Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Increased numbers of revertant colonies were detected in TA 98 after exposure with and without metabolic activation. These observations were, however, not confirmed in a mutagenicity study using mammalian cells. The test material is therefore not considered to present a mutagenic hazard to humans.
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:
disregarded due to major methodological deficiencies
Study period:
2001
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: The test was performed without the modification according to Prival for azo-compounds, although the ligands contain azo-sturcutures. The purity of the test sample is not given
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No modification for azo compounds
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his- ~ his+ and trp· ~ trp+ reversions
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9mix
Test concentrations with justification for top dose:
0; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene,
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
Evaluation criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Strain:  TA 1535              Activation: no

Concentration µg/plate

Revertants/plate

(mean of 3)

SD

Factor

Negative Control

11

2,1

-

Solvent Control

11

0,6

1,0

Positive control

590

11,1

55,3

3

6

1,2

0,5

10

5

1,5

0,4

33

6

1,0

0,6

100

6

1,0

0,6

333

5

1,2

0,4

1000

7

1,2

0,7

2500

5

1,2

0,5

5000

7

1,5

0,6

 

 Strain:  TA 1535              Activation: S9 mix

Concentration µg/plate

Revertants/plate

SD

Factor

Negative Control

10

1,0

-

Solvent Control

12

4,4

1,0

Positive control

151

27,1

12,6

3

8

1,0

0,7

10

9

3,2

0,8

33

10

2,1

0,8

100

7

2,3

0,6

333

6

2,0

0,5

1000

5

3,2

0,4

2500

4

1,2

0,3

5000

7

2,6

0,6

Strain:  TA 1537              Activation: no

Concentration µg/plate

Revertants/plate

(mean of 3)

SD

Factor

Negative Control

6

1,2

-

Solvent Control

5

1,0

1,0

Positive control

43

1,7

8,6

3

4

1,2

0,7

10

3

2,3

0,7

33

3

1,5

0,7

100

5

1,7

1,0

333

4

1,5

0,7

1000

3

0

0,6

2500

10

1,2

2,1

5000

11

3,2

2,3

 

 Strain:  TA 1537              Activation: S9 mix

Concentration µg/plate

Revertants/plate

SD

Factor

Negative Control

6

1,7

-

Solvent Control

5

1,2

1,0

Positive control

80

11,8

17,1

3

4

0,6

0,8

10

5

1,2

1,0

33

5

1,5

1,1

100

5

2,3

1,0

333

7

1,5

1,4

1000

10

2,0

2,1

2500

7

0,6

1,6

5000

7

2,1

1,6

Strain:  TA 98   Activation: no

Concentration µg/plate

Revertants/plate

(mean of 3)

SD

Factor

Negative Control

21

4,6

-

Solvent Control

17

2,5

1,0

Positive control

184

11,6

10,6

3

17

3,1

1,0

10

19

5,9

1,1

33

19

2,3

1,1

100

20

3,1

1,1

333

41

7,5

2,3

1000

80

3,6

4,6

2500

126

9,9

7,2

5000

134

4,5

7,7

 

Strain:  TA 98   Activation: S9 mix

Concentration µg/plate

Revertants/plate

SD

Factor

Negative Control

21

5,0

-

Solvent Control

23

2,1

1,0

Positive control

619

71,6

27,3

3

18

4,0

0,8

10

19

5,5

0,8

33

19

0,6

0,9

100

26

2,3

1,2

333

41

4,5

1,8

1000

63

7,9

2,8

2500

7

4,5

3,3

5000

135

6,0

6,0

 

Strain:  TA 100                Activation: no

Concentration µg/plate

Revertants/plate

(mean of 3)

SD

Factor

Negative Control

120

5

-

Solvent Control

118

12

1,0

Positive control

701         

21

5,9

3

117

4

1,0

10

118

11

1,0

33

115

4

1,0

100

112

15

1,0

333

106

13

0,9

1000

134

11

1,1

2500

161

9

1,4

5000

164

24

1,4

 

