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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:
18 Sep - 1 Oct 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted 08 Jun 2000
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: powder aggregated into lumps
Details on test material:
- Name of test material (as cited in study report): SPM-N
- Physical state: white lumps with powder
- Lot/batch No.: T-9314
- Expiration date of the lot/batch: 29 Aug 2008
- Storage condition of test material: at room temperature in the dark

Method

Target gene:
his operon (S. typhimurium strains), trp operon (E. coli strains)
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:
Cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with phenobarbital (80 mg/kg bw) and beta-naphthoflavone (100 mg/kg bw)
Test concentrations with justification for top dose:
Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate for TA 100 and E. coli WP2 uvr A (with and without metabolic activation)
Experiment 1: 33, 100 and 333, 1000 and 3300 µg/plate for TA 98, TA 1535 and TA 1537 (with and without metabolic activation)
Experiment 2: 33, 100, 330, 1000, 3330 μg/plate for all strains (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, methylmethanesulfonate, 4-nitroquinoline-N-oxide, 2-aminoanthracene (see 'Any other information on materials and methods incl. tables' for details)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction of bacterial background lawn, increase in size of microcolonies, reduction of revertant colonies
Evaluation criteria:
The assays were considered acceptable if they met the following criteria:
a) the negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) the positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least 3 times the concurrent vehicle control group.
c) the selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) the total number of revertants in tester strain TA 100 is not greater than 2 times the concurrent control, and the total number of revertants in tester strains TA 1535, TA 1537, TA 98 or WP2 uvr A is not greater than 3 times the concurrent control.
b) the negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) the total number of revertants in tester strain TA 100 is greater than 2 times the concurrent control, and the total number of revertants in tester strains TA 1535, TA 1537, TA 98 or WP2 uvr A is greater than 3 times the concurrent control.
b) in case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
Statistics:
Mean values and standard deviation were calculated.

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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was observed at the start and the end of the incubation period at concentrations of 3330 and 5000 μg/plate (with and without metabolic activation)

RANGE-FINDING/SCREENING STUDIES: A range-finding study was performed with TA 100 and WP2 uvr A, using 8 concentrations from 33-5000 μg/plate (with and without metabolic activation). The highest concentration of the test substance used in the subsequent mutation assay was the level at which the test substance showed limited solubility (3330 µg/plate). The results were presented as part of experiment 1.

COMPARISON WITH HISTORICAL CONTROL DATA: yes; the results fall within the historical data 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: Results of experiment 1

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates±SD)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

-S9

Dimethyl sulfoxide

107±10

9±4

23±3

18±3

6±3

-S9

3

108±12

-

24±6

-

-

-S9

10

105±5

-

20±3

-

-

-S9

33

100±13

5±1

27±2

20±8

6±1

-S9

100

104±5

7±2

24±3

22±3

5±1

-S9

333

91±5

7±2

23±4

15±1

6±3

-S9

1000

111±4

9±3

20±4

17±4

12±2

-S9

3300 SP

96±10

13±5

24±4

19±5

13±2

-S9

5000 SP

108±15

-

20±1

-

-

Positive controls, –S9

Name

MMS

SA

4-NQO

2-NF

9-AC

Concentrations

(μg/plate)

650

5

10

10

60

 

967±16

987±20

1146±12

963±32

338±16

 

 

 

 

 

 

 

+S9

Dimethyl sulfoxide

81±11

6±2

19±3

23±9

4±1

+S9

3

89±5

-

22±2

-

-

+S9

10

83±15

-

16±3

-

-

+S9

33

90±16

8±3

19±5

27±5

4±1

+S9

100

76±10

8±5

20±4

21±2

4±2

+S9

333

83±10

8±1

20±2

21±8

7±2

+S9

1000

91±10

8±1

20±3

18±3

6±3

+S9

3330 SP

84±14

9±2

21±4

27±2

9±3

+S9

5000 SP

81±16

-

22±3

-

-

Positive controls, +S91

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

1

1

10

1

2.5

 

619±72

221±12

330±10

856±5

338±13

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

2-NF = 2-nitrofluorene

2-AA = 2-aminoanthracene

SP = slight precipitate

1S9-mix contained 5% (v/v) S9-fraction


Table 2: Results of experiment 2

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates±SD)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

-S9

Dimethyl sulfoxide

107±6

13±6

21±3

20±1

4±1

-S9

33

100±5

11±3

25±8

18±1

4±1

-S9

100

99±23

9±1

21±6

14±2

4±1

-S9

333

95±6

11±2

17±2

22±2

8±4

-S9

1000

128±31

8±3

30±3

21±4

11±6

-S9

3300 SP

93±12

10±3

29±2

16±6

12±5

Positive controls, –S9

Name

MMS

SA

4-NQO

2-NF

9-AC

Concentrations

(μg/plate)

650

5

10

10

60

 

976±25

941±25

1026±21

918±83

437±64

 

 

 

 

 

 

 

+S9

Dimethyl sulfoxide

62±3

7±3

22±9

24±3

3±0

+S9

33

60±4

6±2

25±7

19±3

3±1

+S9

100

61±7

5±2

26±3

27±9

3±1

+S9

333

58±2

4±0

25±7

16±5

4±1

+S9

1000

66±5

6±1

26±4

18±6

7±4

+S9

3330 SP

68±10

4±2

27±6

25±9

10±5

Positive controls, +S92

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

2.5

1

10

1

5

 

613±26

138±7

294±24

369±36

259±17

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

2-NF = 2-nitrofluorene

2-AA = 2-aminoanthracene

SP = slight precipitate

2S9-mix contained 10% (v/v) S9-fraction

In the second experiment in strain TA 1537, up to 3.0 and 3.3-fold increases in the number of revertant colonies compared to the vehicle control was noted, with and without metabolic activation. As no increase was noted in any other strain in the 2 experiments, the increase was only noted in 1 experiment and the increase was within the historical data range and only noted at precipitating concentrations, this increase is not considered to be biologically relevant.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative