Registration Dossier

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:
23 May - 15 Jun 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 Jul 1997
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): PM-5927
- Physical state: off-white powder
- Lot/batch No.: 07-02
- Expiration date of the lot/batch: 05 Feb 2008
- Storage condition of test material: at room temperature protected from light

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, 333, 1000 and 3300 µg/plate for TA 98, TA 1535 and TA 1537 (with and without metabolic activation)
Experiment 2: 10, 33, 100, 333, 1000 and 2000 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:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Remarks:
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 background bacterial lawn
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.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test results
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed in the dose-range finding test at the start or at the end of the incubation period in both test strains (TA 100, E. coli WP2 uvrA)

RANGE-FINDING/SCREENING STUDIES:
A range-finding study was performed with TA 100 and E. coli WP2 uvr A, using 8 concentrations ranging 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 cytotoxicity (2000 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. See Table 3 under 'Any other information on results incl. tables'

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the range-finding study, a reduction in bacterial background lawn and/or number of revertant colonies was observed from concentrations of 1000 µg/plate in TA 100 and from concentrations of 3330 µg/plate in WP2 uvrA, with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation method)

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvR

TA98

TA1537

DMSO

118 ± 13

12 ± 4

10 ± 3

24 ± 4

13 ± 4

3

108 ± 13

-

13 ± 3

-

-

10

112 ± 4

7 ± 2

12 ± 1

21 ± 5

10 ± 3

33

99 ± 9

8 ± 2

13 ± 6

18 ± 4

12 ± 1

100

124 ± 4

7 ± 2

10 ± 4

16 ± 2

11 ± 3

333

98 ± 5

8 ± 1

11 ± 3

19 ± 3

7 ± 3

1000

92 ± 19*

4 ± 1*

10 ± 2

13 ± 3

8 ± 1

2000

-

MC

-

1 ± 1*

MC*

3330

0 ± 0*

-

MC*

-

-

5000

0 ± 0*

-

0 ± 0*

-

-

Positive controls, –S9

Name

MMS

SA

4-NQO

NF

9AC

Concentrations

(μg/plate)

650

5

10

10

60

Mean No. of colonies/plate

(average of 3 ± SD)

1062 ± 40

966 ± 23

696 ± 40

1047 ± 7

271 ± 49

+

DMSO

90 ± 7

10 ± 1

26 ± 4

31 ± 2

13 ± 4

+

3

98 ± 8

-

25 ± 6

-

-

+

10

104 ± 13

6 ± 2

24 ± 2

23 ± 5

12 ± 5

+

33

96 ± 11

6 ± 3

29 ± 9

21 ± 5

9 ± 3

+

100

97 ± 7

8 ± 2

29 ± 5

26 ± 3

10 ± 2

+

333

90 ± 4

5 ± 2

22 ± 2

22 ± 5

10 ± 3

+

1000

66 ± 13*

6 ± 2*

22 ± 3

17 ± 3

6 ± 3

+

2000

-

0 ± 0*

-

2 ± 2*

0 ± 0*

+

3330

MC*

-

10 ± 3*

-

-

+

5000

0 ± 0*

-

5 ± 2*

-

-

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1

1

10

1

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

996 ± 40

221 ± 23

311 ± 8

830 ± 48

459 ± 16

MMS: methylmethanesulfonate

SA : sodium azide

4-NQO: 4-nitroquinoline N-oxide

NF: 2-nitrofluorene

9AC: 9-aminoacridine

2-AA: 2 -amino-anthracene

*: slight/moderate/extreme reduction in bacterial background lawn

MC: microcolonies

-: not tested

 


 

Table 2. Test results of experiment 2 (plate incorporation method)

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvR

TA98

TA1537

DMSO

79 ± 9

6 ± 3

14 ± 3

13 ± 1

6 ± 1

10

76 ± 6

5 ± 2

15 ± 2

24 ± 8

9 ± 4

33

78 ± 8

5 ± 0

20 ± 4

22 ± 5

7 ± 2

100

75 ± 3

6 ± 2

19 ± 3

24 ± 2

7 ± 4

333

92 ± 11

8 ± 4

21 ± 2

14 ± 4

4 ± 1

1000

38 ± 70*

6 ± 1*

14 ± 4

18 ± 4

3 ± 2

2000

MC*

MC*

11 ± 1*

9 ± 1*

MC*

Positive controls, –S9

Name

MMS

SA

4-NQO

NF

9AC

Concentrations

(μg/plate)

650

5

10

10

60

Mean No. of colonies/plate

(average of 3 ± SD)

1013 ± 88

871 ± 17

1170 ± 10

978 ± 45

373 ± 90

+

DMSO

69 ± 3

5 ± 2

15 ± 1

17 ± 3

7 ± 1

+

10

65 ± 2

4 ± 1

15 ± 1

14 ± 1

6 ± 1

+

33

50 ± 8

5 ± 1

16 ± 1

15 ± 4

5 ± 2

+

100

47 ± 5

4 ± 2

19 ± 5

13 ± 2

8 ± 3

+

333

57 ± 4

4 ± 2

14 ± 2

15 ± 4

6 ± 2

+

1000

44 ± 12*

2 ± 1*

17 ± 5

15 ± 4

4 ± 3*

+

2000

MC*

4 ± 2*

12 ± 3*

3 ± 3*

MC*

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

1

10

1

5

Mean No. of colonies/plate

(average of 3 ± SD)

786 ± 30

150 ± 4

294 ± 10

381 ± 17

216 ± 51

MMS: methylmethanesulfonate

SA : sodium azide

4-NQO: 4-nitroquinoline N-oxide

NF: 2-nitrofluorene

9AC: 9-aminoacridine

2-AA: 2 -amino-anthracene

*: slight/moderate/extreme reduction in bacterial background lawn

MC: microcolonies

-: not tested

 

 

Table 3. Historical controls

 

Strain

Control

without S9-mix

with S9-mix

 

 

Mean ± 3 x SD

Mean ± 3 x SD

TA100

Solvent

126 ± 79

116 ± 83

TA100

Positive

1038 ± 596

1094 ± 996

TA1535

Solvent

13 ± 14

12 ± 14

TA1535

Positive

1145 ± 819

183 ± 233

WP2 uvR

Solvent

14 ± 17

15 ± 8

WP2 uvR

Positive

643 ± 565

254 ± 343

TA98

Solvent

20 ± 19

25 ± 21

TA98

Positive

1053 ± 748

648 ± 166

TA1537

Solvent

6 ± 8

6 ± 9

TA1537

Positive

315 ± 415

334 ± 433

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative