Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-01-18 to 2005-12-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Juli 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
19 May 2000
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF Aktiengesellschaft, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 fraction
Test concentrations with justification for top dose:
15.625 µg - 5000 µg/plate (STP: Standard plate test)
0.3 µg - 125 µg/plate (PIT: Preincubation test; Salmonella strains)
7.8125 - 125 µg (PIT; Preincubation test; E. coli WP2uvrA)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the Iimited 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 solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: +S-9 mix; 2-aminoanthracene (2-AA): TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA;
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: -S-9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG): TA 1535, TA 100; 4-nitro-o-phenylendiamine (NOPD): TA 98; 9-aminoacridine (AAC): TA 1537; 4-nitroquinoline-N-oxide (4-NQO): E. coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

Standard platetest
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% agar + 0.6% NaCl) and 10 mL amino acid solution (minimal amino acid solution
for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45 °C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S-9 mix (in tests with metabolic activation)
or
0.5 mL phosphate buffer (in tests without metabolic activation)

After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Composition of the minimal glucose agar:
980 mL purified water
20 mL Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.

After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies (his~ revertants) are counted.

Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% agar + 0.6% NaCI) and 10 ml amino acid solution (minimal amino acid solution
for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45 °C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S-9 mix (in tests with metabolic activation)
or
* 0.5 mL phosphate buffer (in tests without metabolic activation)

After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds. Composition of the minimal agar:
The composition of the minimal agar (SAl selective agar) is based on the description of Green, M.H.L. and Muriel, W.J. (5), with the exception of solution E (tryptophan solution),
which has previously been added to the soft agar:
300 mL solution B (agar)
100 mL solution A (sahne solution)
8 mL solution C (glucose solution)
10 mL solution D (casein solution)
After incubation at 370 °C for 48 - 72 hours in the dark, the bacterial colonies (trp~ revertants) are counted.

Preincubation Test:
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S-9 mix or phosphate buffer are incubated at 37 °C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies are counted.

Titer determination
In the standard plate test, 0.1 mL of the overnight cultures is diluted to 10^6 in each case.
Test tubes containing 2-mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 450 °C, and the remaining components are added in the following order:
0.1 mL vehicle (without and with test substance)
0.1 mL fresh bacterial culture (dilution: 10-6)
0.5 mL S-9 mix
In the preincubation test, 0.1 mL of the overnight cultures is diluted to 10^-6 in each case. 0.1 mL vehicle (with and without test substance), 0.1 mL bacterial suspension and 0.5 mL S-9 mix are incubated at 370 °C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 370 °C for 48 - 72 hours in the dark, the bacterial colonies are counted.

NUMBER OF REPLICATIONS: 3

Evaluation criteria:
ACCEPTANCE CRITERIA:
Generally, the experiment is 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 or above.
- The titer of viable bacteria was ≥ 10^8/ mL.

ASSESSMENT CRITERIA:
The test chemical is considered positive in this assay if the following criteria are met:
- 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
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
500 µg - 5000 µg/Plate (+/-S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
100 µg - 5000 µg/Plate (+/-S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
100 µg - 5000 µg/Plate (+/-S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
500 µg - 5000 µg/Plate (+/-S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
500 µg - 5000 µg/Plate (-S9 mix); 2500 µg - 5000 µg/Plate (+S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
30 - 125 µg/plate (+/-S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
30 - 125 µg/plate (+/-S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
30 - 125 µg/plate (+/-S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
30 - 125 µg/plate (+/-S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
125 µg/plate (+/-S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No Precipitation of the test substance was found

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxicity: A strong bacteriotoxic effect was observed under all test conditions.
Remarks on result:
other: Standard plate test

Any other information on results incl. tables

Standard Plate Test (10.02.2005) 

Dose/ Plate

Mean number of revertant colonies/ plate (± SD) with different strains of Salmonella typhimurium and one Escherichia coli strain

