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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:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study according to GLP regulation; full report available

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
pentasodium 1-amino-4-{[4-(N-methylacetamido)-2-sulfonatophenyl]amino}-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate chloride sulfate
EC Number:
944-710-7
Molecular formula:
not available because multi-constituent substance
IUPAC Name:
pentasodium 1-amino-4-{[4-(N-methylacetamido)-2-sulfonatophenyl]amino}-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate chloride sulfate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat Liver S-9
Test concentrations with justification for top dose:
0 - 312,5 - 625 - 1250 - 2500 - 5000 µg/plate
Vehicle / solvent:
water
Controls
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-anthramine (2AM)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 60 min.
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;
Evaluation criteria:
Treatment of results
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtained in the presence of the vehicle), are presented in a table.

Acceptance criteria
This study would be considered valid since the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with the range of our historical data (appendix 2),
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the range of our 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-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
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
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
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
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
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
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
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
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
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
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
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

- Water solubility: 100g/L
- Precipitation: No

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxicity was noted towards all the strains used, both with and without S9 mix.
Remarks on result:
other: strain/cell type: TA 100
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

First experiment with S9

Strain

Dose (µg/plate)

Revertants / Plate

(Mean)

Standard eviation

Ratio

TA 100

0

95

16

1,00

 

312,5

92

5

0,97

 

625

111

11

1,17

 

1250

112

8

1,18

 

2500

105

9

1,11

 

5000

101

22

1,07

 

2AM

2286

94

24,06

 

 

 

 

 

TA102

0

343

33

1,00

 

312,5

352

13

1,03

 

625

351

19

1,02

 

1250

348

8

1,02

 

2500

359

25

1,05

 

5000

338

35

0,99

 

2AM

2139

443

6,24

 

 

 

 

 

TA 98

0

39

8

1,00

 

312,5

47

2

1,21

 

625

45

2

1,15

 

1250

39

4

0,99

 

2500

46

5

1,17

 

5000

54

3

1,39

 

2AM

737

57

18,9

 

 

 

 

 

TA 1535

0

18

6

1,00

 

312,5

21

5

1,21

 

625

26

2

1,45

 

1250

28

10

1,58

 

2500

19

3

1,06

 

5000

19

7

1,09

 

2AM

278

65

15,72

 

 

 

 

 

TA 1537

0

19

8

1,00

 

312,5

24

4

1,26

 

625

19

5

0,98

 

1250

15

5

0,79

 

2500

24

5

1,22

 

5000

21

5

1,07

 

2AM

95

11

4,90

 

Second experiment with S9

Strain

Dose (µg/plate)

Revertants / Plate

(Mean)

Standard Deviation

Ratio

TA 100

0

94

10

1,00

 

312,5

144

9

1,54

 

625

178

24

1,90

 

1250

200

15

2,13

 

2500

271

32

2,90

 

5000

168

4

1,79

 

2AM

1153

32

12,31

 

 

 

 

 

TA102

0

365

27

1,00

 

312,5

423

19

1,16

 

625

403

5

1,10

 

1250

403

16

1,10

 

2500

397

9

1,09

 

5000

374

10

1,02

 

2AM

2670

123

7,31

 

 

 

 

 

TA 98

0

39

4

1,00

 

312,5

49

8

1,26

 

625

54

6

1,38

 

1250

57

5

1,45

 

2500

56

9

1,44

 

5000

44

13

1,13

 

2AM

1098

240

28,16

 

 

 

 

 

TA 1535

0

20

4

1,00

 

500

20

3

0,98

 

1000

19

5

0,95

 

2000

17

1

0,85

 

2500

21

3

1,02

 

5000

18

2

0,90

 

2AM

214

9

10,54

 

 

 

 

 

TA 1537

0

16

2

1,00

 

312,5

17

3

1,02

 

625

20

8

1,24

 

1250

16

1

1,00

 

2500

14

6

0,86

 

5000

10

2

0,63

 

2AM

82

15

5,02

Third Experiment with S9

Strain

Dose (µg/plate)

Revertants / Plate

(Mean)

Standard Deviation

Ratio

TA 100

0

146

8

1,00

 

625

244

7

1,67

 

1250

305

11

2,09

 

2500

341

6

2,34

 

3000

341

32

2,34

 

5000

309

46

2,12

 

2AM

1117

144

7,65

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation only in strain TA 100 after preincubation

The test item showed mutagenic activity in a bacterial reverse mutation test on Salmonella typhimuriumTA 100 strain, with metabolic activation and preincubation method.
Executive summary:

The objective of this study was to evaluate the potential of the test substance to induce reverse mutation in Salmonella typhimurium.


The test item was tested in two independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.


A third experiment was performed with S9 mix.


The first experiment was performed according to the direct plate incorporation method. The second and the third with S9 mix were performed according to the preincubation method (60 minutes, 37C).


Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used.


Each strain was exposed to five dose-levels of the test substance (three plates/dose-level).


After 48 to 72 hours of incubation at 37C, the revertant colonies were scored.


The test substance was dissolved in distilled water.


Results


The test substance was freely soluble in the vehicle at 54.348 mg/ml, corresponding to 50 mg of the active material/ml (purity of the commercial product: 92%). Since the test substance was freely soluble and non-toxic in the preliminary toxicity test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the guidelines.


The selected treatment-levels for the mutagenicity experiments were: 312.5, 625, 1250, 2500 and 5000 µg/plate, for all strains in the first and second experiments, except for the TA 1535 strain in the second experiment with S9 mix where the treatment-levels were: 500, 1000,2000,2500 and 5000 µg/plate.


In the third experiment, the dose-levels were: 625, 1250, 2500, 3000 and 5000 µg/plate.


Slight to strong blue coloration was observed in the Petri plates when scoring the revertants at dose-levels. This coloration did not impede the automatic counting of the bacterial colonies. No precipitate was observed at any dose-level.


No relevant toxicity was noted towards all the strains used, both with and without S9 mix.


The test substance did not induce any significant increase in the number of revertants, with S9 mix and without S9 mix, in any of the five strains using the direct plate incorporation method.


With S9 mix and the preincubation method, a significant increase in the number of revertants, in comparison to the vehicle control, was noted in the TA 100 strain, at the dose-levels of 1250 and 2500 µg/plate. In addition, a dose-related increase was observed in this strain, at all dose-levels tested, except at the highest dose-level of 5000 µg/plate where a slight toxicity could have masked the increase in the number of revertants.


A third experiment was performed under the same conditions, in order to check the reproducibility of the significant increase observed. In this third experiment, the significant induction of the number of revertants was confirmed.


The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria.


The study was therefore considered as valid.


Conclusion


Under our experimental conditions, the test substance showed mutagenic activity in this bacterial reverse mutation test on Salmonella typhimuriumTA 100 strain, with metabolic activation and preincubation method.