Registration Dossier

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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In an in vitro chromosome aberration assay (CA) according to OECD guideline 473, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.

In an in vitro mammalian gene mutation test (HPRT) in CHO-K1 cells according to OECD guideline 476, the test item did not show mutagenic properties.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 June - 09 August, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August, 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, June 2012
Version / remarks:
June 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Date of production: 30.11.2016
Expiration date: 30.11.2021
Target gene:
his/trp
In addition to histidine (his) or tryptophan (trp) mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair.

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:
S9 mix of phenobarbital and β-naphthoflavone-induced rat liver
Test concentrations with justification for top dose:
5000, 1600, 500, 160, 50, and 16 µg/plate
Selection of the concentration range was done on the basis of a solubility test and a concentration range finding test (informatory toxicity test).
Vehicle / solvent:
- Solvents used: ultrapure water (ASTM Type I), Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent: In the study two types of solvent control were used depending on the solubility of the test item and the solubility of positive control reference items.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylenediamine
Remarks:
TA98, without S9 mix, 4 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100 and TA1535, without S9 mix, 2 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537, without S9 mix, 50 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E.coli WP2, without S9 mix, 2 µL/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
All tested Salmonella strains, with S9 mix, 2 µg/plate; E.coli WP2, with S9 mix, 50 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period:
20 min
- Exposure duration:
48 h

NUMBER OF REPLICATIONS:
3

DETERMINATION OF CYTOTOXICITY
- Method: number of revertant colonies, background lawn growth

Rationale for test conditions:
Based on the solubility test, a stock solution/suspension with a concentration of 50 mg/mL was prepared in ultrapure water (ASTM Type I) and diluted accordingly. In the informatory toxicity test a correction factor, based on the active component of the test item (80.2 %) was taken into consideration.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.
In the informatory toxicity test the revertant colony numbers of solvent control plates with and without S9 mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected biological relevant increases in induced revertant colonies in both tester strains.
Evaluation criteria:
The colony numbers on the untreated, solvent control, positive control and the test item treated plates were determined visually by manual counting and the mean values, standard deviations, and the mutation rates were calculated:



* : untreated, solvent or positive control

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the solvent control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control.

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a negative response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
The mean values and appropriate standard deviations and mutation rates were calculated by EXCEL software.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.









Table 4: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichiacoli

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

21.0

1.03

26.0

1.18

93.7

1.05

96.7

1.13

8.3

0.89

11.3

0.87

7.0

0.81

8.3

1.09

26.7

1.00

25.7

0.99

DMSO Control

22.7

1.00

24.3

1.00

–

–

88.3

1.00

–

–

8.7

1.00

8.3

1.00

8.0

1.00

–

–

34.7

1.00

Ultrapure Water Control

20.3

1.00

22.0

1.00

89.0

1.00

85.7

1.00

9.3

1.00

13.0

1.00

8.7

1.00

7.7

1.00

26.7

1.00

26.0

1.00

5000

16.7

0.82

12.0

0.55

69.3

0.78

84.7

0.99

17.0

1.82

18.0

1.38

9.0

1.04

7.3

0.96

30.3

1.14

20.7

0.79

1600

15.3

0.75

14.0

0.64

77.7

0.87

82.0

0.96

8.0

0.86

9.7

0.74

11.0

1.27

10.3

1.35

25.0

0.94

20.3

0.78

500

17.0

0.84

15.3

0.70

80.0

0.90

89.7

1.05

8.7

0.93

10.3

0.79

7.7

0.88

9.3

1.22

29.3

1.10

21.0

0.81

160

16.3

0.80

23.3

1.06

72.3

0.81

90.0

1.05

7.3

0.79

11.0

0.85

9.3

1.08

6.3

0.83

30.7

1.15

26.3

1.01

50

17.3

0.85

29.0

1.32

72.3

0.81

83.3

0.97

10.7

1.14

14.0

1.08

11.7

1.35

6.7

0.87

28.0

1.05

28.3

1.09

16

18.0

0.89

25.7

1.17

80.3

0.90

94.0

1.10

8.3

0.89

11.7

0.90

6.7

0.77

6.0

0.78

27.0

1.01

32.0

1.23

NPD (4mg)

306.0

13.50

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

SAZ (2mg)

–

–

–

–

988.0

11.10

–

–

1428.0

153.00

–

–

–

–

–

–

–

–

–

–

9AA (50mg)

–

–

–

–

–

–

–

–

–

–

–

–

209.0

25.08

–

–

–

–

–

–

MMS (2mL)

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

810.7

30.40

–

–

2AA (2mg)

–

–

1957.3

80.44

–

–

1001.3

11.34

–

–

222.3

25.65

–

–

191.3

23.92

–

–

–

–

2AA (50mg)

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

202.0

5.83

MR:Mutation Rate;          NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA:9-Aminoacridine;MMS:Methyl methanesulfonate;2AA: 2-aminoanthracene

Remarks:           Ultrapure water was applied as vehicle of the test item and the positive control substance: SAZ and MMS; the DMSO was applied as vehicle for positive control substances NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Table 5 : Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

18.3

1.31

20.7

1.09

82.7

1.06

102.3

1.07

15.0

1.05

12.3

1.00

7.0

1.40

9.0

0.93

29.3

0.75

32.7

0.82

DMSO Control

14.7

1.00

20.0

1.00

–

–

92.0

1.00

–

–

14.7

1.00

6.0

1.00

8.7

1.00

–

–

37.3

1.00

Ultrapure Water Control

14.0

1.00

19.0

1.00

77.7

1.00

95.7

1.00

14.3

1.00

12.3

1.00

5.0

1.00

9.7

1.00

39.3

1.00

40.0

1.00

5000

13.0

0.93

14.7

0.77

55.0

0.71

88.3

0.92

17.0

1.19

24.3

1.97

4.7

0.93

7.0

0.72

33.3

0.85

32.0

0.80

1600

12.3

0.88

17.0

0.89

77.7

1.00

80.7

0.84

15.7

1.09

13.3

1.08

6.0

1.20

8.7

0.90

28.0

0.71

36.0

0.90

500

13.7

0.98

21.7

1.14

72.0

0.93

78.3

0.82

12.0

0.84

11.3

0.92

5.0

1.00

10.3

1.07

34.0

0.86

30.3

0.76

160

15.3

1.10

21.3

1.12

77.3

1.00

79.3

0.83

10.7

0.74

12.7

1.03

6.7

1.33

10.7

1.10

46.3

1.18

40.3

1.01

50

15.3

1.10

23.7

1.25

68.3

0.88

89.7

0.94

10.3

0.72

14.7

1.19

5.0

1.00

10.7

1.10

38.0

0.97

33.7

0.84

16

17.7

1.26

31.3

1.65

74.7

0.96

100.7

1.05

11.7

0.81

13.3

1.08

5.0

1.00

7.3

0.76

32.0

0.81

48.0

1.20

NPD (4mg)

304.0

20.73

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

SAZ (2mg)

–

–

–

–

981.3

12.64

–

–

978.7

68.28

–

–

–

–

–

–

–

–

–

–

9AA (50mg)

–

–

–

–

–

–

–

–

–

–

–

–

738.7

123.11

–

–

–

–

–

–

MMS (2mL)

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

1544.0

39.25

–

–

2AA (2mg)

–

–

1411.3

70.57

–

–

2442.7

26.55

–

–

234.0

15.95

–

–

209.7

24.19

–

–

–

–

2AA (50mg)

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

249.7

6.69

MR:Mutation Rate;          NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA:9-Aminoacridine;MMS:Methyl methanesulfonate;2AA: 2-aminoanthracene

Remarks:           Ultrapure water was applied as vehicle of the test item and the positive control substance: SAZ and MMS; the DMSO was applied as vehicle for positive control substances NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Table 6: Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

 

Bacterial strains

Historical control data of untreated control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

21.0

105.0

10.5

8.1

25.4

SD

3.7

25.7

1.4

2.3

5.2

Minimum

9

66

3

2

11

Maximum

39

155

23

19

45

n

226

236

216

214

215

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

27.5

117.1

11.8

9.0

33.9

SD

4.3

18.1

1.4

1.9

5.2

Minimum

12

75

4

2

17

Maximum

46

166

23

20

56

n

226

236

216

214

215

 

Bacterial strains

Historical control data of DMSO

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

20.4

100.1

10.3

7.9

24.7

SD

3.6

24.8

1.3

2.4

4.6

Minimum

10

64

3

2

11

Maximum

38

147

23

20

45

n

226

236

216

214

215

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

26.5

113.8

11.8

8.8

33.7

SD

4.1

18.3

1.5

1.9

5.0

Minimum

15

71

3

3

16

Maximum

47

162

25

20

57

n

226

236

216

214

215

 

Bacterial strains

Historical control data of Water

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

21.9

104.7

10.5

7.6

26.1

SD

3.7

25.9

1.5

2.2

5.5

Minimum

12

68

3

2

12

Maximum

35

154

24

16

48

n

89

236

216

89

215

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

27.4

117.3

11.4

8.7

34.9

SD

4.0

18.5

1.3

2.2

4.9

Minimum

15

83

4

3

18

Maximum

43

167

22

16

57

n

89

152

149

89

148

 

Bacterial strains

Historical control data of positive controls

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

260.1

977.2

847.3

478.6

724.5

SD

31.8

150.6

126.3

104.5

65.0

Minimum

123

521

359

110

320

Maximum

664

1970

1855

1601

1313

n

226

236

216

214

215

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

1222.7

1436.4

164.1

147.0

257.7

SD

274.9

318.3

33.1

20.1

72.5

Minimum

386

583

85

69

140

Maximum

2676

2988

498

399

477

n

226

236

216

214

215

Abbreviations:   TA98, TA100, TA1535, TA1537: Salmonella typhimuriumTA98, TA100, TA1535, TA1537; E. coli:Escherichia coliWP2uvrA

                                     SD: Standard deviation;   DMSO: Dimethyl sulfoxide;  n: number of studies

Conclusions:
In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay (Ames) according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a S9 mix prepared from livers of phenobarbital/beta-naphthoflavone-induced rats. The study included preliminary solubility test, preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding tests the test item was in ultrapure water (ASTM Type I). At the formulation of test item solutions correction of concentrations for active component content (80.2 % dyestuff) was made in the main experiments. Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item (based on 97.4 % dyestuff) were prepared and investigated in the initial and confirmatory mutation tests: 5000, 1600, 500, 160, 50, and 16 µg/plate. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study. The test item did not show unequivocal inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) were considered to be within the biological variability range of the applied test system. The revertant colony numbers of solvent control (ultrapure water (ASTM Type I)) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was solved in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected biologically relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants mostly fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September, 2017 - 03 January, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29 July, 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
14 February, 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: structural chromosome aberrations test in somatic and/or germ cells
Specific details on test material used for the study:
Expiration date: 30 Nov 2021
Target gene:
structural chromosome aberrations in metaphase somatic and/or germ cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ECACC (European Collection of Cells Cultures), Lot. No.: 10H016
- Suitability of cells: The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates
- Doubling time 12 - 14 h
- Sex: male
- Methods for maintenance in cell culture: The cell stocks were kept in liquid nitrogen and were routinely checked for mycoplasma infections. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm² plastic flasks at 37 ± 0.5 °C in a humidified atmosphere in an incubator, set at 5% CO2.
- Modal number of chromosomes: diploid number, 2n=22

MEDIA USED
- Type and identity of media including CO2 concentration: DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM) and 1% of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%). 37 °C, 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: not specified
Cytokinesis block (if used):
colchicine (0.2 μg/mL)
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver.
Test concentrations with justification for top dose:
Experiment A with 3/20 h treatment/sampling time

without S9 mix: 39.1, 78.2, 156.3 and 312.5 µg/mL test item
with S9 mix: 39.1, 78.2, 156.3 and 312.5 µg/mL test item

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 9.8, 19.6, 39.1 and 58.7 µg/mL test item

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 9.8, 19.6, 39.1 and 58.7 µg/mL test item

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 39.1, 78.2, 156.3 and 312.5 µg/mL test item
Vehicle / solvent:
- Solvent used: DME (Dulbecco’s Modified Eagle’s)
- Justification for choice of solvent: DME is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary solubility test, and its suitability is confirmed with the available laboratory’s historical database.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Experiment A, without S9 mix, 1.0 µL/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Experiment B, without S9 mix, 0.4 µL/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Experiment A + B, with S9 mix, 5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 5 x 10E5

DURATION
- Preincubation period:
- Exposure duration: 3 h
- Expression time: 20 h
- Fixation time: 10 min. in 3:1 mixture of methanol: acetic-acid

SPINDLE INHIBITOR (cytogenetic assays):
Cell cultures were treated with colchicine (0.2 μg/mL) 2.5 hours prior to harvesting.

STAIN (for cytogenetic assays):
The preparation was stained with 5 % Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 300; 150 per duplicate

DETERMINATION OF CYTOTOXICITY
- Method: Relative Increase in Cell Counts (RICC)
Evaluation criteria:
Evaluation of Result
- The percentage of cells with structural chromosome aberration(s) was evaluated.
- Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
- Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
- Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
- Individual culture data were summarised in tabular form.
- There were no equivocal results in this study.
-pH and Osmolality data were summarised in tabular form.

Interpretation of Results
Providing that all acceptability criteria are fulfilled, a test item 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 compared with the concurrent negative control
- the increase is dose-related when evaluated with an appropriate trend test
- any of the results are outside the distribution of the laboratory historical negative control data

Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- there is no concentration-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the laboratory historical negative control data

Both biological and statistical significance should be considered together.
There is no requirement for verification of a clearly positive or negative response.
Statistics:
For statistical analysis CHI² test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and
number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the
concurrent negative control.
The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95%
confidence intervals of historical control were calculated with C-chart.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH or osmolality: There were no relevant changes in pH or osmolality after treatment with the test item.
- Water solubility: A clear solution of Solubilised Sulphur Black 1 was obtained in DME (Dulbecco’s Modified Eagle’s) medium up to a concentration of 25 mg/mL.
- Precipitation: There was no precipitation in the medium at any concentration tested.

RANGE-FINDING/SCREENING STUDIES:
A pre-test on cytotoxicity was performed as part of this study to establish an appropriate concentration range for the main chromosome aberration assays (experiment A and B), both in the absence and in the presence of a metabolic activation (rodent S9 mix). Based on cell counts the Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. Based on the results of the cytotoxicity assay the following concentrations were selected for the chromosome aberration assay:

HISTORICAL CONTROL DATA
- Positive historical control data:
Without S9 mix:
Ethyl methanesulfonate (excl. gaps): Mean 31.47; SD 3.77; Lower confidence interval 23.51; upper confidence interval 39.44
With S9 mix:
Cyclophasphamide (excl. gaps): Mean 39.39; SD 2.62; Lower confidence interval 33.85; upper confidence interval 44.93

- Negative (solvent/vehicle) historical control data:
Without S9 mix (excl. gaps): Mean 2.75; SD 0.65; Lower confidence interval 1.39; upper confidence interval 4.11
With S9 mix (excl. gaps): Mean 2.86; SD 0.65; Lower confidence interval 1.50; upper confidence interval 4.22

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

Table 4: 3-hour treatment without and with S9 mix / 20-hour sampling time 

Test group

Concentration
(µg/mL)

Parallels

S9-mix

Cell counts

Mean cell counts

Increase in cell counts

RICC (%)

Cytotoxicity
(%)

First count

Second count

Initial cell count

-

A

–

2050000

2000000

1975000

-

-

-

-

B

–

1900000

1900000

-

C

–

2100000

1850000

-

D

–

1950000

2050000

Solvent control (DME)

