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Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Batch Manufactured By: Clariant

Supplied By: Clariant India Private Limited, BUPI, Product Stewardship-India, Reliable Tech Park, Gut No. 31, Village Elthan, Thane-Belapur Road, Airoli, Navi Mumbai 400708, India

Test Item: Savinyl-Feuerrot 3GLS (Savinyl Fire Red 3GLS)

Synonym: Solvent Red 124 (S.R.124)

CAS No.: 71888-93-2

Physical Appearance: Red powder

Purity as per CoA: 98.8% (w/w) mixture of two comp.1-3 with cobalt (Co3+) as anion and comp. 4 as cation

Batch No.: AAM0021703

Manufactured Date: Unknown

Expiry Date: 16 January 2022

Recommended Storage Condition: Ambient (+15 to +25°C)

pH: about 5

Density: 1.18 g/cm3

Molecular weight: 1154 - 1342 g/mol

Method

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
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.
Controls
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).

Results and discussion

Test results
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'.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.