TA 1535

TA 100

TA 1537

TA 98

E. coli WP2 uvrA

-S9 mix

DMSO

16 ± 1

102 ± 11

13 ± 3

33 ± 7

45 ± 9

20 µg

23 ± 5

95 ± 12

14 ± 6

24 ± 1

47 ± 7

100 µg

16 ± 3

34 ± 6 B

5 ± 1 B

27 ± 4

35 ± 2

500 µg

0B

0B

0B

6 ± 1 B

26 ± 1 B

2500 µg

0B

0B

0B

0B

18 ± 2 B

5000 µg

0B

0B

0B

0B

2 ± 2 B

MNNG 5.0 µg

1218 ± 133

1379 ± 121

-

-

-

AAC

-

-

980 ± 65

-

-

NOPD

-

-

-

1009 ± 95

-

4-NQO

-

-

-

-

1065 ± 106

+S9 mix

DMSO

21 ± 4

104 ± 13

13 ± 1

39 ± 2

44 ± 3

20 µg

23 ± 1

113 ± 5

13 ± 4

38 ± 2

39 ± 3

100 µg

20 ± 3

67 ± 16 B

9 ± 4 B

33 ± 4

35 ± 8

500 µg

0B

0B

0B

6 ± 3 B

18 ± 3

2500 µg

0B

0B

0B

0B

10 ± 4 B

5000 µg

0B

0B

0B

0B

4 ± 1 B

2-AA 2.5 µg

182 ± 20

1564 ± 294

107 ± 6

1544 ± 192

306 ± 16

 

 

Standard Plate Test (18.02.2005) 

Dose/ Plate

Mean number of revertant colonies/ plate (± SD) with different strains of Salmonella typhimurium and one Escherichia coli strain

TA 1535

TA 100

TA 1537

TA 98

E. coli WP2 uvrA

-S9 mix

DMSO

20 ± 3

116 ± 9

10 ± 3

28 ± 2

32 ± 4

15.625 µg

19 ± 2

134 ± 3

9 ± 2

26 ± 2

28 ± 4

31.25 µg

19 ± 3

120 ± 5

13 ± 3

28 ± 4

28 ± 7

62.5 µg

18 ± 1

89 ± 4

8 ± 2

27 ± 3

29 ± 4

125 µg

15 ± 2

48 ± 6

7 ± 1

17 ± 3

25 ± 4

250 µg

11 ± 3

5 ± 3

4 ± 2

20 ± 3

24 ± 4

MNNG 5.0 µg

673 ± 26

625 ± 106

-

-

-

AAC

-

-

578 ± 57

-

-

NOPD

-

-

-

783 ± 30

-

4-NQO

-

-

-

-

594 ± 39

+S9 mix

DMSO

18 ± 1

127 ± 18

13 ± 2

34 ± 4

40 ± 5

15.625 µg

20 ± 3

136 ± 7

10 ± 2

29 ± 5

36 ± 5

31.25 µg

19 ± 1

129 ± 14

10 ± 2

28 ± 4

38 ± 3

62.5 µg

17 ± 3

118 ± 11

10 ± 1

29 ± 2

31 ± 13

125 µg

14 ± 3

104 ± 9

10 ± 2

25 ± 5

32 ± 12

250 µg

16 ± 2

43 ± 43

5 ± 2

25 ± 2

25 ± 4

2-AA 2.5 µg

177 ± 8

1010 ± 129

135 ± 10

857 ± 25

245 ± 27

 

   

Preincubation Test (22.02.2005) 

Dose/ Plate

Mean number of revertant colonies/ plate (± SD) with different strains of Salmonella typhimurium and one Escherichia coli strain

TA 1535

TA 100

TA 1537

TA 98

E. coli WP2 uvrA

-S9 mix

DMSO

18 ± 2

110 ± 3

12 ± 3

32 ± 3

31 ± 2

7.8125 µg

14 ± 1

108 ± 6

11 ± 1

32 ± 6

29 ± 3

 15.625 µg

15 ± 1

103 ± 2

8 ± 1

22 ± 4

31 ± 6

31.25 µg

12 ± 3 B

69 ± 10 B

4 ± 1 B

11 ± 2 B

28 ± 4

62.5 µg

0B

11 ± 3 B

0B

6 ± 3 B

24 ± 3

125 µg

0B

0B

0B

0B

22 ± 7 B

MNNG 5.0 µg

744 ± 26

832 ± 29

-

-

 

AAC

-

-

475 ± 24

-

-

NOPD

-

-

-

954 ± 18

-

4-NQO

-

-

-

-

571 ± 55

+S9 mix

DMSO

17 ± 2

107 ± 3

12 ± 3

37 ± 7

39 ± 5

7.8125 µg

14 ± 2

104 ± 5

11 ± 2

28 ± 3

32 ± 4

 15.625 µg

12 ± 2

110 ± 6

8 ± 1

20 ± 4

36 ± 5

31.25 µg

10 ± 2 B

98 ± 3 B

6 ± 2 B

17 ± 3 B

28 ± 7

62.5 µg

6 ± 1 B

72 ± 3 B

7 ± 0 B

13 ± 2 B

30 ± 2

125 µg

4 ± 2 B

31 ± 5 B

4 ± 1 B

7 ± 2 B

28 ± 3 B

2-AA 2.5 µg

155 ± 17

885 ± 55

158 ± 18

1021 ± 86

240 ± 17

 

 

Preincubation Test (02.03.2005) 

Dose/ Plate

Mean number of revertant colonies/ plate (± SD) with different strains of Salmonella typhimurium and one Escherichia coli strain

TA 1535

TA 100

TA 1537

TA 98

-S9 mix

DMSO

17 ± 2

107 ± 3

9 ± 3

30 ± 5

0.3 µg

16 ± 3

104 ±7

8 ± 2

28 ± 3

1 µg

15 ± 3

111 ± 4

10 ± 2

30 ± 6

3 µg

14 ± 2

100 ± 4

8 ± 2

30 ± 7

10 µg

12 ± 2

87 ± 4

6 ± 2

22 ± 3

30 µg

9 ± 1 B

47 ± 20 B

4 ± 3 B

9 ± 1 B

MNNG 5.0 µg

811 ± 96

750 ± 40

-

-

AAC

-

-

902 ± 58

-

NOPD

-

-

-

635 ± 53

4-NQO

-

-

-

-

+S9 mix

DMSO

18 ± 2

108 ± 10

10 ± 1

31 ± 2

0.3 µg

15 ± 1

104 ± 3

8 ± 2

28 ± 7

1 µg

15 ± 3

107 ± 7

11 ± 3

29 ± 3

3 µg

17 ± 3

103 ± 7

7 ± 1

27 ± 6

10 µg

11 ± 3

100 ± 6

7 ± 2

21 ± 3

30 µg

11 ± 6 B

84 ± 11 B

6 ± 2 B

10 ± 2 B

2-AA 2.5 µg

107 ± 8

902 ± 58

383 ± 41

806 ± 41

SD: Standard deviation

B: Reduced background growth

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions chosen, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay.
Executive summary:

The test substance (in DMSO) was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and in Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2 uvrA according to OECD guideline 471.
The test substance was tested up to concentrations of 20 µg to 5000 µg/plate in the standard plate test (STP). In the preincubation tests concentrations of 0.3 µg to 125 µg/plate (S. typhimurium) and 7.7128 µg to 125 µg/plate (E. coli) were tested. No test substance precipitation was found. A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 100 µg to 500 µg/plate onward. In the preincubation assay bacteriotoxicity (reduced his- background growth, decrease in the number of his+ revertants, reduction in the titer) was observed depending on the Salmonella strain and test conditions from about 30 µg/plate onward. Using E. coli WP2 uvrA toxicity was found at doses ≥ 62.5 µg/plate.
An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.