Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Orange 035 should be regarded as not genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test material: Cibacron Orange P-4R flüssig (FAT 40075/C)
Batch No.: 289637.26
Purity: 21.4 % (active ingredient)
Expiration date: January 1994
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 histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 100, TA 102, TA 1535 and TA 1537) were obtained from Prof. B. Ames, Berkeley, CA., U.S.A.
Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.
The characteristics of the strains were checked monthly. Histidine-auxotrophy of the strains was demonstrated by the requirement for 1-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene (strains TA 98, TA 100, TA 1535 and TA 1537) was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. Strain TA 102 additionally was checked for tetracycline resistance (presence of multicopy plasmid pAQ1). The presence of the uvr+ gene was demonstrated by the resistance of strain TA 102 against UV light. Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Cytotoxicity test: 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg active ingredient/plate
Mutagenicity tests: 61.7, 185.2, 555.55, 1666.7 and 5000 µg active ingredient/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Bidistilled water
- Justification for choice of solvent/vehicle: Based on solubility
Untreated negative controls:
yes
Remarks:
Bidistilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
for TA 100 and TA 1535 without metabolic activation
Untreated negative controls:
yes
Remarks:
bidistilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
for TA 102 without metabolic activation
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
for TA 98 without metabolic activation
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for TA 1537 without metabolic activation
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
for TA 100, TA 102, TA 98 and TA 1537 with metabolic activation
Untreated negative controls:
yes
Remarks:
bidistilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide H20
Remarks:
for TA 1535 with metabolic activation
Details on test system and experimental conditions:
Setting up of the test plates: 0.1 mL of the overnight cultures were mixed with 2 mL of top agar, either 0.5 mL of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 mL of the activation mixture (experiments with activation) and 0.1 mL of a solution of the test substance, the substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 mL of minimal agar (1.5 % agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2 % glucose). The top agar was composed of 0.6 % agar and 0.6 % NaCl. It was supplemented with 10% of 0.5 mM 1-histidine and 0.5 mM (+) biotin dissolved in water.

Preliminary Toxicity/Range-Finding test
A toxicity test (check for reduction in the number of revertant colonies) was carried out with strain TA 100 without and with microsomal activation at six concentrations of the test substance and one negative control according to Standard Operating Procedures of Genetic Toxicology. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of 3. The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration, as well as each negative control was used.

Mutagenicity test: The mutagenicity test was performed with strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 without and with microsomal activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration as well as each positive and negative control with each tester strain. The highest concentration applied in the first and second mutagenicity test was 5000 µg/plate (because of lack of toxicity in the range finding test) and the four lower concentrations were each decreased by a factor of 3. The plates were inverted and incubated for about 48 h at 37 ±1.5 °C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.
Evaluation criteria:
Assay acceptance criteria: A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.
Statistics:
In deviation to the OECD guideline, a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended. No appropriate statistical method is available.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(Only a marginal mutagenic action on strains TA 100 and TA 102)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxicity test/Range finding test: Six concentrations of the test substance ranging from 20.6 to 5000 µg active ingredient/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. Normal back-ground growth was observed at all concentrations. A slight increase in the number of revertant counts was observed at the upper concentrations, followed by a reduction in the number of revertant colonies at the highest concentration, due to toxicity of the test substance. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg active ingredient/plate without and with activation.

Mutagenicity test, original experiment: In the original experiment carried out without metabolic activation, treatment of strain TA 102 with the test substance led to a slight increase in the number of revertant counts at the concentrations of 185.2 to 1666.7 µg active ingredient/plate. In the experiment with activation, a similar effect occurred on strain TA 100 at concentration of 1666.7 µg active ingredient/plate and on strain TA 102 at concentrations of 185.2 and 1666.7 µg active ingredient/plate. No effects were observed with the other strains.

Mutagenicity test, confirmatory experiment: In the confirmatory experiment carried out without metabolic activation, treatment of strain TA 102 with the test substance led to a slight increase in the number of revertant counts at the concentration of 1666.7 µg active ingredient/plate. In the experiment with activation, a similar effect occurred on strains TA 100 and TA 102 at the same concentration. Again, no effects were observed with the other strains. In the mutagenicity tests normal background growth was observed at all concentrations. In the experiments without and with microsomal activation, due to toxicity of the test substance the numbers of revertant colonies occasionally were slightly reduced with strains TA 100 and TA 102 at the upper concentrations.
Conclusions:
Under the study conditions, the test substance is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.
Executive summary:

An in vitro study was performed to investigate the potential of the test substance (of ca. 21.4 % purity) to induce gene mutations according to OECD Guideline 471, EU Method B.14 and EPA OTS 798.5265 in compliance with GLP.

