Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Published in O.J. L 142 (2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test substance name: Acid Brown 235
- Other name: Korostan Brown DGR
- Source and lot/batch No. of test material: 7012/2007
- Expiration date of the lot/batch: May 2018
- Appearance: dark brown powder
Target gene:
gene for histidine or tryptophan synthesis
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 fraction of rat liver homogenate
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 µg per plate
Vehicle / solvent:
water for injection
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-2-phenylenediamine (CAS: 99-56-9); 2-aminofluorene (CAS: 153-78-6); 2-aminoanthracene (CAS: 613-13-8); N-methyl-N´-nitro-N-nitrosoguanidine (CAS: 70-25-7);
Details on test system and experimental conditions:
CHEMICALS AND MEDIA
Solvent:
- water for injection
metabolic activator:
- S9: prepared in-house
Positive controls:
- sodium azide (AS) (CAS: 26647-22-8)
- 4-nitro-2-phenylenediamine (NPD) (CAS: 99-56-9)
- 2-aminofluorene (2-AF) (CAS: 153-78-6)
- 2-aminoanthracene (2-AA) (CAS: 613-13-8)
- N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) (CAS: 70-25-7)
- 9-aminoacridine hydrochloride monohydrate (9-AAc) (CAS: 52417-22-8)
Media:
- Nutrient Broth for microbiology
- Nutrient agar
- Agar-agar

TEST SYSTEM
Bacterial strains:
The bacterial tester strains Salmonella typhimurium TA 1535 (CCM 3814, lot. No. 2101200916917), TA 98 (CCM 3811, lot No. 01022001220053), TA 100 (CCM 3812, lot No. 0102201220054) and TA 1537 (CCM 3815, lot No. 2101200916918) as well as Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732), - were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno. Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagens.
Genotypes of strains:
Genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).
Preparation and using of S9:
The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/mL, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL/g wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70°C. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer.
Each plate in all experiments with metabolic activation contained 0.5 mL of buffer with NADP and glucoso-6-phosphate and 30 or 100 µL S9 (the concentration of S9 in the S9mix was 5.7 or 19%). In experiments without metabolic activation only buffer was added to the top agar.
Controls:
Each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 mL of water. All the control numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers.

PLATE INCORPORATION TEST
Test procedure:
100 µL of the test substance of required concentration, 100 µL of 16-18 h culture of tester strain of density 10^8-10^9 CFU/mL, 0.5 mL relevant buffer and 30, 50 or 100 µL of S9 post mitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45±3°C. After shaking the mixture was poured into a minimal glucose agar plate.
Petri dishes were incubated of 48 - 72 h at 37±1°C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000. For an adequate estimate of variation, triplicate plating was used at each dose level. The toxicity test, which serves for finding of optimal concentrations for the mutagenicity test, was performed in strain TA 98 and two Petri dishes were used for every concentration.
Selection of doses/toxicity:
The test substance was dissolved in water for injection till the maximum recommended concentration 5000 μg per 0.1 mL. For toxicity experiment the highest concentration was diluted to the other 5 concentrations in 3 digit places interval. Although no particles could be observed in higher concentrations due to dark colour of solutions, no particles were observable in top agar and Petri dishes.
The concentration row was tested for toxicity in strain TA 100 without metabolic activation.
No toxicity or precipitation was observed in any dose. The concentration of 5000 µg per 0.1 mL-1 was then used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2· √10. Mutagenicity occurred in some cases in higher doses so lower concentrations were omitted and generally higher concentrations were used in the second mutagenicity experiments. For increase of sensibility, the second experiments were performed with pre-incubation for 30 minutes at 37±1°C and shaking. Fresh solutions of the test substance were prepared before each experiment. Concentrations of the test substance solution were dosed in the volume of 0.1 mL per plate.
Evaluation criteria:
EVALUATION OF RESULTS
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as ”biologically relevant“:
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system. According to OECD TG 471, 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.
Statistics:
1. Maron D. M., Ames B. N., Revised methods for the Salmonella mutagenicity test, Mutation Research, 113, p. 173 - 215 (1983);
2. Dunkel V. C., Chu K.C., Evaluation of methods for analysis of microbial mutagenicity assays, The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, p. 231 - 417 (1980);
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
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:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
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:
not determined
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1: The effect of the test substance

Doses

µg/plate

S9

µL

revertants per

plate

mean

±sd

Rt/Rc

S9

µL

revertants per

plate

mean

±sd

Rt/Rc

Escherichia coli WP2 uvrA

 

experiment I

Sp.rev.

