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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): NovaPK
- Physical state: white powder (Deseription from the Sponsor : Colourless crystalline powder)
- Lot/batch No.: 644
- Expiration date of the lot/batch: 23-Sep-2008
- Storage condition of test material: room temperature
- Other: container -opaque plastic bottle
- Analytical purity: no data

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9, induced with phenobarbitone/ß-naphthoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test: 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate.
Main Study: 5000, 2500, 1250, 625 and 313 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
DMSO (for positive controls 9-aminoacridine, 2-nitrofluorene, 2-aminoanthracene)
Sterile distilled water (for the test item and the positive controls sodium azide and methylmethanesulphonate)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without activation

Migrated to IUCLID6: 1 µg/plate for TA100 and TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without activation

Migrated to IUCLID6: 50 µg/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without activation

Migrated to IUCLID6: 2 µg/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without activation

Migrated to IUCLID6: 500 µg/plate for WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene: 1 µg/plate for TA98 and TA100 (plate incorporation method), TA1535 and TA1537, 2 µg/plate for TA98 and TA100 (pre-incubation method), 10 µg/plate for WP2 uvrA (plate incorporation method, 20 µg/plate for WP2 uvrA (pre-incubation method)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, main test I); preincubation (main test II)

DURATION
- Preincubation period: 30 min
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 3 per concentration and experiment (main test)

DETERMINATION OF CYTOTOXICITY
- Method: decline in the number of spontaneous revertants, thinning of the background lawn or a microcolony formation.
Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
Regression analysis, Student's t-test

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 nor precipitates, but tested up to recommended limit concentrations
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 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
- Precipitation: no


RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed with any tester strain, in the absence or presence of S9 metabolic activation up to 5000 µg/plate.


COMPARISON WITH HISTORICAL CONTROL DATA:
mean plate counts for negative and positive controls fell within the normal historical range.
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, plate incorporation method, without metabolic activation

S9

Concentration (µg/plate)

Number of revertants (mean number of colonies / plate)

TA1535

TA1537

TA98

TA100

WP2uvrA

-

0

18

16

27

138

29

-

313

17

15

26

151

25

-

625

21

15

25

140

29

-

1250

17

18

27

151

24

-

2500

17

22

28

147

22

-

5000

21

18

25

144

24

Negative control

Untreated

18

-

-

138

29

DMSO (100µl/plate)

-

17

26

-

-

Positive controls

Name

SA

9-AA

2-NF

SA

MMS

Concentration (µg/plate)

1

50

2

1

500

No. of revertants (mean)

524

163

174

526

172

SA: Sodium azide

9-AA: 9-aminoacridine

MMS: methylmethane sulfonate

2-NF: 2-Nitrofluorene

 

Table 2: Experiment 1, plate incorporation method, with metabolic activation

S9

Concentration (µg/plate)

Number of revertants (mean number of colonies / plate)

TA1535

TA1537

TA98

TA100

WP2uvrA

+

0

16

19

39

153

33

+

313

19

21

38

161

29

+

625

22

19

36

163

33

+

1250

21

20

38

169

27

+

2500

16

19

37

156

29

+

5000

17

19

36

159

28

Negative control

Untreated

-

-

-

-

-

DMSO (100µl/plate)

20

19

33

151

30

Positive controls

Name

2-AA

2-AA

2-AA

2-AA

2-AA

 

Concentration (µg/plate)

1

1

1

1

10

No. of revertants (mean)

118

100

469

1135

174

2-AA: 2-aminoanthracene

 

 

 

Table 3: Experiment 2, pre-incubation method, without metabolic activation

S9

Concentration (µg/plate)

Number of revertants (mean number of colonies / plate)

TA1535

TA1537

TA98

TA100

WP2uvrA

-

0

18

16

28

152

28

-

313

18

16

29

152

28

-

625

16

16

31

155

28

-

1250

16

17

26

158

25

-

2500

19

18

27

154

27

-

5000

20

17

27

157

26

Negative control

Untreated

17

-

-

152

28

DMSO (50µl/plate)

-

18

30

-

-

Positive controls

Name

SA

9-AA

2-NF

SA

MMS

Concentration (µg/plate)

1

50

2

1

500

No. of revertants (mean)

517

203

162

565

162

SA: Sodium azide

9-AA: 9-aminoacridine

MMS: methylmethane sulfonate

2-NF: 2-Nitrofluorene

 

Table 4: Experiment 2, pre-incubation method, with metabolic activation

S9

Concentration (µg/plate)

Number of revertants (mean number of colonies / plate)

TA1535

TA1537

TA98

TA100

WP2uvrA

+

0

17

19

37

167

31

+

313

19

18

38

167

37

+

625

16

18

36

167

36

+

1250

16

18

40

167

38

+

2500

19

19

36

171

36

+

5000

18

20

38

160

36

Negative control

Untreated

-

-

-

-

-

DMSO (50µl/plate)

16

19

38

126

33

Positive controls

Name

2-AA

2-AA

2-AA

2-AA

2-AA

 

Concentration (µg/plate)

1

1

1

1

10

No. of revertants (mean)

113

106

479

1077

200

2-AA: 2-aminoanthracene

 

Applicant's summary and conclusion

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