Based on the results of a preliminary toxicity/range finding study, the substance was tested for mutagenic effects without and with metabolic activation at 5 doses from 61.7 to 5000 µg active ingredient/plate. An independent repetition of the experiments was performed with the same concentrations. In the preliminary experiment without and with metabolic activation performed on strain TA 100, a slight increase in the number of revertant counts was observed at the upper concentrations, followed by a reduction in the number of revertant colonies at the highest concentration. In the original experiment carried out without metabolic activation, treatment of strain TA 102 with the test substance led to a slight increase in the number of revertant counts at 185.2 to 1666.7 µg active ingredient/plate. In the original experiment with activation, a similar effect occurred on strain TA 100 at 1666.7 µg/plate and on strain TA 102 at 185.2 and 1666.7 µg active ingredient/plate. No effects were observed with the other strains. In the confirmatory experiment carried out without metabolic activation, treatment led to a slight increase in the number of revertant counts at 1666.7 µg active ingredient/plate. In the confirmatory experiment with activation, a similar effect occurred on strains TA 100 and TA 102 at the same concentration. Again, no effects were observed with the other strains. Overall, the test substance exerted only a marginal mutagenic action on strains S. typhimurium TA 100 and TA 102.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Clastogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Name: FAT 40075/D TE
Common Name: NOVACRONE ORANGE P-4R AS Reactive Orange 035
Chemical Name: Trisodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonatonaphthyl]azo]-2,5-dimethylphenyl]azo]benzene-1,4-disulphonate
Batch No.: BS-1113243
CAS No.: 70210-13-8
Physical State / Colour: solid /orange
Storage Conditions: 2°C – 8°C, protected from light
Molecular Weight of Base Form: 745.14 g/mol
Molecular Weight of Salt Form: 814.11 g/mol
Active Components: >65 %
Analysation Date: 03.12.2013
Expiry Date: 14.03.2017
Target gene:
Not available
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE, as large stock cultures allowing the repeated use of the same cell culture batch in experiments.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment:
with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL

Experiment I:
without metabolic activation: 500, 1000 and 1500 µg/mL
with metabolic activation: 750, 1000 and 1500 µg/mL

Experiment II:
without metabolic activation: 15, 20 and 25 µg/mL
with metabolic activation: 900, 1200 and 1900 µg/mL
Vehicle / solvent:
- Vehicle (s)/solvent(s) used: cell culture medium
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 5 mg/mL or 10 mM, whichever is the lowest. Based on the results of the solubility test MEM cell culture medium was used as solvent (MEM + 0 % FBS). The solvent was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation (400 and 900 µg/mL)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation (1.11 µg/mL)
Details on test system and experimental conditions:
TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)

FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SEEDED: 1 x 10E4 - 5 x 10E4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture) except for experiment I, group 3 (1500 µg/mL) without metabolic activation and group C, 3 (750 µg/mL) and 4 (1000 µg/mL) with metabolic activation.
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell count.
Evaluation criteria:
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4.0 % aberrant cells without and 4.3 % with metabolic activation).
Statistics:
A statistical evaluation was used as an aid for interpretation of the results. Statistical significance at the 5 % level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative/solvent control.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Not available

Experiment I and II, without metabolic activation

 

 

 

Dose Group

 

Concentration [µg/mL]

 

Relative Mitotic Index [%]

 

Relative Cell Count [%]

 Mean % Aberrant Cells

Historical Laboratory Negative Control Range

Precipitationa

Statistical Significance (b)

incl.

Gaps

excl.

Gaps

 

 

 

Experiment I

4 h treatment,

20 h preparation interval

C

0

100

100

3.0

2.0

 

0.0% - 4.0%

aberrant cells

-

-

1

500

131

100

2.5

1.0

-

-

2

1000

118

77

6.0

3.0

-

-

3

1500

89

39

7.5

5.3

-

-

EMS

900

99

98

13.5

10.5

-

+

 

 

Experiment II

20 h treatment,

20 h preparation interval

C

0

100

100

1.0

0.0

 

 

0.0% - 4.0%

aberrant cells

-

-

4

15

97

71

3.0

2.5

-

-

6

20

77

68

4.5

2.5

-

-

7

25

60

61

4.0

2.5

-

-

EMS

400

65

69

14.0

12.5

-

+

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

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

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

C: Negative Control (CultureMedium)

EMS: Ethylmethanesulfonate

a: - without precipitation, + with precipitation

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

Experiment I and II, with metabolic activation

 

Dose Group

Concentration [µg/mL]

Relative Mitotic Index [%]

Relative Cell Count [%]

Mean % Aberrant Cells

Historical Laboratory Negative Control Range

Precipitation

Statistical Significance (b)

incl.