0

35

39

31

35± 3

-

100

33

45

29

36± 7

-

water

0

34

28

35

32± 3

-

100

34

35

32

34± 1

-

50

0

34

32

35

34± 1

1.0

100

28

28

39

32± 5

0.9

150

0

30

37

36

34± 3

1.1

100

35

32

33

33± 1

1.0

500

0

33

33

41

36± 4

1.1

100

35

33

37

35± 2

1.0

1500

0

68

62

50

60± 7

1.9

100

39

26

44

36± 8

1.1

5000

0

114

140

131

128±11

4.0

100

96

105

71

91±14

2.7

MNNG/2-AA

0

1038

945

NT

992±47

30.7

100

147

153

NT

150± 3

4.5

 

experiment II

Sp. rev.

0

40

38

32

37± 3

-

100

36

46

49

44± 6

-

water

0

38

44

32

38± 5

-

100

43

42

39

41± 2

-

1000

0

88

58

67

71±13

1.9

100

40

42

36

39± 2

1.0

2000

0

85

90

104

93± 8

2.4

100

47

52

52

50± 2

1.2

3000

0

133

113

100

115±14

3.0

100

64

66

47

59± 9

1.4

4000

0

126

139

169

145±18

3.8

100

99

81

114

98±13

2.4

5000

0

172

176

170

173± 2

4.5

100

126

133

138

132± 5

3.2

MNNG/2-AA

0

1028

1022

NT

1025±3

27.0

100

207

196

NT

202± 6

4.9

Salmonella typhimurium TA 1537

 

experiment I

Sp.rev.

0

12

7

7

9± 2

-

30

15

16

11

14± 2

-

water

0

9

11

11

10± 1

-

30

16

8

14

13± 3

-

50

0

10

18

10

13± 4

1.2

30

19

15

18

17± 2

1.4

150

0

10

9

18

12± 4

1.2

30

16

15

21

17± 3

1.4

500

0

16

11

11

13± 2

1.2

30

18

14

15

16± 2

1.2

1500

0

23

25

21

23± 2

2.2

30

28

25

17

23± 5

1.8

5000

0

71

52

60

61± 8

5.9

30

37

45

44

42± 4

3.3

9-AAc/2-AA

0

980

1109

NT

1045±65

101.1

20

100

117

NT

109± 9

8.6

 

experiment II

Sp. rev.

0

8

13

8

10± 2

-

30

11

18

0

15± 4

-

water

0

10

11

12

11± 1

-

30

10

18

14

14± 3

-

1000

0

18

23

18

20± 2

1.8

30

12

17

17

15± 2

1.1

2000

0

29

29

29

29± 0

2.6

30

21

19

22

21± 1

1.5

3000

0

36

42

28

35± 6

3.2

30

30

41

35

35± 4

2.5

4000

0

48

44

40

44± 3

4.0

30

49

50

47

49± 1

3.5

5000

0

39

41

55

45± 7

4.1

30

66

43

47

52± 10

3.7

9-AAc/2-AA

0

1291

1314

NT

1303±12

118.4

20

201

201

NT

201±0

14.4

Salmonella typhimurium TA 100

 

experiment I

Sp.rev.

0

85

89

111

95±11

-

30

104

105

112

107± 4

-

water

0

89

121

102

104±13

-

30

111

96

118

108± 9

-

50

0

112

111

109

111± 1

1.1

30

108

c

107

108± 1

1.0

150

0

98

87

95

93± 5

0.9

30

88

96

111

98±10

0.9

500

0

101

95

106

101± 4

1.0

30

105

95

127

109±13

1.0

1500

0

114

124

102

113± 9

1.1

30

127

128

121

125± 3

1.2

5000

0

140

128

158

142±12

1.4

30

135

131

184

150±24

1.4

AS/2-AF

0

481

511

NT

496±15

4.8

 

20

1315

1328

NT

1322± 7

12.2

 

experiment II

Sp. rev.

