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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: 7012/2007
Appearance: brown powder
Composition of test substance:
Main component:
2-(4-aminoanilino)-5-nitrobenzenesulphonic acid 5.5 area %

4-[[2,4-dihydroxy-3,5-bis[[4-(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]phenyl]azo]-5-
hydroxynaphtalen-2,7-disulphonate, sodium salts 13.9 area %

4-[[2,4-dihydroxy-3-[(4-methoxyphenyl)azo]-5-[[4-[(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]phenyl]azo]-
5-hydroxynaphthalene-2,7-disulphonate, sodium salts 42.4 area %

4-[[2,4-dihydroxy-3,5-bis[(4-methoxyphenyl)azo]phenyl]azo]-5-
hydroxynaphthalene-2,7-disulphonate, sodium salts 18.2 area %
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

Method

Target gene:
gene for histidine or tryptophan synthesis
Species / strain
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
Controls
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);

Results and discussion

Test resultsopen allclose all
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

Any other information on results incl. tables

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 

Applicant's summary and conclusion

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.