-

A

–

7400000

7250000

7325000

5350000

100.00

0.00

-

B

–

7350000

7300000

Solubilised Sulphur Black 1

39.1

A

–

7200000

7050000

7125000

5150000

96.26

3.74

78.2

A

–

6250000

6550000

6400000

4425000

82.71

17.29

156.3

A

–

5650000

5900000

5775000

3800000

71.03

28.97

312.5

A

–

4500000

4250000

4375000

2400000

44.86

55.14

625

A

–

3750000

3500000

3625000

1650000

30.84

69.16

1250

A

–

3100000

2950000

3025000

1050000

19.63

80.37

2500

A

–

850000

1000000

925000

-1050000*

-19.63**

119.63***

5000

A

–

0

0

0

-1975000*

-36.92**

136.92***

EMS 1 µL/mL

A

–

4850000

4600000

4725000

2750000

51.40

48.60

Solvent control (DME)

-

A

+

7500000

7650000

7575000

5600000

100.00

0.00

-

B

+

7400000

7750000

Solubilised Sulphur Black 1

39.1

A

+

7500000

7500000

7500000

5525000

98.66

1.34

78.2

A

+

6050000

5750000

5900000

3925000

70.09

29.91

156.3

A

+

5350000

5450000

5400000

3425000

61.16

38.84

312.5

A

+

4750000

4550000

4650000

2675000

47.77

52.23

625

A

+

4150000

3850000

4000000

2025000

36.16

63.84

1250

A

+

2600000

2450000

2525000

550000

9.82

90.18

2500

A

+

1000000

1200000

1100000

-875000*

-15.63**

115.63***

5000

A

+

0

0

0

-1975000*

-35.27**

135.27***

Cycl. 5µg/mL

A

+

4700000

4850000

4775000

2800000

50.00

50.00

RICC=Relative Increase in Cell Counts               EMS: Ethyl methanesulfonate (EMS)                    *: cell number decrease          

Cytotoxicity= 100-RICC                                        Cycl: Cyclophosphamide monohydra                    **: zero RICC value,             ***:100% cytotoxicity

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY 

Table 5: 20-hour treatment without S9 mix / 20-hour sampling time 

Test group

Concentration
(µg/mL)

Parallels

S9-mix

Cell counts

Mean cell counts

Increase in cell counts

RICC (%)

Cytotoxicity
(%)

First count

Second count

Initial cell count

-

A

–

2050000

2000000

1975000

-

-

-

-

B

–

1900000

1900000

-

C

–

2100000

1850000

-

D

–

1950000

2050000

Solvent control (DME)

-

A

–

6100000

6250000

6237500

4262500

100.00

0.00

-

B

–

6200000

6400000

Solubilised Sulphur Black 1

39.1

A

–

4650000

4500000

4575000

2600000

61.00

39.00

78.2

A

–

3000000

3000000

3000000

1025000

24.05

75.95

156.3

A

–

1650000

1400000

1525000

-450000*

-10.56**

110.56***

312.5

A

–

700000

500000

600000

-1375000*

-32.26**

132.26***

625

A

–

50000

0

25000

-1950000*

-45.75**

145.75***

1250

A

–

0

0

0

-1975000*

-46.33**

146.33***

2500

A

–

0

0

0

-1975000*

-46.33**

146.33***

5000

A

–

0

0

0

-1975000*

-46.33**

146.33***

EMS 1 µL/mL

A

–

4100000

4300000

4200000

2225000

52.20**

47.80***

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY 

Table 6: 20-hour treatment without S9 mix and 3-hour treatment with S9 mix / 28-hour sampling time 

Test group

Concentration
(µg/mL)

Parallels

S9-mix

Cell counts

Mean cell counts

Increase in cell counts

RICC (%)

Cytotoxicity
(%)

First count

Second count

Initial cell count

-

A

–

2050000

2000000

1975000

-

-

-

-

B

–

1900000

1900000

-

C

–

2100000

1850000

-

D

–

1950000

2050000

Solvent control (DME)

-

A

–

8950000

9050000

8987500

7012500

100.00

0.00

-

B

–

9100000

8850000

Solubilised Sulphur Black 1

39.1

A

–

6150000

6150000

6150000

4175000

59.54

40.46

78.2

A

–

3500000

3750000

3625000

1650000

23.53

76.47

156.3

A

–

1500000

1800000

1650000

-325000*

-4.63**

104.63***

312.5

A

–

450000

300000

375000

-1600000*

-22.82**

122.82***

625

A

–

150000

50000

100000

-1875000*

-26.74**

126.74***

1250

A

–

0

0

0

-1975000*

-28.16**

128.16***

2500

A

–

0

0

0

-1975000*

-28.16**

128.16***

5000

A

–

0

0

0

-1975000*

-28.16**

128.16***

EMS 1 µL/mL

A

–

5900000

5600000

5750000

3775000

53.83**

46.17***

Solvent control (DME)

-

A

+

7750000

7850000

7812500

5837500

100.00

0.00

-

B

+

7800000

7850000

Solubilised Sulphur Black 1

39.1

A

+

7500000

7500000

7500000

5525000

94.65

5.35

78.2

A

+

6350000

6150000

6250000

4275000

73.23

26.77

156.3

A

+

5500000

5700000

5600000

3625000

62.10

37.90

312.5

A

+

4750000

4550000

4650000

2675000

45.82

54.18

625

A

+

2400000

2200000

2300000

325000

5.57

94.43

1250

A

+

1100000

1400000

1250000

-725000

-12.42

112.42

2500

A

+

150000

0

75000

-1900000

-32.55

132.55

5000

A

+

0

0

0

-1975000

-33.83

133.83

Cycl. 5µg/mL

A

+

4950000

5000000

4975000

3000000

51.39

48.61

Table 7: Mean Number of Cells with structural Chromosome Aberrations, Experiment A

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Mean aberrant cells/150cells

incl. gaps

excl. gaps

Negative (Solvent) control

-

3 h

20 h

8

3

Solubilised Sulphur Black 1

39.1 µg/mL

-

3 h

20 h

9

3

78.2 µg/mL

-

3 h

20 h

9

3

156.3 µg/mL

-

3 h

20 h

9

3

312.5 µg/mL

-

3 h

20 h

10

3

Pos. Control
(Ethyl methanesulphonate)

-

3 h

20 h

38**

30**

Negative (Solvent) control

+

3 h

20 h

8

4

Solubilised Sulphur Black 1

39.1 µg/mL

+

3 h

20 h

8

5

78.2 µg/mL

+

3 h

20 h

8

4

156.3 µg/mL

+

3 h

20 h

9

5

312.5 µg/mL

+

3 h

20 h

9

5

Pos. Control (Cyclophosphamide)

+

3 h

20 h

43**

37**

Positive control (-S9): Ethyl methanesulphonate (1.0 µL/mL)

Positive control (+S9): Cyclophosphamide (5.0 µg/mL)

** = p < 0.01 to the concurrent negative control and to the historical control

Table 8: Mean Number of Cells with structural Chromosome Aberrations, Experiment B

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Mean aberrant cells/150cells

incl. gaps

excl. gaps

 

Negative (Solvent) control

-

20 h

20 h

8

3

 

 

Solubilised Sulphur Black 1

 

 

9.8 µg/mL

-

20 h

20 h

8

4

 

 

19.6 µg/mL

-

20 h

20 h

8

3

 

 

39.1 µg/mL

-

20 h

20 h

7

4

 

 

58.7 µg/mL

-

20 h

20 h

8

4

 

 

Pos. Control
(Ethyl methanesulphonate)

-

20 h

20 h

45**

37**

 

 

Negative (Solvent) control

-

20 h

28 h

8

3

 

 

Solubilised Sulphur Black 1

 

 

9.8 µg/mL

-

20 h

28 h

7

3

 

 

19.6 µg/mL

-

20 h

20 h

9

4

 

 

39.1 µg/mL

-

20 h

28 h

7

2

 

 

58.7 µg/mL

-

20 h

28 h

9

4

 

 

Pos. Control
(Ethyl methanesulphonate)

-

20 h

28 h

45**

38**

 

Positive control (-S9): Ethyl methanesulphonate (0.4 µL/mL)

** = p < 0.01 to the concurrent negative control and to the historical control

Table 9: Mean Number of Cells with structural Chromosome Aberrations, Experiment B 

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Mean aberrant cells/150cells

 

incl. gaps

excl. gaps

 

Negative (Solvent) control

+

3 h

28 h

8

4

Solubilised Sulphur Black 1

39.1 µg/mL

+

3 h

28 h

8

4

78.2 µg/mL

+

3 h

28 h

8

3

156.3 µg/mL

+

3 h

28 h

9

4

312.5 µg/mL

+

3 h

28 h

9

4

Pos. Control (Cyclophosphamide)

+

3 h

28 h

46**

40**

Cyclophosphamide: 5.0 µg/mL

** = p < 0.01 to the concurrent negative control and to the historical control

 

Table 10: Number of polyploidy Cells and endoreduplicated Cells, Experiment A 

Concentration
(µg/mL)

S9 mix

Treatment/Harvesting
time

Polyploid Cells (mean)

Endoredup-lication (mean)

Negative (Solvent) control

-

3/20 h

0.0

0.0

Solubilised Sulphur Black 1

39.1 µg/mL

-

3/20 h

0.0

0.0

78.2 µg/mL

-

3/20 h

0.0

0.0

156.3 µg/mL

-

3/20 h

0.0

0.0

312.5 µg/mL

-

3/20 h

0.0

0.0

Pos. Control
(Ethyl methanesulphonate)

-

3/20 h

0.0

0.0

Negative (Solvent) control

+

3/20 h

0.0

0.0

Solubilised Sulphur Black 1

39.1 µg/mL

+

3/20 h

0.0

0.0

78.2 µg/mL

+

3/20 h

0.0

0.0

156.3 µg/mL

+

3/20 h

0.0

0.0

312.5 µg/mL

+

3/20 h

0.0

0.0

Pos. Control (Cyclophosphamide)

+

3/20 h

0.0

0.0

Ethyl methanesulphonate: 1.0 µmL/mL

Cyclophosphamide: 5.0 µg/mL

 The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.


Table 11: Number of polyploidy Cells and endoreduplicated Cells, Experiment B 

Concentration
(µg/mL)

S9 mix

Treatment/Harvesting
time

Polyploid Cells (mean)

Endoredup-lication (mean)

Negative (Solvent) control

-

20/20 h

0.0

0.0

Solubilised Sulphur Black 1

9.8 µg/mL

-

20/20 h

0.0

0.0

19.6 µg/mL

-

20/20 h

0.0

0.0

39.1 µg/mL

-

20/20 h

0.0

0.0

58.7 µg/mL

-

20/20 h

0.0

0.0

Pos. Control

-

20/20 h

0.0

0.0

Negative (Solvent) control

-

20/28 h

0.0

0.0

Solubilised Sulphur Black 1

9.8 µg/mL

-

20/28 h

0.0

0.0

19.6 µg/mL

-

20/28 h

0.0

0.0

39.1 µg/mL

-

20/28 h

0.0

0.0

58.7 µg/mL

-

20/28 h

0.0

0.0

Pos. Control

-

20/28 h

0.0

0.0

Positive control (-S9):Ethyl methanesulphonate (0.4 µL/mL), The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.

Table 12: Number of polyploidy Cells and endoreduplicated Cells, Experiment B

 

Concentration
(µg/mL)

S9 mix

Treatment/Harvesting
time

Polyploid Cells (mean)

Endoredup-lication (mean)

Negative (Solvent) control

+

3/28 h

0.0

0.0

Solubilised Sulphur Black 1

39.1 µg/mL

+

3/28 h

0.0

0.0

78.2 µg/mL

+

3/28 h

0.0

0.0

156.3 µg/mL

+

3/28 h

0.0

0.0

312.5 µg/mL

+

3/28 h

0.0

0.0

Pos. Control

+

3/28 h

0.0

0.0

Cyclophosphamide: 5.0 µg/mL

 

The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.

 

Table 13: pH AND Osmolality Data, Experiment A

 

Concentration
(µg/mL)

pH

Osmolality (mmol/kg)

3-hour treatment without S9 Mix / 20-hour sampling time

Negative (solvent) Control

8.09

339

Solubilised Sulphur Black 1

39.1

8.34

350

78.2

8.40

354

156.3

8.32

347

312.5

8.37

355

EMS

8.32

349

3-hour treatment with S9 Mix / 20-hour sampling time

Negative (solvent) Control

8.17

345

Solubilised Sulphur Black 1

39.1

8.22

350

78.2

8.26

352

156.3

8.19

345

312.5

8.14

347

Cycl.

8.20

346

EMS: Ethyl methanesulphonate(1 µL/mL)

Cycl.: Cyclophosphamide (5.0 µg/mL)


Table 14: pH AND Osmolality Data, Experiment B

 

Concentration
(µg/mL)

pH

Osmolality (mmol/kg)

20-hour treatment without S9 Mix / 20-hour and 28-hor sampling times

Negative (solvent) Control

8.16

347

Solubilised Sulphur Black 1

9.8

8.35

345

19.6

8.37

342

39.1

8.42

350

58.7

8.40

347

EMS

8.36

352

3-hour treatment with S9 Mix / 28-hour sampling time

Negative (solvent) Control

8.20

347

Solubilised Sulphur Black 1

39.1

8.26

350

78.2

8.22

352

156.3

8.25

344

312.5

8.18

347

Cycl.

8.17

344

EMS:Ethyl methanesulphonate(0.4 µL/mL )

Cycl.:Cyclophosphamide (5.0 µg/mL)

Conclusions:
In an in vitro chromosome aberration assay (CA) according to OECD guideline 473, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.
Executive summary:

The clastogenic potential of the test item was tested in an in vitro chromosome aberration (CA) assay according to OECD guideline 473 in V79 (Chinese hamster lung cellline) in two independent experiments.For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix)

Experiment A with 3/20 h treatment/sampling time

without S9 mix:                39.1, 78.2, 156.3 and 312.5 µg/mL test item

with S9 mix:                   39.1, 78.2, 156.3 and 312.5 µg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix:              9.8, 19.6, 39.1 and 58.7 µg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix:              9.8, 19.6, 39.1 and 58.7 µg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix:                   39.1, 78.2, 156.3 and 312.5 µg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained (Giemsa). In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.

Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts. No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.

In experiment A in the presence of metabolic activation, three values were slightly above the 95% control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. 

The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µL/mL) (without S9 mix) or cyclophosphamide (5.0 µg/mL) (with S9 mix) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were comparable with the historical positive control data. Thus, the study is considered valid.

In conclusion, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 October 2017- 03 January 2018
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)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: In vitro mammalian gene mutation assay using the Hprt gene.
Specific details on test material used for the study:
Expiration date: 30 Nov 2021
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) located on the X chromosome.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
K1 subline
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)
- Methods for maintenance in cell culture: The cell stocks are kept in liquid nitrogen. For each experiment the cells were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air. Growing cells were subcultured in an appropriate number of flasks. The CHO K1 cells for this study were grown in Ham's F12 medium (F12-10) supplemented with 1 % antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %).

MEDIA USED
- Type and identity of media including CO2 concentration: Ham´s F12 Medium, 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of phenobarbital and β-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
Mutation Assay 5-hour treatment period without S9-mix:
1250, 2500, 2750, 3000, 3500, and 4000 µg/mL

Mutation Assay 5-hour treatment period with S9-mix:
312.5, 625, 1250, 2500, and 5000 µg/mL

The results of the pre-test on cell toxicity were used for dose selection of the test item concentrations used in the main mutation assay.
Vehicle / solvent:
- Solvent used: Ham's F12 medium
- Justification for choice of solvent: This solvent was chosen based on the results of the preliminary solubility test and its suitability is confirmed with the available laboratory’s historical database.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9 mix, 1.0 µL/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With S9 mix, 20 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;
- Cell density at seeding: 10E6 cells/dish

DURATION
- Preincubation period: 24 h
- Exposure duration: 5 h
- Expression time (cells in growth medium): 8 d

SELECTION AGENT (mutation assays): thioguanine (6-TG)

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The colonies were fixed with methanol for five minutes, stained with Giemsa

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Evaluation of Results
Providing that all acceptability criteria are fulfilled, the test item 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 compared with the concurrent negative control
•any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit)
•the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
•none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
•there is no concentration-related increase when evaluated with an appropriate trend test
•all results are inside the distribution of the historical negative control data (based 95% control limit)
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
Statistical Analysis was performed with SPSS PC+ software for the following data:
•mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups
•mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control
ite treated groups
•The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH or osmolality: The osmolality and pH values of test item solutions did not show any relevant alterations when compared to the concurrent control groups in the pre-test
- Precipitation: There was no precipitation of the test item at any dose level tested.

RANGE-FINDING/SCREENING STUDIES:
A study for cytotoxicity and solubility was performed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (95%)
- Positive historical control data:
Without S9 mix (EMS)
Mean: 1517.44; SD 29.86; Lower confidence interval 1455.15; upper confidence interval 1579.73
With S9 mix (DMBA)
Mean: 745.11; SD 16.86; Lower confidence interval 709.95; upper confidence interval 780.27

- Solvent historical control data:
Without S9 mix
Mean: 6.72; SD .066; Lower confidence interval 5.35; upper confidence interval 8.10
With S9 mix
Mean: 6.89; SD 0.81; Lower confidence interval 5.20; upper confidence interval 8.58

Summarized Results of the PRE-TEST ON TOXICITY (CONCENTRATION SELECTION)

Table 1: 5-hour treatment with and without S9-mix

Test group

Dose
µg/mL

S9-mix

Treatment/
time/ hour

Number of colonies/200cells/dish

Mean

Relativea
survival
in percent

dish 1

dish 2

dish 3

Solvent Control (Ham's F12 medium)

–

–

5

205

206

201

204,0

100

Solubilised Sulphur Black 1

39.1

–

5

203

202

206

203,7

100

78.2

–

5

200

201

199

200,0

98

156.3

–

5

204

204

200

202,7

99

312.5

–

5

203

204

205

204,0

100

625

–

5

200

199

201

200,0

98

1250

–

5

198

200

197

198,3

97

2500

–

5

160

162

158

160,0

78

5000

–

5

0

0

0

0,0

0

Solvent Control Ham's F12 medium)

–

+

5

200

204

204

202,7

100

Solubilised Sulphur Black 1

39.1

+

5

205

202

199

202,0

100

78.2

+

5

203

201

203

202,3

100

156.3

+

5

197

202

201

200,0

99

312.5

+

5

201

201

205

202,3

100

625

+

5

198

199

203

200,0

99

1250

+

5

204

202

200

202,0

100

2500

+

5

198

203

199

200,0

99

5000

+

5

201

197

202

200,0

99

a Relative to Solvent Control

 

CHO/HPRT MUTAGENESIS ASSAY RESULTS, MAIN MUTATION ASSAY/a, b, c and d

Table 2: 5-hour Treatment without S9-Mix 

Study code:

805-476-2715

 

 

Test item:

Solubilised Sulphur Black 1
(without S9-mix)

Batch number:

2401003784

Test date of Main Mutation Assay:

October 10, 2017 - October 26, 2017

Expression period:

8 days

Solvent:

Ham's F12 medium

Selective agent:

3.4 µg/mL 6-thioguanine

Cells seeded for analysis:

2x105cells /dish for mutant selection: 200 cells/dish for C.E.

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

 

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

 

Solvent control a

201.3

±

2.31

100

100

1

2

3

1

1

8

101

7.92

 

Pos. control
(EMS 1.0µL/mL) a

53.3

±

2.08

26

71

198

203

200

195

207

1003

72

1393.06**

 

TEST ITEM

 

 

1250g/mL a

201,7

±

1,53

100

100

1

0

3

3

0

7

100

7.00

 

2500g/mL a

161,3

±

1,53

80

101

0

2

2

1

1

6

102

5.88

 

2750g/mL a

151,0

±

1,00

75

100

2

2

1

2

0

7

101

6.93

 

3000g/mL a

127,3

±

1,15

63

99

1

3

1

1

3

9

100

9.00

 

3500g/mL a

36,3

 

2,08

18

99

1

3

2

3

2

11

100

11.00*

 

a = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

EMS=Ethyl methanesulfonate

* = p < 0.05 to the historical control

** = p < 0.01 to the concurrent negative control and to the historical control

 

 

Table 3: 5-hour Treatment without S9-Mix 

Study code:

805-476-2715

 

 

Test item:

Solubilised Sulphur Black 1
(without S9-mix)

Batch number:

2401003784

Test date of Main Mutation Assay:

October 10, 2017 - October 26, 2017

Expression period:

8 days

Solvent:

Ham's F12 medium

Selective agent:

3.4 µg/mL 6-thioguanine

Cells seeded for analysis:

2x105cells /dish for mutant selection: 200 cells/dish for C.E.

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

 

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

 

Solvent control b

202.7

±

0.58

100

100

1

1

2

2

1

7

101

6.93

 

Pos. control
(EMS 1.0µL/mL) b

53.0

±

2.65

26

71

210

205

195

194

199

1003

72

1393.06**

 

TEST ITEM

 

 

1250g/mL b

199,7

±

0,58

99

99

1

3

0

2

1

7

101

6.93

 

2500g/mL b

160,0

±

1,00

79

100

1

2

1

2

0

6

101

5.94

 

2750g/mL b

151,0

±

1,00

75

99

2

2

2

0

0

6

100

6.00

 

3000g/mL b

129,0

±

1,00

64

100

3

1

1

2

1

8

101

7.92

 

3500g/mL b

34,0

±

1,00

17

99

3

3

0

2

2

10

100

10.00

 

b = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

EMS=Ethyl methanesulfonate

** = p < 0.01 to the concurrent negative control and to the historical control


 Table 4: 5-hour Treatment without S9-Mix 

Study code:

805-476-2715

 

 

Test item:

Solubilised Sulphur Black 1
(without S9-mix)

Batch number:

2401003784

Test date of Main Mutation Assay:

October 10, 2017 - October 26, 2017

Expression period:

8 days

Solvent:

Ham's F12 medium

Selective agent:

3.4 µg/mL 6-thioguanine

Cells seeded for analysis:

2x105cells /dish for mutant selection: 200 cells/dish for C.E.

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

 

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

 

Solvent control c

202.0

±

3.46

100

100

2

1

1

1

2

7

101

6.93

 

Pos. control
(EMS 1.0µL/mL) c

55.0

±

1.73

28

66

207

202

192

197

206

1004

67

1498.51**

 

TEST ITEM

 

 

1250g/mL c

199,0

±

2,00

99

99

0

3

0

3

1

7

100

7.00

 

2500g/mL c

162,3

±

2,08

80

98

1

1

2

2

1

7

99

7.07

 

2750g/mL c

154,0

±

1,00

76

98

1

1

3

0

2

7

99

7.07

 

3000g/mL c

127,3

±

2,52

63

99

4

1

1

3

0

9

100

9.00

 

3500g/mL c

37,0

±

1,00

18

99

4

0

2

1

3

10

100

10.00

 

c = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

EMS=Ethyl methanesulfonate

** = p < 0.01 to the concurrent negative control and to the historical control

 

Table 5: 5-hour Treatment without S9-Mix

Study code:

805-476-2715

 

 

Test item:

Solubilised Sulphur Black 1
(without S9-mix)

Batch number:

2401003784

Test date of Main Mutation Assay:

October 10, 2017 - October 26, 2017

Expression period:

8 days

Solvent:

Ham's F12 medium

Selective agent:

3.4 µg/mL 6-thioguanine

Cells seeded for analysis:

2x105cells /dish for mutant selection: 200 cells/dish for C.E.

