Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material showed mutagenic effects in 1 strain of Salmonella typhimurium (TA 98) with metabolic activation.

The test item did not induce gene mutations inthe hprt locus in CHO cells in vitro.

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:
2014-08-26 till 2014-08-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
Vehicle:
- Vehicle(s)/solvent(s) used: suspended in DMSO
- Justification for choice of solvent/vehicle: Better than others
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
Details on test system and conditions:
METHOD OF APPLICATION: plate incorporation


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
in strain TA 98 with metabolic activation
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Water solubility: unsoluble
- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. No precipitation of the test item was observed on the incubated agar plates. The undissolved particles had no influence on the data recording.

- Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: performed
The data in the untreated control of strain TA 1535 were slightly above our historical control range in the absence of metabolic activation. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal back¬ground growth up to 5000 µg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol¬ic activation.

Study Name: 1652800

Study Code: Harlan CCR 1652800

Experiment: 1652800 VV Plate

Date Plated: 26/08/2014

Assay Conditions:

Date Counted: 29/08/2014

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

26 ± 4B M

7 ± 2

21 ± 7

174 ± 10

35 ± 13

Untreated

 

 

28 ± 3B M

10 ± 3

22 ± 6

162 ± 30

48 ± 5

Fat Blue B 01

3 µg

 

27 ± 4B M

10 ± 4

28 ± 1

177 ± 2

42 ± 0

 

10 µg

 

26 ± 2B M

8 ± 2

19 ± 4

166 ± 9

39 ± 5

 

33 µg

 

30 ± 2B M

8 ± 1

22 ± 3

191 ± 2

44 ± 6

 

100 µg

 

26 ± 1B M

7 ± 1

17 ± 2

216 ± 16

41 ± 4

 

333 µg

 

28 ± 2B M

9 ± 1

19 ± 9

185 ± 16

54 ± 9

 

1000 µg

 

28 ± 1B D M

7 ± 1D

21 ± 1D

173 ± 10D

44 ± 9D

 

2500 µg

 

26 ± 5B D M

6 ± 2D M

21 ± 4D M

174 ± 11D M

32 ± 4D M

 

5000 µg

 

28 ± 2B D M

6 ± 1D M

17 ± 2D M

176 ± 13D M

27 ± 4D M

NaN3

10 µg

 

2459 ± 235

 

 

1835 ± 57

 

4-NOPD

10 µg

 

 

 

395 ± 13

 

 

4-NOPD

50 µg

 

 

80 ± 8

 

 

 

MMS

2.0 µL

 

 

 

 

 

1107 ± 49

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

13 ± 6

15 ± 2

30 ± 10

123 ± 23

55 ± 4

Untreated

 

 

14 ± 7

12 ± 5

38 ± 6

175 ± 4

57 ± 14

Fat Blue B 01

3 µg

 

19 ± 5

20 ± 1

41 ± 1

155 ± 7

41 ± 4

 

10 µg

 

18 ± 2

18 ± 5

32 ± 3

146 ± 5

54 ± 6

 

33 µg

 

17 ± 0

19 ± 4

37 ± 13

155 ± 4

53 ± 2

 

100 µg

 

16 ± 4

16 ± 1

42 ± 2

163 ± 16

51 ± 5

 

333 µg

 

13 ± 5

22 ± 4

73 ± 3

148 ± 16

50 ± 2

 

1000 µg

 

14 ± 3D

20 ± 5D

117 ± 13D

151 ± 18D

46 ± 5D

 

2500 µg

 

13 ± 2D M

21 ± 2D M

172 ± 33D M

168 ± 6D M

49 ± 10D M

 

5000 µg

 

13 ± 3D M

18 ± 2D M

192 ± 7D M

179 ± 5D M

34 ± 3D M

2-AA

2.5 µg

 

454 ± 13

310 ± 18

2167 ± 35

4272 ± 129

 

2-AA

10.0 µg

 

 

 

 

 

212 ± 10

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

D

M

B

Densely coloured plate

Manual count

Extensive bacterial growth

 

Conclusions:
Interpretation of results (migrated information):
positive in strain TA 98 with metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 98 with S9 mix.
Executive summary:

The test item Fat Blue B 01 was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmo­nella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and theEscheri­chia colistrain WP2 uvrA.

The assay was performed with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. The test item was tested at the following concentrations:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. No precipitation of the test item was observed on the incubated agar plates. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol­ic activation.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with Fat Blue B 01 at any dose level in the absence of metabolic activation (S9 mix).