Gaps

excl.

Gaps

 

 

 

Experiment I

4 h treatment,

20 h preparation interval

C

0

100

100

6.0

3.7

 

 

 

0.0% - 4.3%

aberrant cells

-

-

3

750

97

81

6.3

4.0

-

-

4

1000

93

62

7.8

4.5

-

-

6

1500

97

44

3.5

1.5

-

-

CPA

1.11

66

81

15.5

11.5

-

+

 

 

Experiment II

4 htreatment,

20 h preparation interval

C

0

100

100

3.0

1.5

 

 

0.0% - 4.3%

aberrant cells

-

-

3

900

123

85

1.5

1.5

-

-

4

1200

154

68

2.0

0.5

-

-

7

1900

167

49

1.5

1.0

-

-

CPA

1.11

134

88

9.0

8.0

-

+

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

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

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

C: Negative Control (Culture Medium)

EMS: Ethylmethanesulfonate

a:- without precipitation, + with precipitation

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

Conclusions:
In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item FAT 40075/D did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Thus, the test item FAT 40075/D is considered to be non-clastogenic in this chromosome aberration test using V79 cells in the absence and the presence of metabolic activation.
Executive summary:

In an in vitro chromosome aberration assay, the test item FAT 40075/D was investigated for the potential to induce structural chromosomal aberrations in Chinese hamster V79 cells in the absence and presence of metabolic activation with S9 homogenate. This test was conducted in accordance to OECD guideline 473, EPA OPPTS 870.5375 and EU Method B.10. The metaphases were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation in experiment I. In experiment II, the treatment interval was 20 h without and 4 h with metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations. Based on the results of the solubility test MEM cell culture medium was used as solvent (MEM + 0 % FBS). The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 500, 1000 and 1500 µg/mL

with metabolic activation: 750, 1000 and 1500 µg/mL

Experiment II:

without metabolic activation: 15, 20 and 25 µg/mL

with metabolic activation: 900, 1200 and 1900 µg/mL

No precipitation of the test item was observed without and with metabolic activation in all dose groups evaluated in experiment I and II. In experiment I (short-term treatment) without metabolic activation, cytotoxic effects of the test item were determined at a concentration of 1500 µg/mL considering the relative cell count. However, considering the relative mitotic index no cytotoxic effects were observed. With metabolic activation cytotoxic effects of the test item were determined at a concentration of 1000 µg/mL and higher considering the relative cell count. However, considering the relative mitotic index no cytotoxic effects were observed. In experiment II (long-term treatment) without metabolic activation, cytotoxic effects of the test item were observed at a concentration of 25 µg/mL considering the relative mitotic index. Cytotoxicity was also determined by the decrease of relative cell count at concentrations of 20 µg/mL and higher. With metabolic activation, no cytotoxic effects of the test item considering the relative mitotic index were observed. However, considering the relative cell count cytotoxic effects were observed at concentrations of 1200 µg/mL and higher. In all experiments, no biologically relevant increase of the aberration rates was observed after treatment with the test item without and with metabolic activation. No concentration-related increase of structural chromosome aberrations were determined in experiment I and II. On the basis of the results of the present study, the test substance did not lead to any biologically relevant increase in the number of structural chromosome aberrations at all sampling times. In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was observed after treatment with the test item as compared to the negative controls. The distinct increase in the number of structural chromosome aberrations induced by the positive controls (EMS, CPA) clearly demonstrates the sensitivity of the test system.

There was no evidence of chromosome aberration induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data. In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item FAT 40075/D did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Thus, the test item FAT 40075/D is considered to be non-clastogenic in this chromosome aberration test using V79 cells in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the "Read across justification" attached in section 13 of this dossier.
Reason / purpose:
read-across source
Specific details on test material used for the study:
None
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I without S9: ≥ 1000 μg/mL; experiment I with S9: ≥ 2500 μg/mL; Experiment II with S9:≥ 3000 μg/mL
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Biologically relevant growth inhibition was observed in experiment I with and without metabolic activation and in experiment II with metabolic activation. No biologically relevant growth inhibition was observed in experiment II without metabolic activation. In experiment I without metabolic activation the relative growth was 27.1 % for the highest concentration (5000 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 21.1 %.
In experiment II without metabolic activation the relative growth was 72.5 % for the highest concentration (5000 µg/mL) evaluated. In experiment II with metabolic activation the highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 34.9 %. In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.37 was found at a concentration of 500 µg/mL with a relative growth of 76.2 %. In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 2.04 was found at a concentration of 2500 µg/mL with a relative growth of 54.1 %. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 0.74 was found at a concentration of 1000 µg/mL with a relative growth of 77.5%. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 2.88 was found at a concentration of 4000 µg/mL with a relative growth of 52.4 %. The positive controls did induce the appropriate response.  There was no evidence of a concentration related positive response of induced mutant colonies over background.
Conclusions:
The read across substance, FAT 40171/Y is considered to be non-mutagenic in the HPRT locus assay using V79 cells of the Chinese Hamster.
Executive summary:

In a mammalian cell gene mutation assay (HPRT locus), V79 cells cultured in vitro were exposed to the read across substance, FAT 40171/Y at concentrations of 5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL (with and without metabolic activation, Experiment I), 100, 250, 500, 750, 1000, 2000, 3000, 4000 and 5000 µg/mL (without metabolic activation, Experiment II) and 45, 90, 180, 375, 750, 1500, 3000, 4000 and 5000 µg/mL (with metabolic activation, Experiment II). FAT 40171/Y was tested up to cytotoxic concentrations. Biologically relevant growth inhibition was observed in experiment I with and without metabolic activation and in experiment II with metabolic activation. No biologically relevant growth inhibition was observed in experiment II without metabolic activation In experiment I without metabolic activation the relative growth was 27.1 % for the highest concentration (5000 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 21.1 %. In experiment II without metabolic activation the relative growth was 72.5 % for the highest concentration (5000 µg/mL) evaluated. In experiment II with metabolic activation the highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 34.9 %. In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.37 was found at a concentration of 500 µg/mL with a relative growth of 76.2 %. In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 2.04 was found at a concentration of 2500 µg/mL with a relative growth of 54.1 %. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 0.74 was found at a concentration of 1000 µg/mL with a relative growth of 77.5 %. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 2.88 was found at a concentration of 4000 µg/mL with a relative growth of 52.4 %. The positive controls did induce the appropriate response.  There was no evidence of a concentration related positive response of induced mutant colonies over background. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The genetic toxicity database of Reactive Orange 035 consists of a bacterial reverse mutation assay and an in vitro mammalian chromosomal aberration assay. However, to complete the data set required at this tonnage level as well as genetic toxicity assessment of the target chemical, a study investigating mutagenic potential in mammalian cells is required. The genetic toxicity database for the salts of Reactive Black 039 consists of bacterial reverse mutation assays (with both salts), an in vitro mammalian cell gene mutation assay with pentasodium salt and an in vitro chromosomal aberration assay with lithium sodium salt. Hence, the studies with the source chemical were used to the genetic toxicity assessment of the target substance.

Bacterial reverse mutation assay with Reactive Orange 035

A bacterial reverse mutation assay was performed to investigate the potential of Reactive Orange 035 (of ca. 21.4 % purity) to induce gene mutations according to OECD Guideline 471, EU Method B.14 and EPA OTS 798.5265 in compliance with GLP. Based on the results of a preliminary toxicity/range finding study, the substance was tested for mutagenic effects without and with metabolic activation at 5 doses from 61.7 to 5000 µg active ingredient/plate. An independent repetition of the experiments was performed with the same concentrations. In the original experiment carried out without metabolic activation, treatment of strain TA 102 with the test substance led to a slight increase in the number of revertant counts at 185.2 to 1666.7 µg active ingredient/plate. In the original experiment with activation, a similar effect occurred on strain TA 100 at 1666.7 µg/plate and on strain TA 102 at 185.2 and 1666.7 µg active ingredient/plate. No effects were observed with the other strains. In the confirmatory experiment carried out without metabolic activation, treatment led to a slight increase in the number of revertant counts at 1666.7 µg active ingredient/plate. In the confirmatory experiment with activation, a similar effect occurred on strains TA 100 and TA 102 at the same concentration. Again, no effects were observed with the other strains. Overall, the test substance exerted only a marginal mutagenic action on strains S. typhimurium TA 100 and TA 102.

Bacterial reverse mutation assays with Reactive Black 039

The source substance, Reactive Black 039 pentasodium salt was evaluated for the potential to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP. The assay was performed in two independent experiments, both with and without liver metabolic activation. Experiment I was performed as a plate incorporation assay and experiment II as a pre-incubation assay using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. Each concentration, including the controls, was tested in triplicate. The substance was tested up to 5000 µg/plate. A slight toxic effect, evidenced by a reduction in the number of revertants, occurred in strain TA 1537 without metabolic activation at the highest concentration in experiment I. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed at any dose level. Hence Reactive Black 039 pentasodium salt was considered to be not mutagenic in this Salmonella typhimurium reverse mutation assay. The lithium sodium salt of Reactive Black039 was also found to be not mutagenic in a similar bacterial reverse mutation assay.