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

 

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

 

Solvent control d

202.0

±

2.00

100

100

1

2

2

1

2

8

101

7.92

 

Pos. control
(EMS 1.0µL/mL) d

54.0

±

1.00

27

67

208

204

197

193

198

1000

68

1470.59**

 

TEST ITEM

 

 

1250g/mL d

198,0

±

1,73

98

99

0

3

2

1

0

6

100

6.00

 

2500g/mL d

161,0

±

1,73

80

99

1

1

2

3

0

7

100

7.00

 

2750g/mL d

154,0

±

0,00

76

99

1

2

0

2

2

7

100

7.00

 

3000g/mL d

127,0

±

2,00

63

100

1

1

2

3

2

9

100

9.00

 

3500g/mL d

37,3

±

2,08

18

99

3

3

2

2

2

12

100

12.00##

 

d = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

EMS=Ethyl methanesulfonate

** = p < 0.01 to the concurrent negative control and to the historical control

##== p < 0.01 to the historical control

 

Table 6: 5-hour Treatment with S9 -Mix 

Study code:

805-476-2715

 

 

Test item:

Solubilised Sulphur Black 1
(with S9-mix)

Batch number:

2401003784

Test date of Main Mutation Assay:

October 10, 2017 - October 26, 2017

Expression period:

8 days

Solvent:

Ham's F12 medium

Selective agent:

3.4 µg/mL 6-thioguanine

Cells seeded for analysis:

2x105cells /dish for mutant selection: 200 cells/dish for C.E.

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

 

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

 

Solvent control a

202.0

±

2.00

100

100

2

1

1

1

1

6

100

6.00

 

Pos. control
(DMBA20 µg/mL) a

125.0

±

1.00

62

77

112

110

117

108

105

552

77

716.88**

 

TEST ITEM

 

 

312.5g/mL a

201,7

±

2,52

100

99

0

2

2

2

0

6

99

6.06

 

625g/mL a

202,3

±

2,08

100

99

1

2

3

0

1

7

99

7.07

 

1250g/mL a

203,3

±

2,52

101

98

0

1

2

1

2

6

98

6.12

 

2500g/mL a

198,7

±

1,15

98

99

2

0

2

1

2

7

99

7.07

 

5000g/mL a

199,0

±

2,00

99

98

1

3

2

0

1

7

98

7.14

 

a = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

DMBA=7,12-Dimethyl benzanthracene

** = p < 0.01 to the concurrent negative control and to the historical control

 

 

Table 7: 5-hour Treatment with S9 -Mix  

Study code:

805-476-2715

 

 

Test item:

Solubilised Sulphur Black 1
(with S9-mix)

Batch number:

2401003784

Test date of Main Mutation Assay:

October 10, 2017 - October 26, 2017

Expression period:

8 days

Solvent:

Ham's F12 medium

Selective agent:

3.4 µg/mL 6-thioguanine

Cells seeded for analysis:

2x105cells /dish for mutant selection: 200 cells/dish for C.E.

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

 

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

 

Solvent control b

201.7

±

1.53

100

100

2

0

1

1

3

7

100

7.00

 

Pos. control
(DMBA20 µg/mL) b

125.3

±

0.58

62

76

119

111

109

114

106

559

76

735.53**

 

TEST ITEM

 

 

312.5g/mL b

199,7

±

0,58

99

99

0

3

0

1

3

7

99

7.07

 

625g/mL b

201,3

±

1,53

100

99

1

1

2

1

2

7

99

7.07

 

1250g/mL b

201,0

±

1,00

100

99

2

0

1

1

2

6

99

6.06

 

2500g/mL b

198,7

±

1,15

99

99

2

2

1

1

1

7

100

7.00

 

5000g/mL b

199,7

±

1,53

99

99

0

3

1

0

3

7

99

7.07

 

b = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

DMBA=7,12-Dimethyl benzanthracene

** = p < 0.01 to the concurrent negative control and to the historical control

 

 

 

Table 8: 5-hour Treatment with S9 -Mix 

Study code:

805-476-2715

 

 

Test item:

Solubilised Sulphur Black 1
(with S9-mix)

Batch number:

2401003784

Test date of Main Mutation Assay:

October 10, 2017 - October 26, 2017

Expression period:

8 days

Solvent:

Ham's F12 medium

Selective agent:

3.4 µg/mL 6-thioguanine

Cells seeded for analysis:

2x105cells /dish for mutant selection: 200 cells/dish for C.E.

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

 

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

 

Solvent control c

200.7

±

2.31

100

100

1

3

0

2

0

6

100

6.00

 

Pos. control
(DMBA20 µg/mL) c

126.3

±

3.21

63

70

107

114

106

102

110

539

70

770.00**

 

TEST ITEM

 

 

312.5g/mL c

203,3

±

1,53

101

99

3

3

0

0

0

6

99

6.06

 

625g/mL c

201,0

±

2,00

100

100

1

1

2

2

1

7

100

7.00

 

1250g/mL c

199,3

±

1,53

99

98

1

2

0

2

2

7

98

7.14

 

2500g/mL c

201,3

±

1,53

100

98

1

2

2

1

0

6

98

6.12

 

5000g/mL c

200,0

±

2,00

100

99

1

1

1

3

0

6

99

6.06

 

c = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

DMBA=7,12-Dimethyl benzanthracene

** = p < 0.01 to the concurrent negative control and to the historical control

 

 


Table 9: 5-hour Treatment with S9 -Mix 

Study code:

805-476-2715

 

 

Test item:

Solubilised Sulphur Black 1
(with S9-mix)

Batch number:

2401003784

Test date of Main Mutation Assay:

October 10, 2017 - October 26, 2017

Expression period:

8 days

Solvent:

Ham's F12 medium

Selective agent:

3.4 µg/mL 6-thioguanine

Cells seeded for analysis:

2x105cells /dish for mutant selection: 200 cells/dish for C.E.

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

 

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

 

Solvent control d

202.3

±

0.58

100

100

1

1

1

2

1

6

100

6.00

 

Pos. control
(DMBA20 µg/mL) d

124.3

±

2.31

61

70

115

108

105

109

101

538

70

768.57**

 

TEST ITEM

 

 

312.5g/mL d

201,0

±

1,00

99

99

1

2

2

1

1

7

99

7.07

 

625g/mL d

200,7

±

2,08

99

99

3

0

1

2

1

7

100

7.00

 

1250g/mL d

200,0

±

1,73

99

98

2

1

2

0

2

7

98

7.14

 

2500g/mL d

201,3

±

0,58

100

99

1

2

1

1

1

6

99

6.06

 

5000g/mL d

199,3

±

1,15

99

99

1

1

2

3

0

7

99

7.07

 

d = parallel for mutation.

abs.C.E. = Absolute Cloning Efficiency

DMBA=7,12-Dimethyl benzanthracene

** = p < 0.01 to the concurrent negative control and to the historical control

Table 10: Day 1 Cloning efficiencies -Main Mutation Assay (5-hour treatment without And with S9-Mix) 

Test Group a and b

Concentration
µg/mL

S9-mix

Treatment
time/ hour

Number of colonies/200cells/dish

Cloning
efficiency (%)

% of Control

dish 1

dish 2

dish 3

Mean

Negative (Solvent Control)

-

–

5

204

200

200

201,3

101

100

Solubilised Sulphur Black 1- Group a

1250

–

5

200

202

203

201,7

101

100

2500

–

5

160

161

163

161,3

81

80

2750

–

5

150

151

152

151,0

76

75

3000

–

5

126

128

128

127,3

64

63

3500

–

5

38

34

37

36,3

18

18

4000

–

5

0

0

0

0,0

0

0

EMS (1µL/mL)

–

5

54

51

55

53,3

27

26

Negative (Solvent Control)

-

–

5

203

202

203

202,7

101

100

Solubilised Sulphur Black 1 - Group b

1250

–

5

199

200

200

199,7

100

99

2500

–

5

159

161

160

160,0

80

79

2750

–

5

151

150

152

151,0

76

75

3000

–

5

130

129

128

129,0

65

64

3500

–

5

33

34

35

34,0

17

17

4000

–

5

0

0

0

0,0

0

0

EMS (1µL/mL)

–

5

55

50

54

53,0

27

26

EMS= Ethyl methanesulfonate

Table 11: Day 1 Cloning efficiencies -Main Mutation Assay (5-hour treatment without And with S9-Mix) 

Test Group c and d

Concentration
µg/mL

S9-mix

Treatment
time/ hour

Number of colonies/200cells/dish

Cloning
efficiency (%)

% of Control

dish 1

dish 2

dish 3

Mean

Negative (Solvent Control)

-

–

5

198

204

204

202,0

101

100

Solubilised Sulphur Black 1- Group c

1250

–

5

197

199

201

199,0

100

99

2500

–

5

164

160

163

162,3

81

80

2750

–

5

155

154

153

154,0

77

76

3000

–

5

130

127

125

127,3

64

63

3500

–

5

38

37

36

37,0

19

18

4000

–

5

0

0

0

0,0

0

0

EMS (1µL/mL)

–

5

53

56

56

55,0

28

27

Negative (Solvent Control)

-

–

5

204

200

202

202,0

101

100

Solubilised Sulphur Black 1 - Group d

1250

–

5

197

197

200

198,0

99

98

2500

–

5

160

160

163

161,0

81

80

2750

–

5

154

154

154

154,0

77

76

3000

–

5

129

127

125

127,0

64

63

3500

–

5

39

38

35

37,3

19

18

4000

–

5

0

0

0

0,0

0

0

EMS (1µL/mL)

–

5

55

54

53

54,0

27

27

EMS= Ethyl methanesulfonate


Table 12: Day 1 Cloning efficiencies -Main Mutation Assay (5-hour treatment without And with S9-Mix) 

Test group a and b

Concentration
µg/mL

S9-mix

Treatment
time/ hour

Number of colonies/200cells/dish

Cloning
efficiency (%)

% of Control

dish 1

dish 2

dish 3

Mean

Negative (Solvent Control)

-

+

5

200

204

202

202,0

101

100

Solubilised Sulphur Black 1- Group a

312.5

+

5

204

202

199

201,7

101

100

625

+

5

203

204

200

202,3

101

100

1250

+

5

206

203

201

203,3

102

101

2500

+

5

200

198

198

198,7

99

98

5000

+

5

199

201

197

199,0

100

99

DMBA (20µg/mL)

+

5

125

124

126

125,0

63

62

Negative (Solvent Control)

-

+

5

200

202

203

201,7

101

100

Solubilised Sulphur Black 1-Groupb

312.5

+

5

200

200

199

199,7

100

99

625

+

5

201

203

200

201,3

101

100

1250

+

5

202

200

201

201,0

101

100

2500

+

5

200

198

198

198,7

99

99

5000

+

5

200

201

198

199,7

100

99

DMBA (20µg/mL)

+

5

125

125

126

125,3

63

62

DMBA= 7,12-Dimethylbenzanthracene

Table 13: Day 1 Cloning efficiencies -Main Mutation Assay (5-hour treatment without And with S9-Mix) 

Test group c and d

Concentration
µg/mL

S9-mix

Treatment
time/ hour

Number of colonies/200cells/dish

Cloning
efficiency (%)

% of Control

dish 1

dish 2

dish 3

Mean

Negative (Solvent Control)

-

+

5

202

198

202

200,7

100

100

Solubilised Sulphur Black 1- Group c

312.5

+

5

203

202

205

203,3

102

101

625

+

5

199

201

203

201,0

101

100

1250

+

5

198

201

199

199,3

100

99

2500

+

5

200

201

203

201,3

101

100

5000

+

5

202

200

198

200,0

100

100

DMBA (20µg/mL)

+

5

124

125

130

126,3

63

63

Negative (Solvent Control)

-

+

5

202

203

202

202,3

101

100

Solubilised Sulphur Black 1-Groupd

312.5

+

5

201

200

202

201,0

101

99

625

+

5

200

199

203

200,7

100

99

1250

+

5

199

202

199

200,0

100

99

2500

+

5

201

201

202

201,3

101

100

5000

+

5

200

198

200

199,3

100

99

DMBA (20µg/mL)

+

5

123

123

127

124,3

62

61

DMBA= 7,12-Dimethylbenzanthracene

Table 14: Day 8 Cloning efficiencies -Main Mutation Assay (5-hour treatment without and with S9-Mix) 

Test Group a and b

Concentration
µg/mL

S9-mix

Treatment
time/ hour

Number of colonies/200cells/dish

Cloning
efficiency (%)

% of Control

dish 1

dish 2

dish 3

Mean

Negative (Solvent Control)

-

–

5

202

200

203

201,7

101

100

Solubilised Sulphur Black 1- Group a

1250

–

5

203

200

199

200,7

100

100

2500

–

5

202

204

204

203,3

102

101

2750

–

5

202

199

202

201,0

101

100

3000

–

5

199

198

203

200,0

100

99

3500

–

5

197

201

199

199,0

100

99

4000

–

5

0

0

0

0,0

0

0

EMS (1µL/mL)

–

5

142

147

142

143,7

72

71

Negative (Solvent Control)

-

–

5

200

204

203

202,3

101

100

Solubilised Sulphur Black 1 - Group b

1250

–

5

201

202

200

201,0

101

99

2500

–

5

200

203

202

201,7

101

100

2750

–

5

200

200

201

200,3

100

99

3000

–

5

201

200

203

201,3

101

100

3500

–

5

198

200

200

199,3

100

99

4000

–

5

0

0

0

0,0

0

0

EMS (1µL/mL)

–

5

143

144

143

143,3

72

71

EMS= Ethyl methanesulfonate


Table 15: Day 8 Cloning efficiencies -Main Mutation Assay (5-hour treatment without and with S9-Mix)

Test Group c and d

Concentration
µg/mL

S9-mix

Treatment
time/ hour

Number of colonies/200cells/dish

Cloning
efficiency (%)

% of Control

dish 1

dish 2

dish 3

Mean

Negative (Solvent Control)

-

–

5

203

202

203

202,7

101

100

Solubilised Sulphur Black 1- Group c

1250

–

5

203

198

198

199,7

100

99

2500

–

5

196

198

202

198,7

99

98

2750

–

5

197

195

203

198,3

99

98

3000

–

5

200

197

202

199,7

100

99

3500

–

5

200

200

200

200,0

100

99

4000

–

5

0

0

0

0,0

0

0

EMS (1µL/mL)

–

5

120

122

117

119,7

60

59

Negative (Solvent Control)

-

–

5

201

202

202

201,7

101

100

Solubilised Sulphur Black 1 - Group d

1250

–

5

202

200

199

200,3

100

99

2500

–

5

199

200

202

200,3

100

99

2750

–

5

198

199

201

199,3

100

99

3000

–

5

199

201

202

200,7

100

100

3500

–

5

199

199

200

199,3

100

99

4000

–

5

0

0

0

0,0

0

0

EMS (1µL/mL)

–

5

119

121

118

119,3

60

59

EMS= Ethyl methanesulfonate

 

 

Table 16: Day 8 Cloning efficiencies -Main Mutation Assay (5-hour treatment without and with S9-Mix)

Test group a and b

Concentration
µg/mL

S9-mix

Treatment
time/ hour

Number of colonies/200cells/dish

Cloning
efficiency (%)

% of Control

dish 1

dish 2

dish 3

Mean

Negative (Solvent Control)

-

+

5

200

202

197

199,7

100

100

Solubilised Sulphur Black 1- Group a

312.5

+

5

198

199

197

198,0

99

99

625

+

5

195

200

198

197,7

99

99

1250

+

5

197

194

199

196,7

98

98

2500

+

5

195

199

197

197,0

99

99

5000

+

5

195

194

196

195,0

98

98

DMBA (20µg/mL)

+

5

155

151

157

154,3

77

77

Negative (Solvent Control)

-

+

5

199

202

201

200,7

100

100

Solubilised Sulphur Black 1-Groupb

312.5

+

5

199

199

198

198,7

99

99

625

+

5

196

199

198

197,7

99

99

1250

+

5

198

197

199

198,0

99

99

2500

+

5

198

199

200

199,0

100

99

5000

+

5

197

198

200

198,3

99

99

DMBA (20µg/mL)

+

5

151

152

155

152,7

76

76

DMBA= 7,12-Dimethylbenzanthracene


 

Table 17: Day 8 Cloning efficiencies -Main Mutation Assay (5-hour treatment without and with S9-Mix)

Test group c and d

Concentration
µg/mL

S9-mix

Treatment
time/ hour

Number of colonies/200cells/dish

Cloning
efficiency (%)

% of Control

dish 1

dish 2

dish 3

Mean

Negative (Solvent Control)

-

+

5

200

201

199

200,0

100

100

Solubilised Sulphur Black 1- Group c

312.5

+

5

195

199

200

198,0

99

99

625

+

5

197

200

201

199,3

100

100

1250

+

5

194

197

195

195,3

98

98

2500

+

5

195

199

191

195,0

98

98

5000

+

5

198

196

197

197,0

99

99

DMBA (20µg/mL)

+

5

140

141

138

139,7

70

70

Negative (Solvent Control)

-

+

5

202

200

199

200,3

100

100

Solubilised Sulphur Black 1-Groupd

312.5

+

5

197

199

199

198,3

99

99

625

+

5

198

198

201

199,0

100

99

1250

+

5

195

197

197

196,3

98

98

2500

+

5

199

200

195

198,0

99

99

5000

+

5

197

199

197

197,7

99

99

DMBA (20µg/mL)

+

5

139

141

140

140,0

70

70

DMBA= 7,12-Dimethylbenzanthracene

 

Table 18: pH and Osmolality Data in Pre-test on Toxicity (Concentration selection)

Concentration (µg/mL)

pH

Osmolality (mmol/kg)

Treatment period (hours): 5/without S9-Mix

Negative (solvent) Control

7.68

292

Solubilised Sulphur Black 1

39.1

7.63

297

78.2

7.61

297

156.3

7.60

296

312.5

7.64

299

625

7.65

302

1250

7.66

316

2500

7.76

321

5000

7.84

330

Treatment period (hours): 5/with S9-Mix

Negative (solvent) Control

7.31

300

Solubilised Sulphur Black 1

39.1

7.29

300

78.2

7.28

299

156.3

7.32

299

312.5

7.34

301

625

7.39

305

1250

7.48

312

2500

7.55

319

5000

7.73

330

 

Table 19: pH and Osmolality Data in Main Mutation Assay

Concentration (µg/mL)

pH

Osmolality (mmol/kg)

Treatment period (hours): 5/without S9-Mix

Negative (solvent) Control

7.58

309

Solubilised Sulphur Black 1

1250

7.77

307

2500

7.79

311

2750

7.78

314

3000

7.70

316

3500

7.79

317

4000

7.71

321

EMS

1.00 µL/mL

7.71

299

Treatment period (hours): 5/with S9-Mix

Negative (solvent) Control

7.42

301

Solubilised Sulphur Black 1

312.5

7.48

304

625

7.48

305

1250

7.52

304

2500

7.56

306

5000

7.63

311

DMBA

20 µg/mL

7.45

558

 

EMS= Ethyl methanesulfonate,

DMBA= 7,12-Dimethylbenzanthracene



Conclusions:
In an in vitro mammalian gene mutation test (HPRT) in CHO-K1 cells according to OECD guideline 476, the test item did not show mutagenic properties.
Executive summary:

The mutagenic potential of the test item was tested in an mammalian gene Mutation test in CHO-K1 cells according to OECD guideline 476. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver:

Mutation Assay: 5-hour treatment period without S9-mix:

1250, 2500, 2750, 3000, 3500, and 4000 µg/mL 

Mutation Assay: 5-hour treatment period with S9-mix:

312.5, 625, 1250, 2500 and 5000 µg/mL

Phenotypic expression was evaluated up to 8 days following exposure. There was no precipitation of the test item observable at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. In the absence of metabolic activation clear cytotoxicity (survival ca. 20%) of the test item was observed at 4000 µg/mL, whereas in the presence of metabolic activation, no cytotoxicity was observed up to the highest tested concentration of 5000 µg/mL.

The mutation frequency found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulfonate (1.0 µL/mL) and 7, 12-dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency when compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In both experimental parts, there were no statistically significant increases in mutation frequency when compared to the concurrent solvent control at any concentration tested in the absence and presence of metabolic activation. In the presence of S9 mix, there were no statistically significant differences to the historical control data and no dose-dependency was observed. In the absence of S9 mix, in the cultures treated with 3000 and 3500 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (3 of 4 and 4 of 4 cultures, respectively). Statistically significant differences to the historical control data were observed at concentration 3500 µg/mL in 2 of 4 cultures. These findings were considered not to be biologically relevant since no dose-response relationships were noted, all values were within the normal range of mutation frequency and no statistical difference to the concurrent controls were observed. 

The test item was tested up to the maximum recommended concentration with and without metabolic activation over a 5 hour treatment period did not induce biologically relevant increases in mutant frequency. 

It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test under the given conditions.

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

Additional information

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay (Ames) according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a S9 mix prepared from livers of phenobarbital/beta-naphthoflavone-induced rats. The study included preliminary solubility test, preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding tests the test item was solved in ultrapure water( ASTM Type I). At the formulation of test item solutions correction of concentrations for active component content (80.2 % dyestuff) was made in the main experiments. Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000, 1600, 500, 160, 50, and 16 µg/plate. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study. The test item did not show unequivocal inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) were considered to be within the biological variability range of the applied test system. The revertant colony numbers of solvent control (ultrapure water (ASTM Type I)) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected biologically relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants mostly fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

The clastogenic potential of the test item was tested in an in vitro chromosome aberration (CA) assay according to OECD guideline 473 in V79 (Chinese hamster lung cellline) in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test (without and with metabolic activation using rodent S9 mix).

Experiment A with 3/20 h treatment/sampling time

without S9 mix:                39.1, 78.2, 156.3 and 312.5 µg/mL test item

with S9 mix:                   39.1, 78.2, 156.3 and 312.5 µg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix:              9.8, 19.6, 39.1 and 58.7 µg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix:              9.8, 19.6, 39.1 and 58.7 µg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix:                   39.1, 78.2, 156.3 and 312.5 µg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained (Giemsa). In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item. Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts. No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.

In experiment A in the presence of metabolic activation, three values were slightly above the 95% control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. 

The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µL/mL) (without S9 mix) or cyclophosphamide (5.0 µg/mL) (with S9 mix) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were comparable with the historical positive control data. Thus, the study is considered valid.

In conclusion, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

The mutagenic potential of the test item was tested in an mammalian gene mutation test in CHO-K1 cells according to OECD guideline 476. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver:

Mutation Assay: 5-hour treatment period without S9-mix:

1250, 2500, 2750, 3000, 3500, and 4000 µg/mL 

Mutation Assay: 5-hour treatment period with S9-mix:

312.5, 625, 1250, 2500 and 5000 µg/mL

Phenotypic expression was evaluated up to 8 days following exposure. There was no precipitation of the test item observable at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. In the absence of metabolic activation clear cytotoxicity (survival ca.20%) of the test item was observed at 4000 µg/mL, whereas in the presence of metabolic activation, no cytotoxicity was observed up to the highest tested concentration of 5000 µg/mL.

The mutation frequency found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulfonate (1.0 µL/mL) and 7, 12-dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency when compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In both experimental parts, there were no statistically significant increases in mutation frequency when compared to the concurrent solvent control at any concentration tested in the absence and presence of metabolic activation. In the presence of S9 mix, there were no statistically significant differences to the historical control data and no dose-dependency was observed. In the absence of S9 mix, in the cultures treated with 3000 and 3500 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (3 of 4 and 4 of 4 cultures, respectively). Statistically significant differences to the historical control data were observed at concentration 3500 µg/mL in 2 of 4 cultures. These findings were not considered to be biologically relevant since no dose-response relationships were noted, all values were within the normal range of mutation frequency and no statistical difference to the concurrent controls were observed. 

The test item was tested up to the maximum recommended concentration with and without metabolic activation over a 5 hour treatment period and did not induce biologically relevant increases in mutant frequency. 

It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test under the given conditions.

The test item did not induce gene mutation in a bacterial or mammalian test system. Furthermore, the test item did not induce chromosome aberration in another mammalian test system. According to these results, the test item is considered to be non-genotoxic.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the test substance is considered not to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.