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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 May 1998 to 12 June 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, performed in accordance with valid guidelines and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrasodium 7-[[4-[[4,6-bis[(3-sulfonatopropyl)thio]-1,3,5-triazin-2-yl]amino]-3-methoxyphenyl]azo]naphthalene-1,3-disulfonate
EC Number:
428-480-0
EC Name:
Tetrasodium 7-[[4-[[4,6-bis[(3-sulfonatopropyl)thio]-1,3,5-triazin-2-yl]amino]-3-methoxyphenyl]azo]naphthalene-1,3-disulfonate
Cas Number:
214559-61-2
Molecular formula:
C26H24N6O13S6Na4
IUPAC Name:
tetrasodium 7-{2-[4-({4,6-bis[(3-sulfopropyl)sulfanyl]-1,3,5-triazin-2-yl}amino)-3-methoxyphenyl]diazen-1-yl}naphthalene-1,3-disulfonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Y-1189
- Physical state: solid
- Appearance: orange granulous powder
- Storage condition of test material: at room temperature and protected from light

Method

Target gene:
S. typhimurium: Histidine locus

E. coli: Tryptophan locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0, 10, 100, 500, 1000, 2500, 5000 μg/plate (prelminary toxicity test with TA 98, TA 100 and WP2 uvrA, with and without S9 mix)

0, 312.5, 625, 1250, 2500, 5000 μg/plate (mutagenicity test 1 and 2, with and without S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-anthramine
Details on test system and experimental conditions:
- Experiment 1 and Experiment 2 without S9 mix
METHOD OF APPLICATION: in agar (plate incorporation)
The day before treatment, cultures were inoculated from frozen permanents. A scrape was taken under sterile conditions and put into approximately 6 mL of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37 °C for about 14 hours.
0.05 to 0.1 mL of the test material solution, 0.5 mL of S9 mix (with S9 mix) and 0.1 mL of the strain were mixed with 2 mL of overlay agar (containing traces of the relevant amino acid and biotin and maintained at 45 °C). After rapid homogenisation, the mixture was overlaid onto a Petri plate containing minimum medium.

- Experiment 2 with S9 mix
METHOD OF APPLICATION: preincubation
The day before treatment, cultures were inoculated from frozen permanents. A scrape was taken under sterile conditions and put into approximately 6 mL of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37 °C for about 14 hours.
0.05 to 0.1 mL of the test material solution, 0.5 mL of S9 mix (with S9 mix) and 0.1 mL of the strain were incubated for 60 minutes at 37 °C prior to the addition of overlay agar. The mixture was then poured onto the surface of a minimum agar plate.

DURATION
- Exposure duration: Plates were incubated inverted at 37 °C for 48 to 72 hours in the dark.

NUMBER OF REPLICATIONS: Test concentrations were performed in triplicate (mutagenicity experiments) (one dose/plate in preliminary toxicity test)

EVALUATION PROCEDURE: Following the total incubation period, revertants were scored with an automatic counter.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was assessed as a reduction in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.
Evaluation criteria:
The study was considered valid if the following were met:
- The number of revertants of the controls was within the range of historical control data
- The number of revertants of the positive controls was higher than that of the controls and within the range of historical data

Evaluation criteria:
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose level and/or evidence of a dose-response relationship was considered as a positive result.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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:
not applicable
Positive controls validity:
valid
Additional information on results:
- Preliminary Toxicity Test
The test material was found to be freely soluble in the vehicle (distilled water) at 50 mg/mL. Consequently, with a maximum dose volume of 100 µL/plate, the dose levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at all dose levels. A slight orange-yellow colouration was noted in the plates at dose levels at and above 500 µg/plate. No toxicity was noted towards the three strains used, with and without S9 mix.

- Mutagenicity Experiments
The numbers of revertants of the vehicle and positive controls was as specified in the acceptance criteria; the study was therefore considered valid.
Since the test material was freely soluble and non-toxic, the highest dose-level was 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at all dose levels. A moderate orange-yellow colouration was noted in the plates at 5000 µg/plate. A slight colourations was observed at the remaining dose levels

- Experiments without S9 mix:
In the first experiment, a slight to moderate decrease in revertants was observed in both TA 1537 and TA 98 strains at dose levels at and above 2500 µg/plate. In the TA 1535 strain, a slight decrease in the number of revertants was noted in the plates at 5000 µg/plate. In the second experiment, except for slight decreases in the number of revertants noted in the TA 100 strain at dose levels at and above 1250 µg/plate, no notable toxicity was observed.
Overall, no noteworthy increase in the number of revertants was induced in all tester strains, in both experiments.