Strain:  TA 100                Activation: S9 mix

Concentration µg/plate

Revertants/plate

SD

Factor

Negative Control

167

15

-

Solvent Control

140

20

1,0

Positive control

1135

196

8,1

3

143

7

1,0

10

132

5

0,9

33

120

12

0,9

100

139

18

1,0

333

164

5

1,2

1000

178

6

1,3

2500

185

22

1,3

5000

172

14

1,2

 

 

Strain:  WP2 uvrA         Activation: no

Concentration µg/plate

Revertants/plate

(mean of 3)

SD

Factor

Negative Control

32

5,5

-

Solvent Control

37

5,3

1,0

Positive control

573

16,6

15,5

3

34

8,1

0,9

10

33

1,2

0,9

33

41

9,3

1,1

100

36

2,1

1,0

333

30

2,9

0,8

1000

34

2,5

0,9

2500

3

1,2

0,8

5000

32

5,9

0,9

  

Strain:  WP2 uvrA         Activation: S9 mix

Concentration µg/plate

Revertants/plate

SD

Factor

Negative Control

42

3,5

-

Solvent Control

39

6,1

1,0

Positive control

175

6,4

4,5

3

33

6,7

0,9

10

39

1,

1,0

33

36

7,0

0,9

100

43

0,6

1,1

333

40

5,5

1,0

1000

47

3,2

1,2

2500

43

2,1

1,1

5000

33

2,0

0,8

 

Conclusions:
Interpretation of results (migrated information):
positive

Under the experimental conditions reported, the test item induced gene mutations by frameshifts in strain TA 98 at precipitating concentrations with and without metabolic activaation.
Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed with and without rat liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 3, 1 0; 33; 1 00; 333; 1 000; 2500; and 5000 µg/plate The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation. No relevant toxic effects, evident as a dose dependent reduction of the number of revertants below 0.5 times the corresponding solvent control, occurred in the test groups with and without metabolic activation. A dose dependent increase in revertant colony numbers was observed following treatment with the test item i

n strain TA 98 with and without metabolic activation. The threshold of twice the colony count of the corresponding solvent control was exceeded at 333 µg/plate and above in the absence - and at 1 000 µg/plate and above in the presence of metabolic activation. Precipitation of the test item occurred at the concentrations producing mutagenic effects above the threshold mentioned above. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item induced gene mutations by frameshifts in the genome of the strain T A 98 in the precipitating concentration range. Therefore,

the test item i

s considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
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
Target gene:
This mammalian cell mutation assay system detects point mutations involving base substitutions, deletions, frameshifts and rearrangements within the locus. The enzyme hypoxanthine guanine phosphoribosyl transferase (hprt) catalyses phosphorylation of purines in one of the purine salvage pathways. The selective agent used in this assay, 6-Thioguanine (6-TG), is also a substrate for this enzyme and cells that retain the functional hprt enzyme are susceptible to the cytotoxic effects of 6-TG. Forward mutations that result in the loss of the functional hprt gene render cells resistant to 6-TG. These mutant cells can be quantitated after an expression period by cloning in culture medium supplemented with 6-TG, the selective agent.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 homogenate
Test concentrations with justification for top dose:
A) 4 B) 15 C) 58 and D) 230 µg/mL (factor of 4)

Vehicle / solvent:
DMSO was used as the vehicle control.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Details on test system and experimental conditions:
- Source of the Test System:
American Type Culture Collection, P. O. Box 1549, Manassas, VA 20108, USA

- Storage of Test System
Stock cultures of the CHO-K1 cell line were stored in the test facility as frozen permanents in liquid nitrogen.

Evaluation criteria:
- Evaluation and Interpretation : When all the validity criteria are fulfilled:

a.A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

•At least one of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
•The increase is dose-dependent when evaluated with an appropriate trend test
•Any of the results are outside the distribution of the historical vehicle control data

b.A test chemical is considered to be clearly negative if, in all experimental conditions examined:

•None of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
•There is no concentration-related increase when evaluated with an appropriate trend test
•All results are inside the distribution of the historical vehicle control data

c.The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
A power transformation procedure (Snee and Irr, 1981) was used with which, the observed mutant frequency was transformed.
Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p < 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p < 0.05).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

TABLE 3.         Determination of pH of Test Medium

Treatment (mg/mL)

pH at the beginning of exposure to treatment

pH at the end of exposure to treatment

With S9

Without S9

With S9

Without S9

DMSO

7.10

7.03

7.00

7.20

4

7.07

7.10

7.09

7.15

8

7.11

7.12

7.10

7.13

20

7.15

7.09

7.00

7.14

50

7.19

7.14

7.12

7.10

128

7.20

7.18

7.15

7.07

320

7.15

7.21

7.13

7.09

800

7.13

7.20

7.12

7.10

2000

7.21

7.18

7.15

7.12

 

Note: The test item precipitated in the test medium at and above 800 µg/mL both in the presence and absence of metabolic activation at 3 hours post-treatment.

TABLE 2.         Determination of Osmolality of Test Medium

Treatment (mg/mL)

Osmolality at the beginning of exposure to treatment

(OSMOL/kg)

Osmolality at the end of exposure to treatment

(OSMOL/kg)

With S9

Without S9

With S9

Without S9

DMSO

0.470

0.463

0.483

0.456

320

-

-

0.471

0.452

800

-

-

0.459

0.455

2000

0.439

0.440

0.460

0.434

 

TABLE 3.         Results of Preliminary Cytotoxicity Test

 

Treatment

(mg/mL)

3-hour exposure with metabolic activation

3-hour exposure without metabolic activation

Cloning Efficiency

(CE)

Cells at end of treatment

(x105/flask)

Adjusted Cloning Efficiency (ACE)

Relative Survival (%RS)

Cloning Efficiency

(CE)

Cells at end of treatment

(x105/flask)

Adjusted Cloning Efficiency (ACE)

Relative Survival (%RS)

DMSO

0.93

14.70

0.83

100

0.94

14.05

0.80

100

4

0.88

14.05

0.75

90

0.86

13.40

0.70

88

8

0.66

13.70

0.55

66

0.82

11.85

0.59

74

20

0.63

11.00

0.42

51

0.67

11.10

0.45

56

50

0.60

9.85

0.36

43

0.61

10.50

0.39

49

128

0.58

7.35

0.26

31

0.48

9.00

0.26

33

320

0.10

6.25

0.04

5

0.09

6.85

0.04

5

800

No colony growth

No colony growth

2000

Note: Baseline cell count (No. of cells at the beginning of treatment): 16.5 x 105cells/mL                      

          CE and ACE values are rounded of 2 decimal points and RS values are rounded off to the nearest whole number

TABLE 4.         Parallel Cytotoxicity Test Results from Experiment 1

Treatment

µg/mL

No. of Colonies /Flask

CE*

ACE

RS

%

1

2

3

DMSO

191

182

190

0.95

0.93

100

196

184

193

4

180

175

170

0.88

0.81

87

178

181

172

15

131

128

130

0.67

0.56

60

141

137

132

58

117

120

115

0.59

0.40

43

122

112

114

230

60

65

62

0.32

0.18

19

71

59

61

3-MCA

152

150

147

0.77

0.55

59

157

161

152

TABLE 5.         Parallel Cytotoxicity Test Results from Experiment 2

Treatment

µg/mL

No. of Colonies /Flask

CE*

ACE

RS

%

1

2

3

DMSO

184

192

190

0.95

0.95

100

182

195

198

4

170

184

169

0.88

0.79

83

179

182

164

15

141

137

140

0.69

0.56

59

132

145

128

58

110

118

112

0.58

0.40

42

117

120

115

230

61

65

75

0.34

0.19

20

78

64

60

TABLE 6.         Summary Results of the Gene Mutation Assay in the Presence of Metabolic Activation (Experiment 1)

Treatment

µg/mL

Mutation Assay Flasks

Cloning Efficiency of Mutant Colonies

Cloning Efficiency Flasks

6-TG Mutants

per 106Clonable Cells

(MF)

TG Colonies/Flask

No. of Colonies/Flask

1

2

3

4

5

Total

1

2

3

CE*

DMSO

3

0

3

1

2

17

0.0000085

189

191

184

0.92

9.24

1

3

1

1

2

178

175

185

4

3

1

1

0

2

16

0.000008

171

182

176

0.89

8.99

3

2

2

1

1

182

180

174

15

0

2

3

2

3

21

 

0.000011

170

168

184

0.86

12.21

3

2

0

4

2

182

167

165

58

3

1

3

1

1

21

0.000011

180

171

174

0.85

12.35

2

1

3

5

1

172

161

160

230

4

1

2

0

2

18

 

0.000009

158

160

157

0.80

11.25

2

2

3

2

0

164

155

161

3-MCA

37

34

36

35

28

340

0.000170

147

156

155

0.77

220.78

36

30

38

32

34

153

160

157

TABLE 7.         Summary Results of the Gene Mutation Assay in the Absence of Metabolic Activation (Experiment 2)

Treatment

µg/mL

Mutation Assay Flasks

Cloning Efficiency of Mutant Colonies

Cloning Efficiency Flasks

6-TG Mutants

per 106Clonable Cells

(MF)

TG Colonies/Flask

No. of Colonies/Flask

1

2

3

4

5

Total

1

2

3

CE*

DMSO

2

3

2

5

0

22

0.0000110

190

187

191

0.94

11.70

2

3

2

3

0

185

183

186

4

2

2

1

2

3

22

0.000011

177

180

174

0.88

12.50

2

3

2

2

3

184

170

172

15

3

2

2

1

1

16

0.000008

167

179

165

0.87

9.20

2

2

1

1

1

178

181

177

58

0

3

2

1

4

22

0.000011

160

172

158

0.83

13.25

2

3

2

4

1

177

162

164

230

2

0

4

4

3

20

0.000010

167

158

155

0.80

12.50

2

1

3

1

0

160

154

169

Vehicle Control: DMSO              CE: Cloning Efficiency        MF: Mutant Frequency                                        

* calculated from the mean values of the replicates of each group and rounded off to two decimal places

 

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that the test item, Savinyl-Feuerrot 3GLS (Savinyl Fire Red 3GLS) does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.
Executive summary:

The genotoxic potential of the test item Savinyl-Feuerrot 3GLS (Savinyl Fire Red 3GLS) to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.

 

The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

Savinyl-Feuerrot 3GLS (Savinyl Fire Red 3GLS) was soluble in Dimethyl sulphoxide (DMSO) at 200 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the test item showed evidence of significant cell growth inhibition as Relative Survival between 128 and 320 µg/mL both in the presence and absence of metabolic activation. There was no colony formation at 800 and 2000 µg/mL both in the presence and absence of metabolic activation, due to test item toxicity. The test item precipitated in the test medium at and above 800 µg/mL, but did not show any appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 230 µg/mL was tested in the gene mutation assay. 

 

In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 4, 15, 58 and 230 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) was also tested in duplicate.

 

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. In each of these experiments, the respective positive controls produced a statistically significant increase in the frequencies of mutants, under identical conditions.

 

The results of the forward gene mutation test at thehprtlocus with Savinyl-Feuerrot 3GLS (Savinyl Fire Red 3GLS) indicated that the test item was non-mutagenic under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

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

Additional information

Additional information from genetic toxicity in vitro:

The test item showed mutagenic activity in TA 98 with and without metabolic activation by rat liver S9 mix. The substance is considered a mutagen in bacterial cells. As however, these observations were not confirmed in a mutagenicity study using mammalian cells. The test material is therefore not considered to present a mutagenic hazard.


Justification for selection of genetic toxicity endpoint
All available valid studies.

Justification for classification or non-classification

No classification

According to 1272/2008 EU a single positive result in a bacterial test alone is not suffcient for classification.