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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:
2009-11-11 till 2010-02-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: "Kanpoan No. 287 -- Environment Protection Agency" "Eisei No. 127 -- Ministry of Health &Welfarew "Heisei 09110131 Kikyoku No. 2 -- Ministry of International Trade & Industry"
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- public name of test material : Coupling reaction on diazotized (aminonaphthalen)sulfonyl)ethanol polysulfanate with 6-chloro-N-ethyl-N-(3-(ethylsulfonyl)phenyl)-N'-naphthalen-1-yl-1,3,5-triazine polysulfonate, polyhydroxide, polyamine, sodium and potassium salts
- Substance type: textile dyestuff
- Physical state: red powder
- Analytical purity: ca. 63 % of all colored components
- Lot/batch No.: BOP 07-09
- Expiration date of the lot/batch: 2014-07-31
- Storage condition of test material: At room temperature at about 20 °C

Method

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:
Phenobarbital/beta-Naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
deionised water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: without metabolic activation: sodium azide (TA1535, TA100), 4-nitro-o-phenylene-diamine (TA 1537, TA 98), methyl methane sulfonate (WP2 uvrA); with mitabolic activation: 2-aminoanthracene (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: approx 72h

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method:reduction in the number of revertants (below the indication factor of 0.5)
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
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
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
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
valid
Positive controls validity:
valid
Additional information on results:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

A biologically relevant increase in revertant colony numbers were observed following treatment with the test material in strains TA 98 and TA 100 (table 1).
The number of colonies in strain TA 98 exceeded the threshold of twice the number of the corresponding solvent control at concentrations 1000 µg/plate and above in the absence of metabolic activation and at 5000 µg/plate in the presence of metabolic activation.
In strain TA 100, the number of colonies exceeded the threshold of twice the number of the corresponding solvent control at concentrations 1000 µg/plate and above in the absence of metabolic activation.
A slight and dose dependent increase in the number of revertants was also detected at 5000 µg/plate in strain TA 1537 in the absence of metabolic activation and in strain TA 100 with metabolic activation but the threshold was not quite reached.

With metabolic activation, the number of colonies did not quite reach the lower limit of our historical control data (table 2) in strain TA 1537 (solvent control). Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of results

Metabolic activation

Test group

Dose level [µg/plate]

Revertant colony counts (mean ± SD)

TA1535

TA1537

TA98

TA100

WP2uvrA

without

water

 

12±5

8±2

30±4

122±12

65±12

untreated

 

15±2

7±3

25±4

141±3

57±8

test material

3

12±3

10±5

23±1

122±18

56±4

10

15±3

11±3

30±4

136±18

64±12

33

13±1

12±3

28±4

145±24

57±1

100

11±3

10±3

28±2

151±18

56±1

333

15±4

8±1

33±5

197±17

62±10

1000

12±2

9±1

76±4 *

300±7 *

59±8

2500

14±2 d

11±3 d

112±8 d *

411±30 d *

79±2 d

5000

11±2 d

19±5 dm

145±10 dm *

408±18 dm *

83±6 dm

NaN3

10

1919±43 *

 

 

2033±44 *

 

4-NOPD

10

 

 

234±2 *

 

 

4-NOPD

50

 

145±27 *

 

 

 

MMS

3.0

 

 

 

 

1373±35 *

with

water

 

13±3

8±2

35±7

163±11

72±8

untreated

 

21±1

11±2

40±3

153±6

67±8

test material

3

15±5

13±2

33±3

136±13

52±4

10

19±4

12±4

41±6

145±12

64±21

33

15±3

11±2

36±7

164±23

66±7

100

17±3

9±5

39±4

145±12

75±12

333

18±3

8±1

32±2

174±22

69±10

1000

11±6

8±2

36±5

186±6

76±10

2500

12±2 d

10±3 d

54±3 d

235±17 d

76±7 d

5000

12±4 d

13±1 dm

88±8 dm *

303±17 dm

81±9 dm

2-AA

2.5

449±18 *

300±45 *

2223±66 *

2602±105 *

 

2-AA

10

 

 

 

 

381±184 *

NaN3: sodium azide, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS: methyl methane sulfonate

2-AA: 2-aminoanthracene,

d: densely coloured plate

m: manual count

* Value exceeds the respective threshold of twice (TA98, TA100, WP2uvrA) or thrice (TA1535, TA1537) the respective solvent control


Table 2: Laboratory’s historical control data from January 2008 until October 2008 representing approx. 600 experiments (WP2 uvrA the historical data are based on approx. 300 experiments).

Strain

Control

without S9

with S9

Mean

SD

min

max

Mean

SD

min

max

TA1535

solvent

17

5.17

9

39

21

5.82

8

41

negative

17

5.33

9

38

20

6.23

10

46

positive

2024

315.8

1041

3138

294

140.0

102

945

TA1537

solvent

13

3.12

6

25

17

3.90

9

35

negative

13

3.38

5

26

18

4.05

8

31

positive

116

30.52

68

407

204

69.54

72

454

TA98

solvent

30

5.59

13

59

39

6.34

20

60

negative

31

5.45

16

55

39

6.53

19

59

positive

489

169.8

211

1694

1455

463.0

200

3553

TA100

solvent

130

18.79

89

224

155

22.54

92

218

negative

139

17.30

93

205

147

21.78

92

234

positive

2160

342.7

588

3379

1839

621.27

404

3868

WP2uvrA

solvent

49

6.02

33

82

60

8.76

34

89

negative

49

8.58

30

79

60

8.71

31

84

positive

986

482.0

187

2367

414

158.56

164

1597

 

Applicant's summary and conclusion

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

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes and frameshifts in the genome of strains TA 98 and TA 100.
Therefore, the test material is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of the test material to induce gene mutations in the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA according to OECD Guideline 471 and EC method B13/14.

The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

A biologically relevant increase in revertant colony numbers were observed following treatment with the test material in strains TA 98 and TA 100. The number of colonies in strain TA 98 exceeded the threshold of twice the number of the corresponding solvent

control at concentrations 1000 µg/plate and above in the absence of metabolic activation and at 5000 µg/plate in the presence of metabolic activation. The number of colonies in strain TA 100 exceeded the threshold of twice the number of the corresponding solvent control at concentrations 1000 µg/plate and above in the absence of metabolic activation. A slight and dose dependent increase in the number of revertants was also detected at 5000 µg/plate in strain TA 1537 in the absence of metabolic activation and in strain TA 100

with metabolic activation but the threshold was not quite reached.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes and frameshifts in the genome of strains TA 98 and TA 100.

Therefore, the test material is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.