will not be scored as an aberration but were considered part of the incomplete exchange. Pulverized cells and severely damaged cells (counted as 10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY vernier for each cell with a structural aberration was recorded. The percentage of cells with numerical aberrations (polyploid and endoreduplicated cells) was evaluated for 150 cells per culture (a total of 300 per dose level). The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.
Evaluation criteria:
The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤0.05), and
• the increase was concentration-related (p ≤0.05), and
• results were outside the 95 % control limit of the historical negative control data.

The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.
Statistics:
Statistical analysis was performed using the Fisher's exact test (p ≤0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the preliminary toxicity assay, CHO cells were exposed to nine dose levels of FAT 20004/J, ranging from 0.2 to 2000 µg/mL, as well as vehicle controls, in both the absence and presence of an Aroclor-induced S9 metabolic activation system for 4 hours, or continuously for 20 hours in the absence of S9 activation. The test substance formed clear solutions in water from 0.002 to 20 mg/mL.

Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as follows:

Treatment

Condition

Treatment

Time

Recovery

Time

Dose levels

(µg/mL)

 Non-activated

4 hr

16 hr

10, 25, 50, 60, 70, 80, 90, 100, 125, 150, 175

20 hr

0 hr

10, 25, 50, 60, 70, 80, 90, 100, 125, 150, 175

 S9-activated

4 hr

16 hr

25, 50, 100, 150, 200, 250, 400

In the initial assay, the test substance formed clear solutions in water from 0.1 to 4 mg/mL. Visible precipitate was observed in treatment medium at the following dose levels:

Treatment

Condition

Treatment

Time

Visible precipitate

At the beginning of Treatment period

At the conclusion of Treatment period

Non-activated

4 hr

None

175 µg/mL

20 hr

None

175 µg/mL

S9-activated

4 hr

None

³ 200 µg/mL

Conclusions:
Under the conditions of the assay described in this report, FAT 20004/J was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.
Executive summary:

FAT 20004/J was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor‑induced rat liver S9 metabolic activation system. This study was conducted according to OECD test guideline 473, in a GLP-certified laboratory. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, CHO cells were treated for 4 and 20 hours in the non‑activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Water was used as the vehicle. In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 µg/mL. Cytotoxicity ( ≥50 % reduction in cell growth index relative to the vehicle control) was observed at doses 200 µg/mL in the non‑activated 4 and 20-hour exposure groups, and at dose levels 600 µg/mL in the S9‑activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels 200 µg/mL in all three treatment conditions. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 10 to 175 µg/mL for the non‑activated 4 and 20-hour exposure groups, and from 25 to 400 µg/mL for the S9‑activated 4-hour exposure group. In the initial chromosome aberration assay, 55 ± 5 % cytotoxicity (reduction in-cell growth index relative to the vehicle control) was observed at dose levels ≥125 µg/mL in the non-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at 175 µg/mL in the non‑activated 4 and 20-hour exposure groups; and at dose levels  ≥200 µg/mL in the S9‑activated 4-hour exposure group. Due to lack of requisite cytotoxicity, the chromosome aberration assay was repeated in the non-activated 20-hour exposure group at dose levels ranging from 25 to 175 µg/mL. In the repeat assay, 55 ± 5 % cytotoxicity (reduction in-cell growth index relative to the vehicle control) was observed at dose levels ≥140 µg/mL in the non-activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at 175 µg/mL. The dose levels selected for microscopic analysis were 50, 100, and 125 µg/mL for the non‑activated 4-hour exposure group; 100, 150, and 200 µg/mL for the S9‑activated 4-hour exposure group, and 100, 125, and 140 µg/mL for the non‑activated 20-hour exposure group. No significant or dose‑dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in any of the treatment groups (p >0.05; Fisher’s Exact and Cochran-Armitage tests). All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤0.01). Thus, all criteria for a valid study were met. Under the conditions of the assay described in this report, FAT 20004/J was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assays:


In a study performed in 1984 (Project No. 840957; Deparade, E.), FAT 20004/G was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. The study included original and confirmatory experiments with and without metabolic activation. None of the tested concentrations of FAT 20004/G with or without metabolic activation led to an increase in the incidence of histidine-prototrophic mutants in comparison with the controls.


There were two more bacterial reverse mutation assays conducted with 20004/F and 20004/E (both conducted by Fouillet, X; Gutty, D. and Barret, J. in 1983, KL 3) which investigated the mutagenic potential in Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation. No mutagenic response was seen in both studies with strain TA 100, TA 1535 and TA 1537 with and without metabolic activation and with TA 98 without metabolic activation. In both the studies, the evidence of mutagenicity was reported with TA 98 and only in the presence of the metabolic activation. In the study with FAT 20004/F, cytotoxicity was reported with the highest concentration of 5120 ug. No confirmatory experiments were conducted in these studies to confirm the findings of the original experiments. Also, the increase obtained in these studies was nearly similar to the spontaneous reversions reported for this strain by the conducting lab i.e. spontaneous reversion reported was 50, while the increased counts were 64, 50 and 15 with concentrations of 320, 1280 and 5120 ug respectively for FAT 20004/F; while the increased counts were 25 and 48 against the spontaneous conversions of 43 for the study with FAT 20004/E. However, owing to the absence of the confirmatory experiments and the increased counts being similar to the spontaneous reversion rates reported, a definite conclusion could not be drawn from these studies.


 


In vitro mammalian cell gene mutation assay:


In order to further investigate whether FAT 20004/F is capable of causing mutations in mammalian cells, a GLP compliant study according to OECD guideline 476 was performed with, where it was tested to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in three independent experiments, using identical procedures, both with and without presence of an artificial metabolic activation system (rat liver S9 mix). Up to the highest investigated concentration no relevant increase in mutant colony numbers was obtained in two independent experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant 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 gene mutations at the HPRT locus in V79 cells. Therefore, FAT 20004/J is considered to be non-mutagenic in HPRT assay.


 


In vitro mammalian chromosomal aberration assay:


Another key study was performed following OECD guideline 473 to test the chromosome aberration assay of FAT 20004/J using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor induced rat liver S9 metabolic activation system. No significant or dose‑dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in any of the treatment groups (p >0.05). All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p>0.01). Thus, all criteria for a valid study were met. Under the conditions of the assay described in this report, FAT 20004/J was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.


 


 


Conclusion on genetic toxicity
Acid Yellow 219 showed negative results in the key Salmonella typhimurium reverse mutation assay (OECD 471) and in the in vitro chromosome aberrations test (OECD 473) and in vitro in a mammalian cell gene mutation assay (OECD 476). The substance is therefore considered to have no mutagenic potential in vitro.

Justification for classification or non-classification

Based on the available genotoxicity studies, the test substance does not need to be classified for genotoxicity according to Directive 67/548/EEC and according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.