In the presence of metabolic activation a substantial and dose dependent increase was observed in strain TA 98.The number of colonies exceeded the threshold of twice the number of the corresponding solvent control at concentrations from 333 µg/plate to 5000 µg/plate

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

The data in the untreated control of strain TA 1535 were slightly above our historical control range in the absence of metabolic activation. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

 

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
Reference:
Composition 0
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 information:
Composition 1
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:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 homogenate
Test concentrations with justification for top dose:
A) 250 B) 500 C) 1000 and D) 2000 µg/mL (factor of 2)

Vehicle:
DMSO was used as the vehicle control.
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Details on test system and 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).
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

TABLE 1.         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

7.09

7.22

7.01

7.07

8

7.11

7.31

7.00

7.07

16

7.12

7.26

6.99

7.12

32

7.14

7.26

7.02

7.25

64

7.14

7.27

7.02

7.21

128

7.13

7.28

7.00

7.14

256

7.14

7.28

7.04

7.14

512

7.15

7.27

7.03

7.18

1024

7.14

7.30

7.02

7.20

2000

7.14

7.33

7.04

7.17

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

0.467

0.466

0.464

0.469

64

-

-

0.455

0.456

128

-

-

0.452

0.464

2000

0.437

0.437

0.430

0.433

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

(1x105/mL)

Adjusted Cloning Efficiency

Relative Survival (%RS)*

Cloning Efficiency

(CE)

Cells at end of treatment

(1x105/mL)

Adjusted Cloning Efficiency

Relative Survival (%RS)

DMSO

0.96

12.4

1.39

100

0.95

13.35

1.48

100

8

0.89

11.80

1.22

88

0.86

12.45

1.25

84

16

0.84

11.45

1.12

81

0.78

12.15

1.11

75

32

0.76

9.90

0.88

63

0.75

10.75

0.94

64

64

0.67

9.20

0.72

52

0.66

10.05

0.77

52

128

0.60

9.50

0.66

47

0.55

10.00

0.64

43

256

0.52

9.05

0.55

40

0.58

9.45

0.64

43

512

0.49

8.80

0.50

36

0.52

8.35

0.51

34

1024

0.43

8.05

0.40

29

0.37

7.90

0.34

23

2000

0.34

7.45

0.30

22

0.32

7.80

0.29

20

TABLE 4.         Parallel Cytotoxicity Test Results from Experiment 1

Treatment

µg/mL

No. of Colonies /Flask

CE*

ACE

RS

%

1

2

3

DMSO

198

195

199

0.99

1.03

100

199

196

198

250

99

109

106

0.53

0.52

50

108

101

110

500

88

81

86

0.42

0.40

39

79

84

82

1000

64

70

72

0.35

0.29

28

71

74

69

2000

58

54

56

0.29

0.22

21

60

58

59

3-MCA

130

123

126

0.64

0.46

45

121

135

127

CE: Cloning EfficiencyACE: Adjusted CE                 RS: Relative Survival   

TABLE 5.         Parallel Cytotoxicity Test Results from Experiment 2

Treatment

µg/mL

No. of Colonies /Flask

CE

ACE

RS

%

1

2

3

DMSO

194

193

196

0.96

0.95

100

194

186

191

250

104

109

100

0.51

0.48

51

106

100

98

500

98

91

89

0.45

0.42

44

88

90

85

1000

74

74

78

0.37

0.32

34

76

69

72

2000

68

70

62

0.32

0.22

23

59

65

64

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

1

2

1

2

18

0.000009

186

192

190

0.96

9.38

1

2

2

2

2

194

191

193

250

2

0

1

3

1

13

0.000007

176

184

186

0.89

7.86

1

1

2

1

1

169

179

177

500

0

2

1

1

1

12

0.000006

158

149

162

0.79

7.59

1

3

2

1

0

163

159

160

1000

2

1

1

0

1

11

0.000006

148

131

141

0.71

8.45

1

1

2

1

1

142

147

140

2000

2

1

0

1

1

11

0.000006

126

111

127

0.60

10.00

3

1

0

1

1

123

110

119

3-MCA

30

33

31

35

33

322

0.000161

138

146

139

0.72

223.61

32

31

31

34

32

154

139

145

Positive Control: 8 µg/mL 3-MCA             Vehicle Control: DMSO               CE: Cloning Efficiency                MF: Mutant Frequency

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

1

2

2

2

3

21

0.000011

186

188

184

0.93

11.83

2

1

3

4

1

190

185

187

250

2

1

1

2

3

16

0.000008

169

174

177

0.86

9.30

1

3

1

1

1

171

170

173

500

2

1

1

2

1

14

0.000007

149

158

153

0.76

9.21

0

2

2

1

2

138

149

159

1000

2

3

0

1

2

14

0.000007

132

136

123

0.64

10.94

2

1

1

1

1

128

129

125

2000

1

1

2

1

1

13

0.000007

111

123

119

0.60

11.67

1

2

1

1

2

126

120

117

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

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that the test item, Fat Blue B01 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 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).

 

The test item formed a free flowing suspension 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 Relative Survival was 22 and 20% at the top concentration of 2000 µg/mL, in the presence and absence of metabolic activation, respectively. The test item precipitated in the test medium at and above 128 µg/mL, but did not show any appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of
2000 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 250, 500, 1000 and 2000 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) were 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. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.

 

The results of the forward gene mutation test at the hprt-locus 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

Additional information

Additional information from genetic toxicity in vitro:

The test item did induce gene mutations by frameshifts in the genome of strain TA 98 with S9 mix. However, no mutagenic effects were detected in the hprt-locus in CHO cells.


Justification for selection of genetic toxicity endpoint
more recent studies and according to GLP and guideline

Justification for classification or non-classification

not classified;

positive findings in an Ames test are not sufficient for classification. Additionally the findings could not be confirmed in a genemutation assay in CHO cells at the hprt locus.