0

83

78

75

79± 3

-

30

84

108

91

94±10

-

water

0

84

106

82

91±11

-

30

97

89

105

97± 7

-

1000

0

105

113

127

115± 9

1.3

30

90

96

86

91± 4

0.9

2000

0

139

135

132

135± 3

1.5

30

110

107

106

108± 2

1.1

3000

0

138

161

136

145±11

1.6

30

110

131

120

120± 9

1.2

4000

0

129

134

140

134± 4

1.5

30

132

122

140

131± 7

1.4

5000

0

180

183

171

178± 5

2.0

30

134

154

135

141± 9

1.5

AS/2-AF

0

492

492

NT

492± 0

5.4

20

950

1034

NT

992±42

10.2

Salmonella typhimurium TA 1535

 

experiment I

Sp.rev.

0

21

17

25

21± 3

-

30

18

23

16

19± 3

-

water

0

19

20

25

21± 3

-

30

12

19

12

14± 3

-

50

0

22

27

29

26± 3

1.2

30

14

15

12

14± 1

1.0

150

0

20

24

21

22± 2

1.0

30

16

16

12

15± 2

1.0

500

0

15

28

27

23± 6

1.1

30

18

12

11

14± 3

1.0

1500

0

19

24

24

22± 2

1.0

30

18

15

10

14± 3

1.0

5000

0

27

20

23

23± 3

1.1

30

25

14

24

21± 5

1.5

AS/2-AA

0

410

419

NT

415± 5

19.4

20

184

150

NT

167±17

11.7

 

experiment II

Sp. rev.

0

28

18

18

21± 5

-

30

17

22

19

19± 2

-

water

0

23

21

24

23± 1

-

30

20

15

24

20± 4

-

1000

0

21

17

16

18± 2

0.8

30

15

24

19

19± 4

1.0

2000

0

18

24

19

20± 3

0.9

30

k

15

21

18± 3

0.9

3000

0

21

27

20

23± 3

1.0

30

15

18

24

19± 4

1.0

4000

0

25

20

22

22± 2

1.0

30

25

19

17

20± 3

1.0

5000

0

24

24

19

22± 2

1.0

30

21

25

32

26± 5

1.3

AS/2-AA

0

414

444

NT

429±15

18.9

20

191

196

NT

194±3

9.8

Salmonella typhimurium TA 98

 

experiment I

Sp.rev.

0

31

25

31

29± 3

-

30

30

39

39

36± 4

-

water

0

25

30

25

27± 2

-

30

40

35

36

37± 2

-

50

0

26

29

29

28± 1

1.1

30

35

40

25

33± 6

0.9

150

0

26

22

23

24± 2

0.9

30

38

40

29

36± 5

1.0

500

0

27

29

30

29± 1

1.1

30

38

33

26

32± 5

0.9

1500

0

24

27

30

27± 2

1.0

30

32

25

23

27± 4

0.7

5000

0

31

36

30

32± 3

1.2

30

39

36

34

36± 2

1.0

NPD/2-AF

0

664

817

NT

741±77

27.8

20

1796

1871

NT

1834±38

49.6

 

experiment II

Sp.rev.

0

32

31

24

29± 4

-

30

35

48

38

40± 6

-

water

0

30

33

34

32± 2

-

30

38

44

41

41± 2

-

1000

0

38

46

32

39± 6

1.2

30

41

39

34

38± 3

0.9

2000

0

35

31

34

33± 2

1.0

30

37

33

28

33± 4

0.8

3000

0

43

38

37

39± 3

1.2

30

42

37

36

38± 3

0.9

4000

0

56

115

36

69±34

2.1

30

36

34

38

36± 2

0.9

5000

0

55

75

77

69±10

2.1

30

56

56

43

52± 6

1.3

NPD/2-AF

0

615

658

NT

637±22

19.7

20

1571

1578

NT

1575± 4

38.4

- S9        without metabolic activation

+ S9       with metabolic activation

CFU        colony forming units, number of live bacteria in suspension

sd          standard deviation

Rt/Rc    ratio of number of revertants at tested dose to number of revertants in negative control

S9          amount of supernatant of rat liver homogenate per plate

Sp.rev. spontaneous reversion (untreated control)

AS         sodium azide

NPD      4-nitro-2-phenylenediamine

2-AF     2-aminofluorene

2-AA     2-aminoanthracene

9-AAc   9-aminoacridine hydrochloride monohydrate

MNNG   N-methyl-N´-nitro-N-nitrosoguanidine

NT          not tested

c             contamination 

Conclusions:
Under the test conditions, the test substance, Acid Brown 235, was mutagenic for the Salmonella typhimurium TA 1537, Salmonella typhimurium TA 100 and Escherichia coli WP2uvrA in experiments with as well as without metabolic activation.
The test substance was non-mutagenic for Salmonella typhimurium TA 98 and TA 1535 in experiments with as well as without metabolic activation.
Executive summary:

The test substance, Acid Brown 235, was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria,which is analogous to the OECD Test Guideline No. 471.

Four indicator, Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and one indicator, Escherichia coliWP2 uvrA strain, were used. The test substance was diluted in water for injection and assayed in doses of 50 - 5000 mg per plate, which were applied to plates in volume of 0.1 mL. The first mutagenicity experiments were performed without and with metabolic activation using a supernatant of rat liver (30 μL or 100 μL per plate) and a mixture of cofactorsby the plate in corporation test with a dose range of 50 – 5000mg per plate. Some signs of a positive response were observed in some tester strains, so, to increase the sensitivity of the assay, the second mutagenicity experiments were performed with pre-incubation and concentrations were shifted towards expected mutagenic doses to obtain a dose-response.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Mean revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

Under the test conditions, the test substance, Acid Brown235, was mutagenic for the Salmonella typhimurium TA 1537, Salmonella typhimurium TA 100 and Escherichia coli WP2uvrA in experiments with as well as without metabolic activation.

The test substance was non-mutagenic for Salmonella typhimurium TA 98 and TA 1535 in experiments with as well as without metabolic activation.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
Adopted 29th July, 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
other: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: certified medical laboratory (MeDiLa)
- Sex, age and number of blood donors if applicable: healthy non smoking females up to 35 years of age
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver homogenate and mixture of cofactors
Test concentrations with justification for top dose:
first experiment: 125, 250, 500, 1000 and 2000 µg/mL
second experiment: 250, 500, 1000 and 2000 µg/mL
third experiment: 62.5 and 125 µg/mL
Vehicle / solvent:
Vehicle(s)/solvent(s) used: none
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: colchicine (CAS: 64-86-8)
Details on test system and experimental conditions:
TEST METHOD
The test for genotoxicity was performed according to OECD TG 487 and Protocol Cytokinesis-block micronucleus cytome assay.
The genotoxic potential is indicated by doubling of number of binucleated cells with micronuclei in comparison to the negative control (two-fold increase rule) and/or by dependence of increasing number of binucleated cells with micronuclei on dose (dose-response relationship).

CHEMICALS AND MEDIA
Positive controls:
- colchicine (CAS: 64-86-8) (Lot. No. SLBL0409V)
- cyclophosphamide monohydrate (CAS: 6055-19-2) (Lot. No. 120M1253V)
Media:
- RPMI 1640 with L-Glutamine (with sodium bicarbonate and Phenol Red as a pH indicator) (Lot. No. 6MB165)
- Foetal Bovine Serum (Lot. No. 032M3395, Lot. No. 124M3337)
- RPMI-M (RPMI 1640 + Foetal Bovine Serum + Penicilin-Streptomycin + PHA-M)
Other chemicals:
- PHA-M (Phytohaemagglutinin M, lectin extracted from red kidney beans) (Lot. No. 1624475)
- Penicilin-Streptomycin (Lot. 095M4773V)
- Cytochalasin B (Lot. No. 125M4047V)
- MgCl2 . 6H2O (Lot. No. SLBP9770V)
- NADP disodium salt (Lot. No. 076240809)
- D-Glucose-6-phosphate disodium salt (Lot. No. AS435477)
- DMSO (Lot. No. 2103180316)
- KCl (Lot. No. PP/2015/02174)
- Methanol (Lot. No. PP/2016/08749 Lot. No. 2017/0008)
- Acetic acid (Lot. No. PP/2016/01773 Lot. No. PP/2016/12357)
- Giemsa-Romanowski (Lot. No. 1708120816V)
- S9 (Lot. No. II)

TEST SYSTEM
The human peripheral blood lymphocytes used for testing were obtained from healthy non-smoking females (up to 35 years of age). Peripheral blood (heparinized) is taken from donors in certified medical laboratory (MeDiLa) in the morning and transported into the test facility as soon as possible.

MICRONUCLEUS TEST
Schedule of the test
Day 1: Blood sampling. Blood was taken and then stored in the fridge until the next day.
Day 2 and 3: Cultivation. The whole human peripheral blood was transferred to the growth medium and mitogenic stimulator (phytohaemagglutinin M) was added. These operations were carried out in a laminar box at room temperature. The cultivation runs without interrupting for 48 hours (37°C ± 1°C; 5% CO2).
Day 4: Exposition. The test substance, positive and negative control substances were added to the individual cultures (in a laminar box at room temperature). In the first experiment without and with metabolic activation (S9-mix), the cultures were washed (after 3 hours of continuous exposure to the test substance in 37°C ± 1°C; 5% CO2) and transferred to fresh culture medium with cytokinesis blocker (cytochalasin B). Washing and transfer were carried out in a laminar box at room temperature. Then the lymphocytes were cultured (37°C ± 1°C; 5% CO2) for remaining period (total 23 hours from the start of exposure).
Day 5: Harvesting of cultures. All cultures were processed in a laminar box at room temperature, (with hypotonia, fixation solution). Suspensions were dropped on clear microspcopic slides. The slides were allowed to dry at least overnight.
Day 6: Staining of slides. Slides were stained by Giemsa-Romanowski staining solution.

EXPERIMENTAL PROCEDURE
Selection of concentrations:
The test substance was suspended in culture medium. The starting suspension (2000 µg/mL - according OECD TG 487 paragraph 31) was diluted and concentration series (2000, 1000, 500, 250, 125 and 62.5 µg/mL) was prepared. The concentration series was prepared also according OECD TG 487 (concentration intervals of 2 to 3 fold). At first the cytotoxicity of test substance was determined by measuring of cell proliferation. For the highest concentration of the test substance used for analysis of genotoxic effect the cytotoxicity should not be higher than 55±5 %. Fresh solutions of the test substance were prepared before each experiment.
In the highest prepared concentration (2000 µg/mL) the pH was measured.

Controls:
Each experiment included corresponding positive controls (known clastogen - cyclophosphamide; and known aneugen – colchicine) and negative controls (untreated culture; in case of experiment with metabolic activation system – untreated culture with S9 mix).
The aneugenicity control (colchicine) serves also as the positive control without S9-mix, and the clastogenicity control (cyclophosphamide) is used to test of the adequacy of the metabolic activation system used. The final concentration of colchicine in cultures was 0.7 µg/mL (eventually 0.007 µg/mL at 23h exposure of the test substance) and the final concentration of cyclophosphamide in cultures is 7.8 µg/mL.

Preparation and using of S9-mix:
The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/mL, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15 M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL/g wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70°C. Fresh solution of the cofactors (1.6 mM MgCl2.6H2O, 0.8 mM NADP and 1 mM glucose-6-phosphate) was prepared before each experiment with metabolic activation. Each culture in experiments with metabolic activation contained 18.5 µL of S9 and 18.5 µL of cofactors solution (S9-mix).

Cell cycle length determination:
The proliferation of lymphocytes was evaluated by doubling time experiment. Four separate tubes with blood in the medium were prepared in day 2. Every following day (day 3, day 4 and day 5) lymphocytes were isolated and quantified. Then the proliferation curve was constructed and cell cycle length was determined. The lymphocyte`s cell cycle length was 16 hours. This is suitable to experimental design described above according to OECD TG 487. The cell cycle length determination is done once per 6 months. Results of the experiment are archived in the laboratory.

Treatment and cultivation:
50 µL of an appropriate concentration of test substance solution in medium was added to lymphocyte culture (2.5 mL growth medium RPMI-M + cca 150-210 µL human peripheral blood) in the presence and absence of a metabolic activation system (S9-mix). Duplicate cultures were used for each concentration and control. It was carried out in a laminar box at room temperature. Cultures were treated with the test substance 48 hours after mitogenic stimulation. The cultivation runs without interrupting for 48 hours (37°C ± 1°C; 5% CO2) after mitogenic stimulation.
In the first experiment without and with metabolic activation (S9-mix) the concentrations 125, 250, 500, 1000 and 2000 µg/mL were used. After shaking the cultures were cultivated with the test substance for 3 hours. After that they were rinsed up by RPMI-M and then transported to the fresh growth medium (RPMI-M) with 11.25 µL of cytochalasin B (the final concentration in cultures was 4.5 µg/mL). Cultures were then cultivated and sampled after 23 hours since the beginning of treatment. The first experiments gave negative results, so the second experiment without metabolic activation had to be done with extended exposure.
In the second experiment (prolonged exposition without activation) the concentrations 250, 500, 1000 and 2000 µg/mL were used. Cultures were treated with test substance without metabolic activation for 23 hours in the presence of cytochalasin B (7.5 µL; final concentration in cultures was 3 µg/mL). No transport to fresh medium was needed in that experimental design. The second experiment gave cytotoxic results in the two highest concentrations (2000 and 1000 µg/mL). So the third experiment without metabolic activation had to be done with extended exposure, because of meeting acceptability criteria (chapter 2.7, paragraph V) – the cytotoxicity of at least the three highest concentrations should not be higher than 55±5 % for genotoxicity evaluation.
In the third experiment (prolonged exposition without activation) the concentrations 62.5 and 125 µg/mL were used. Cultures were treated with test substance without metabolic activation for 23 hours in the presence of cytochalasin B (7.5 µL; final concentration in cultures was 3 µg/mL). No transport to fresh medium was needed in that experimental design.

Preparation of slides:
Cultures were harvested 23 hours after the beginning of treatment (after about 1.5 to 2 cell cycles). Cultures were treated by hypotonic solution (RT, ca 5 min.) and then they were centrifuged (1200 rpm, 10 min.). After removing of hypotonic solution, fixation solution was added to cultures and cultures were centrifuged again (1200 rpm, 10 min.). The addition of fixation solution and centrifugation were repeated three times. Suspensions were then dropped on clear microscopic slides. Preparations were let to dry at laboratory temperature and then slides were stained by Giemsa Romanowski staining solution.

Scoring of cells for cytotoxicity and genotoxicity evaluation:
All slides were coded before microscopic analysis. At least 1000 cells were scored per each concentration and controls divided equally between the duplicates for determination of cytotoxicity. CBPI index was calculated from ratio of mononucleated, binucleated and multinucleated cell at each culture. The cytotoxic effect was characterized as % of cytotoxicity. 2000 binucleated cells were analysed per each concentration and control divided equally between the duplicates for determination of genotoxicity. The genotoxic effect is characterized by numbers of binucleated cells with micronuclei.

Concentrations evaluated in analysis of cytotoxicity:
In the first experiment without and with metabolic activation (S9-mix) the concentrations 125, 250, 500, 1000 and 2000 µg/mL have been analysed for cytotoxic effect.
In the second experiment (prolonged exposition without activation) the concentrations 250, 500, 1000 and 2000 µg/mL have been analysed for cytotoxic effect.
In the third experiment (prolonged exposition without activation) the concentrations 62.5 and 125 µg/mL have been analysed for cytotoxic effect.

Concentrations evaluated in analysis of genotoxic effect:
For genotoxic effect analysis the concentrations with low or without cytotoxicity were used.
In the first experiment without and with metabolic activation (S9-mix) the concentrations 500, 1000 and 2000 µg/mL were analysed.
In the second experiment (prolonged exposition without activation) the concentrations 250 and 500 µg/mL were analysed.
In the third experiment (prolonged exposition without activation) the concentrations 62.5 and 125 µg/mL were analysed.
Evaluation criteria:
see Any other information on materials and methods
Key result
Species / strain:
other: human peripheral blood lymphocytes
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:
not valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table No. 1: Evaluation ofgenotoxicity

Treatment/Test substance concentration

Number of binucleated cells with MN

Number of MN

Average number of BN cells with MN per 1000 binucleated cells

Average number of MN per 1000 binucleated cells

Mt/Mc

3h exposure, without metabolic activation

UTC

11

12

5.5

6

1.0

2000mg/mL

20

20

10

10

1.82

1000mg/mL

12

12

6

6

1.09

500mg/mL

20

22

10

11

1.82

colchicine

76

90

38

45

6.91

3h exposure, with metabolic activation

S9-mix

(positive control)