- Experiments with S9:
Except for a slight decrease in the number of revertants noted in the first experiment with the TA 98 strain at 5000 µg/plate, no notable toxicity was observed.

The test material did not induce any relevant increase in the number of revertants, in all tester strains in both experiments.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment 1 Results

Strain

Dose

Without S9

With S9

Mean revertants/ plate

Ratio

Mean revertants/ plate

Ratio

TA 1535

0

14

-

 11

-

30

10

0.76

15

1.44

100

12

0.88

12

1.09

300

11

0.80

12

1.13

1000

11

0.83

10

0.94

3000*

8

0.61

9

0.88

NaN3 / 2AM

323

23.63

214

20.03

TA 1537

0

7

-

11

-

30

6

0.90

12

1.13

100

8

1.14

11

1.00

300

6

0.90

12

1.13

1000

4

0.62

15

1.44

3000*

3

0.38

13

1.25

9AA / 2AM

334

47.71

268

25.16

TA 98

0

27

-

27

-

30

18

0.67

21

0.77

100

21

0.77

27

0.98

300

30

1.10

20

0.73

1000

17

0.61

22

0.79

3000*

12

0.45

17

0.61

2NF / 2AM

178

6.52

2340

85.61

TA 100

0

93

-

110

-

30

110

1.18

106

0.96

100

110

1.18

120

1.09

300

90

0.96

106

0.96

1000

95

1.02

99

0.90

3000*

69

0.74

108

0.98

NaN3 / 2AM

529

5.66

2902

26.30

E. coli WP2 uvr A

0

46

-

33

-

30

48

1.04

28

0.84

100

45

0.98

29

0.88

300

44

0.96

32

0.96

1000

43

0.93

25

0.77

3000*

39

0.84

28

0.84

4NQO / 2AM

1738

37.78

323

9.80

Table 2: Experiment 2 Results

Strain

Dose

Without S9

With S9

Mean revertants/ plate

Ratio

Mean revertants/ plate

Ratio

TA 1535

0

8

-

11

-

30

15

1.80

12

1.16

100

11

1.32

11

1.06

300

9

1.12

13

1.22

1000

12

1.44

12

1.13

3000*

8

1.00

15

1.41

NaN3 / 2AM

404

48.48

314

29.44

TA 1537

0

7

-

14

-

30

6

0.81

11

0.79

100

12

1.67

11

0.81

300

7

1.05

15

1.05

1000

10

1.38

9

0.64

3000*

9

1.33

12

0.83

9AA / 2AM

289

41.24

195

13.93

TA 98

0

14

-

20

-

30

15

1.12

24

1.20

100

18

1.32

23

1.19

300

17

1.24

24

1.20

1000

15

1.12

22

1.14

3000*

16

1.15

21

1.07

2NF / 2AM

146

10.66

1664

84.63

TA 100

0

97

-

106

-

30

94

0.97

143

1.35

100

80

0.82

134

1.26

300

64

0.66

132

1.24

1000

58

0.60

120

1.14

3000*

55

0.57

109

1.03

NaN3 / 2AM

546

5.63

2459

23.20

E. coli WP2 uvr A

0

33

-

53

-

30

31

0.93

44

0.83

100

26

0.79

47

0.88

300

26

0.78

39

0.73

1000

25

0.76

41

0.76

3000*

29

0.88

40

0.74

4NQO / 2AM

1369

41.49

214

4.01

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), response in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli strain WP2 uvrA both in the presence and absence of metabolic activation. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.
Executive summary:

The potential of the test material to cause gene mutation in bacterial strains was determined in accordance with standardised guidelines OECD 471 and EU Method B.13/14. Four trains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and one strain of Escherichia coli (WP2 uvrA) were treated in the presence and absence of a metabolic activation system (S9 mix). In two separate assays, the test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the tester strains used in the absence or absence of metabolic activation.