In vitro mammalian cell gene mutation assay with Reactive Orange 035

In a mammalian cell gene mutation assay (HPRT locus), V79 cells cultured in vitro were exposed to FAT 40171/Y at concentrations of 5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL (with and without metabolic activation, Experiment I), 100, 250, 500, 750, 1000, 2000, 3000, 4000 and 5000 µg/mL (without metabolic activation, Experiment II) and 45, 90, 180, 375, 750, 1500, 3000, 4000 and 5000 µg/mL (with metabolic activation, Experiment II). FAT 40171/Y was tested up to cytotoxic concentrations. In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.37 was found at a concentration of 500 µg/mL with a relative growth of 76.2 %. In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 2.04 was found at a concentration of 2500 µg/mL with a relative growth of 54.1 %. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 0.74 was found at a concentration of 1000 µg/mL with a relative growth of 77.5 %. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 2.88 was found at a concentration of 4000 µg/mL with a relative growth of 52.4 %. There was no evidence of a concentration related positive response of induced mutant colonies over background. In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item FAT 40171/Y is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

In vitro chromosomal aberration assay with Reactive Orange 035

In anin vitrochromosome aberration assay, Reactive Orange 035 (FAT 40075/D) was investigated for the potential to induce structural chromosomal aberrations in Chinese hamster V79 cells in the absence and presence of metabolic activation with S9 homogenate. This test was conducted in accordance to OECD Guideline 473, EPA OPPTS 870.5375 and EU Method B.10. In all experiments, no biologically relevant increase of the aberration rates was observed after treatment with the test item without and with metabolic activation. No concentration-related increase of structural chromosome aberrations were determined in experiment I and II. On the basis of the results of the present study, the test substance did not lead to any biologically relevant increase in the number of structural chromosome aberrations at all sampling times. In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was observed after treatment with the test item as compared to the negative controls.Based on the findings of the study, Reactive Orange 035 (FAT 40075/D) is considered to be not clastogenic in this chromosome aberration test using V79 cells in the absence and the presence of metabolic activation.

In vitro chromosomal aberration assay with Reactive Black 039

The read-across chemical, Reactive Black 039 lithium sodium salt (FAT 45168/A) was assessed for its potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamsterin vitro in two independent experiments. The following concentrations were evaluated:

Experiment I

without S9 mix: 18 h: 100; 300; 1000 µg/ml 28 h: 1000 µg/ml

with S9 mix: 18 h: 300; 2500; 5000 µg/ml 28 h: 5000 µg/ml

Experiment II

without S9 mix: 18 h: 300; 1500; 2000 µg/ml 28 h: 300 µg/ml

with S9 mix: 18 h: 300; 2500; 5000 µg/ml 28 h: 2500 µg/ml

In both independent experiments, there were no biologically relevant increases in cells with structural aberrations after treatment with the test article. In both experiments, no biologically relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the negative controls. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosomal aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, FAT 45168/A is considered to be non-clastogenic in this chromosome aberration test.

Conclusion:

Reactive Orange 035 was found to have exerted a marginal mutagenic action with Salmonella typhimurium TA 100 and TA 102 strains. Reactive Orange 035 is a diazo compound, and as widely known, the high levels of reductase enzymes present in bacterial cells compared to mammalian cells, are responsible for converting the azo compounds to electrophilic compounds and thereby may lead to false positive results in the bacterial reverse mutation assay. Hence, the observed marginal action was considered to be the false positive owing to the diazo structure present in the dye. Both Reactive Black 039 pentasodium as well as lithium sodium salt, were not mutagenic in the bacterial reverse mutation assays. Further, the pentasodium salt of the source chemical was not mutagenic in the in vitro mammalian cell gene mutation assay conducted using Chinese hamster V79 cells. Taking the above information into account, Reactive Orange 035 is regarded as not mutagenic. Both Reactive Orange 035 and Reactive Black 039 lithium sodium salt were not clastogenic in the in vitro chromosomal aberration assays. Thus, the target as well as the source chemical can be considered to neither mutagenic nor clastogenic, and hence not genotoxic.

Justification for classification or non-classification

Reactive Orange 035 has been found to be not genotoxic, hence it does not warrant classification as the Regulation (EC) No. 1272/2008.