20

25

10

12.5

1.0

2000 mg/mL +

S9-mix

16

18

8

9

0.80

1000mg/mL +

S9-mix

13

13

6.5

6.5

0.65

500mg/mL +

S9-mix

13

13

6.5

6.5

0.65

UTC

11

12

5.5

6

0.55

cyclophosphamide + S9-mix

46

49

23

24.5

2.30

23h exposure, without metabolic activation (second experiment)

UTC

15

16

7.5

8

1.00

500mg/mL

24

24

12

12

1.60

250mg/mL

22

24

11

12

1.47

colchicine

31

34

15.5

17

2.07

23h exposure, without metabolic activation (third experiment)

UTC

38

42

19

21

1.00

125mg/mL

49

52

24.5

26

1.29

62.5mg/mL

49

55

24.5

27.5

1.29

colchicine

76

83

38

41.5

2.00

Mt/Mc  ratio of number of binucleated cells with micronuclei at tested dose to number of binucleated cells with micronuclei in negative control

UTC        untreated culture

S9          amount of supernatant of rat liver homogenate per plate

MN        mononucleated cells

BN          binucleated cells

Conclusions:
Under the experimental conditions, the test substance, Acid Brown 235, had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation.
Executive summary:

In Vitro Mammalian Cell Micronucleus Test assayed genotoxicity of the test substance, Acid Brown 235. The testwas performed according to OECD Test Guideline No. 487 - In Vitro Mammalian Cell Micronucleus Test, Adopted 29th July, 2016.

The human peripheral blood lymphocytes from healthy donors were used for testing. The test substance was suspended in RPMI medium and assayed in five concentrations 62.5 - 2000 µg/mL, which were applied to cultures in volume of 50 µL. Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

Under the experimental design, the test substance, Acid Brown 235, had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation. The result of micronucleus test was negative, the test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16/05/2018-15/10/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
no impact on the outcome of study (see Any other information ...)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell transformation assay
Target gene:
hypoxanthine-guaninephosphoribosyl-transferase (HPRT) gene
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: The lung fibroblasts V79 from male Chinese hamster were used for testing. Frozen permanent cell cultures were obtained from European Collection of Cell Cultures (ECACC). V79 used for experiments: Lot. No.: 15H003. ECACC Certificate of Analysis is a part of archived study documentation. Sponsor declared identity, sterility and absence of Mycoplasma in provided cultures.

- Number of passages if applicable: max. 5

- Methods for maintenance in cell culture if applicable:
Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants. Cleansing was not performed before cytotoxicity experiments.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
DMEM : FBS : Atb = 500 : 55 : 5.5, prepared in laboratory
Fresh solutions of the test substance were prepared for every experiment

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
As new frozen stock is used for every mutagenicity experiment, no contamination is presumed at the start of experiments. Mycoplasma determination was then performed in samples withdrawn in the end of experiment - usually at withdrawal of mutants - from several plates of random concentration / duplicate in both experiments.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat liver homogenate and mixture of cofactors
Test concentrations with justification for top dose:
Based on the cytotoxicity test: 0.25, 0.5, 1.0 and 2.0 mg.mL-1 with/without
Vehicle / solvent:
DMEM, Sigma-Aldrich, RNBG 5578, RNBG 6521, RNBG 7734
Negative solvent / vehicle controls:
yes
Remarks:
9ml of complete medium + 1 ml of DMEM
Positive controls:
yes
Remarks:
9.9 or 9.95 mL of complete medium and 100 or 50 µL of relevant positive control diluted in DMSO
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

- Cell density at seeding (if applicable):
After treatment, approximately 2×10e6 cells were transferred to suitable number of dishes to seed enough cells. At the same time, cells were seeded for detection of number of cells (PE estimation).

DURATION
- Preincubation period: 5 days before treatment
- Exposure duration: 3 h
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 6 days
Survival and plating efficiency plates were incubated for at least 6 days (37±1ºC, 5% CO2, moistened) prior to scoring.
Mutant plates were incubated for an appropriate period to ensure adequate colony size (about 10 days).

SELECTION AGENT (mutation assays): 6-thioguanine 98%, CAS No. 154-24-7, Sigma , diluted in 0,5% Na2CO3 (Penta); 5 µg.mL-1 (final concentration) for selection of mutants

STAIN: Methylene blue, CAS No. 122965-43-9, AlfaAesar, 0.1 % solution

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Determination of survival
After treatment period, the cultures were trypsinised and an aliquot (0.3 mL of 10e3/mL cell suspension) was diluted and plated to 6 cm Petri dishes to estimate the viability of the cells.

Subculturing
A sufficient number of live cells (but never less than 2 millions, with respect to cytotoxicity results) were further cultured in 10 cm Petri dishes.
Also on the 3rd, 6th and 8th day the cell populations were sub cultured in order to maintain them in exponential growth.
The number of cells taken forward was adjusted according to the expected viability, to give two millions viable cells seeded in 10 cm Petri dishes.

Incubation, staining and scoring
Survival and plating efficiency plates were incubated for at least 6 days (37±1ºC, 5% CO2, moistened) prior to scoring.
Mutant plates were incubated for an appropriate period to ensure adequate colony size (about 10 days). After incubation, the plates were stained with methylene blue and colonies were scored.

NUMBER OF CELLS EVALUATED: 220000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 300

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Concentratios used in toxicity test were: 2.0, 1.0, 0.5, 0.25, 0.1 and 0.05 mg.mL- 1.
No cytotoxicity was observed.
Evaluation criteria:
Each experiment is separately evaluated using modified two-fold increase rule according to Claxton L.D. et al, Mutat. Res.,189, 83-91, 1987 (2).
The mutagenic potential is indicated by increasing number of mutants in treated groups in comparison to the negative solvent control (modified two-fold increase rule and any of the results outside the distribution of the historical negative control data) and/or by dependence of increasing number of mutants on dose (dose-response relationship).
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive than a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions i.e. S9 concentration or S9 origin) could be performed.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other:
Remarks:
Negative control = Solvent control
Positive controls validity:
valid
Conclusions:
Under the experimental conditions the test item, Acid Brown 235, was non-mutagenic for V79 cells with and without metabolic activation.
Executive summary:

The test substance, Acid Brown 235R, was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test. The study was performed according to OECD Test Guideline No. 476 – In Vitro Mammalian Cell Gene Mutation Test using the Hprt and Xprt genes (2016), which is analogous to the EU method B.17.

Chinese hamster V79 fibroblasts were used for testing.

The test item is water soluble, so it was dissolved in assay medium (DMEM) and it can be dosed till the highest recommended concentration 2.0 mg per plate.

This concentration was used as maximum for cytotoxicity test without metabolic activation performed in advance. Other concentrations were prepared by dilution and concentrations used were 2.0, 1.0, 0.5, 0.2, 0.1 and 0.01 mg per mL.

No cytotoxicity was observed in the cytotoxicity experiment so concentrations of 0.25, 0.5, 1.0 and 2.0 were used for mutagenicity experiments.

The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors.

Cytotoxicity about 25% was observed in mutagenicity experiments in the highest concentration. No signs of mutagenicity were observed either in experiment with or without metabolic activation.

According to OECD Test Guideline No. 476, UVCB substances should be tested up to 5 mg per mL therefore an extra concentration 5 mg per mL was tested beyond in aditional experiments with and without metabolic activation.

In experiment with metabolic activation the Mt/Msc above two was observed in the second duplicate in concentration of 5.0 (2) mg per mL. No increase was observed in the first duplicate of the same concentration. In experiment without metabolic activation, no increase was observed.

The concurent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations.

Under the experimental conditions indicated above the test item, Acid Brown 235, was non-mutagenic for V79 cells with and without metabolic activation

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

According to the bacterial reverse mutation test (Ames test), the test substance, Acid Brown 235, was mutagenic for the Salmonella typhimurium TA 1537, Salmonella typhimurium TA 100 and Escherichia coli WP2uvrA in experiments with as well as without metabolic activation, at the same time, the test substance was non-mutagenic for Salmonella typhimurium TA 98 and TA 1535 in experiments with as well as without metabolic activation.

In accordance withIn VitroMammalian Cell Micronucleus Test, the test substance, Acid Brown 235, had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation. The result of micronucleus test was negative, the test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

In accordance withIn VitroMammalian Cell Gene Mutation Test, the test substance, Acid Brown 235, had no cytotoxicity effects in Chinese hamster V79 fibroblasts in experiments both without and with metabolic activation.

Consistently with the classification rules under Regulation (EC) No. 1272/2008, the test substance can not currently be classified as a mutagen only on the basis of in vitro tests. Appropriate in vivo mutagenicity studies will be considered.