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:
1999-03-17 to 2000-04-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenates (S9 fraction) from male Sprague-Dawly rats, induced with 500 mg/kg bw Aroclor 1254 (single injection intraperitoneal)
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenates (S9 fraction) from male Sprague-Dawly rats, induced with 500 mg/kg bw Aroclor 1254 (single injection intraperitoneal)
Test concentrations with justification for top dose:
All concentrations in µg/plate:
Experiment 1:
0; 20; 100; 500; 2,500; 5,000
Experiment 2:
0 ; 20; 100; 500; 2,500; 5,000
Experiment 3:
Salmonella strains: 0; 0.8; 4; 20; 100; 500
E. coli: 0; 4; 20; 100; 500; 1,000
Experiment 4:
Salmonella strains: 0; 3.125; 6.25; 12.5; 25; 50
E. coli: 0; 4; 20; 100; 500; 1,000
Vehicle:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
With metabolic activation; strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without metabolic activation; strains: TA 1535, TA 100
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine (NOPD)
Remarks:
Without metabolic activation; strains: Ta 98
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation; strains: TA 1537
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without metabolic activation; strains: E. coli WP2 urvA
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer
Evaluation criteria:
Acceptance criteria:
Generally, the experiment is to be considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.
- The titer of viable bacteria was > 10^9 / mL.

Evaluation criteria:
The test item is considered positive in this assay if:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

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:
yes
Remarks:
depending on the strain and conditions from about 20 - 500 µg/plate onward
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
depending on the strain and conditions from about 20 - 500 µg/plate onward
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: There was a limited solubility of the test substance in water.
- Precipitation: No test substance precipitation was found.

ADDITIONAL INFORMATION ON CYTOTOXICITY: A bacteriotoxic effect (reduced background growth, decrease in the number of revertants, reduction in the titer) was observed depending on the strain and test conditions from about 20 µg - 500 µg/plate onward.

Any other information on results incl. tables

Experiment 1 & 2 (standard plate test)

Dose (µg/plate)

Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium and E. coli

 

TA 100

TA 98

TA 1535

TA 1537

WP2 uvrA

 

 

 

Results without S9

DMSO

142 ± 8

27 ± 3

21 ± 4

10 ± 2

30 ± 5

20

120 ± 10

23 ± 3

16 ± 3

10 ± 1

28 ± 6

100

115 ± 10

19 ± 3

16 ± 3

8 ± 3

27 ± 1

500

64 ± 13

13 ± 3

12 ± 2

5 ± 2

24 ± 4

2500

 0

0

0

0

24 ± 3

5000

 0

0

0

0

13 ± 3

MNNG (5)

 684 ± 145

602 ± 27

NOPD (10)

926 ± 27

AAC (100)

818 ± 81

4-NQO (5)

991 ± 61

 

 

 

Results with S9 (1:9)

DMSO

133 ± 3

34 ± 1

20 ± 3

11 ± 2

37 ± 4

20

142 ± 16

20 ± 3

16 ± 1

8 ± 2

37 ± 3

100

117 ± 36

17 ± 3

16 ± 3

9 ± 1

35 ± 4

500

 23 ± 2

16 ± 2

9 ± 4

6 ± 3

28 ± 2

2500

0

0

9 ± 2

0

27 ± 8

5000

0

0

0

0

16 ± 3

2- AA (2.5)

1087 ± 90

1038 ± 23

168 ± 21

145 ± 14

241 ± 11

Experiment 3 (standard plate test)

Dose (µg/plate)

Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium and E. coli

 

TA 100

TA 98

TA 1535

TA 1537

WP2 uvrA

 

 

 

Results without S9

DMSO

110 ± 9

27 ± 3

20 ± 3

12 ± 3

25 ± 1

0.8

97 ± 6

27 ± 2

15 ± 3

9 ± 2

-

4

90 ± 9

22 ± 2

16 ± 3

10 ± 2

23 ± 4

20

99 ± 6

17 ± 4

16 ± 3

8 ± 2

22 ± 4

100

52 ± 7

10 ± 1

12 ± 2

3 ± 2

28 ± 3

500

6 ± 3

8 ± 2

11 ± 2

1 ± 0

21 ± 2

1000

-

-

-

-

22 ± 3

MNNG (5)

697 ± 72

842 ± 89

NOPD (10)

799 ± 60

AAC (100)

845 ± 41

4-NQO (5)

822 ± 101

 

 

 

Results with S9 (1:9)

DMSO

112 ± 10

36 ± 3

19 ± 3

12 ± 2

34 ± 3

0.8

98 ± 8

39 ± 2

16 ± 3

10 ± 1

-

4

90 ± 2

28 ± 2

16 ± 4

9 ± 1

31 ± 2

20

77 ± 3

36 ± 2

13 ± 2

9 ± 2

30 ± 1

100

32 ± 19

24 ± 4

8 ± 3

6 ± 1

31 ± 3

500

8 ± 4

13 ± 2

7 ± 2

2 ± 2

30 ± 3

1000

-

-

-

-

26 ± 4

2- AA (2.5)

1100 ± 59

922 ± 15

202 ± 22

113 ± 10

22 ± 19

Experiment 4 (Preincubation test)

Dose (µg/plate)

Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium

 

TA 100

TA 98

TA 1535

TA 1537

 

 

 

Results without S9

DMSO

112 ± 7

23 ± 4

18 ± 2

9 ± 2

3.125

108 ± 14

21 ± 3

14 ± 5

6 ± 3

6.25

100 ± 9

23 ± 6

15 ± 2

8 ± 1

12.5

101 ± 14

20 ± 2

17 ± 2

8 ± 2

25

89 ± 14

20 ± 3

15 ± 1

8 ± 2

50

90 ± 8

14 ± 2

5 ± 1

0

MNNG (5)

1177 ± 22

1027 ± 43

NOPD (10)

1158 ± 40

AAC (100)

717 ± 11

 

Results with S9 (1:9)

DMSO

109 ± 8

32 ± 8

17 ± 2

8 ± 2

3.125

117 ± 18

30 ± 2

16 ± 4

11 ± 2

6.25

105 ± 16

30 ± 2

14 ± 4

10 ± 2

12.5

113 ± 10

33 ± 3

15 ± 1

9 ± 2

25

116 ± 35

28 ± 5

9 ± 4

7 ± 1

50

94 ± 7

30 ± 4

14 ± 3

8 ± 2

2- AA (2.5)

644 ± 19

687 ± 38

138 ± 20

106 ± 11

Dose (µg/plate)

Mean number of revertant colonies of 3 replicates (± S.D.) for E. coli

 

WP2 uvrA

 

 

 

Results without S9

DMSO

24 ± 4

4

24 ± 5

20

18 ± 3

100

19 ± 3

500

18 ± 7

1000

16 ± 2

4-NQO (5)

746 ± 39

 

 

 

Results with S9 (1:9)

DMSO

33 ± 4

4

27 ± 4

20

24 ± 11

100

20 ± 5

500

16 ± 7

1000

16 ± 1

2- AA (2.5)

221 ± 27

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative