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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 August 1986 till 8 September 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
OECD TG 471

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
with and without pre-incubation
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: PPF/U.K.
- Sample Number: S15100-T01
- Test item: N 2,3-Trimethyl-2-isopropyl butanamide (WS23),

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Stock solutions of DMSO - 50 mg/ml.





Method

Target gene:
TA 1535 hisG46, rfa-, uvrB-
TA 1537 hisC3076, rfa-, uvrB-
TA 1538 hisD3052, rfa-, uvrB-, pKM101
TA 100 hisG46, rfa-, uvrB-, pKM101
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine-dependent bacteria strains
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: histidine-dependent bacteria strains
Metabolic activation:
with and without
Metabolic activation system:
S9 derived from Aroclor-induced rat liver
Test concentrations with justification for top dose:
Number of dose levels and concentration (mg/plate): 1.58, 2.5, 5, 7,5 and 10 (concentrations spaced at log10 intervals).

The concentrations of WS-23, which were used in the mutation assay were determined from a dose range finding toxicity assay using 5 strains of S. typhimurium. The toxicity assay was performed with 5 concentrations of WS-23 spaced at log10 intervals.
Vehicle / solvent:
Vehicle: DMSO.
WS23 was found to be readily soluble in DMSO.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Remarks:
Positive controls: 2-aminoanthracene - stock solution in DMSO Sodium azide - stock solutions in distilled water and filter sterilised 2-nitrofluorene - stock solution in DMSO 9-aminoacridine - stock solution in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Bacteria, test material and metabolising system were incorporated into the top agar of the Petri dishes. The strains were tested routinely for sensitivity to crystal violet, ultraviolet light and, where applicable, resistance to ampicillin.

Negative controls were treated only with the solvent - DMSO.
The petri dishes were incubated for 2 to 3 days, as appropriate, and the number of revertants was determined for each treatment.

Cultures of bacteria of known concentrations were diluted using PBS to about 5x10E3 cells/ml. 0.1 ml of diluted bacteria was added to 2 ml molten top agar followed by 0.1 ml test solution or solvent and 0.5 ml S9 mix or cofactor mix. The top agar was allowed to solidify at room temperature before inversion of the plates for incubation at 37 degrees C. Three plates were prepared for each concentration tested.

After 2-3 days, the plates were removed from the incubator and the number of colonies on each plate were determined.



Rationale for test conditions:
Certain auxotrophic histidine requiring strains of Salmonella exist which readily undergo reversion to histidine independence under the action of chemical mutagens. The test consists of treating histidine-dependent bacteria with the test material plating out on selective medium (i.e. medium not containing histidine), incubating for 2-3 days and finally counting the mutant colonies.
Evaluation criteria:
The average number of mutant colonies per plate is compared with the average number of spontaneous revertants in the control. A dose-related increase in the number of colonies which reaches at least a doubling of the control values is usually considered to be a positive response.
Statistics:
Thee number of bacterial colonies on each plate were determined using Artek 880 automatic colony counter. The number of colonies of bacteria which grew following each treatment is expressed as a percentage of the number of colonies which grew on control plates. The mean number of colonies and standard deviation for each set of plates were calculated, as were the percentage survival including 95% confidence limits.

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:
7,5 and 10 mg/plate with metabolic activation
Vehicle controls validity:
valid
Remarks:
DMSO
Untreated negative controls validity:
other: untreated cells
Positive controls validity:
valid
Remarks:
2-aminoanthracene, sodium azide
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10 mg/plate without and 7,5 and 10 mg/plate with metabolic activation
Vehicle controls validity:
valid
Remarks:
DMSO
Untreated negative controls validity:
other: untreated cells
Positive controls validity:
valid
Remarks:
2-aminoanthracene, 9-aminoacridine
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 7,5 and 10 mg/plate with metabolic activation
Vehicle controls validity:
valid
Remarks:
DMSO
Untreated negative controls validity:
other: untreated cells
Positive controls validity:
valid
Remarks:
2-aminoanthracene, sodium azide
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 7,5 and 10 mg/plate with metabolic activation
Vehicle controls validity:
valid
Remarks:
DMSO
Untreated negative controls validity:
other: untreated cells
Positive controls validity:
valid
Remarks:
2-aminoanthracene, 2-nitrofluorene
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 7,5 and 10 mg/plate with metabolic activation
Vehicle controls validity:
valid
Remarks:
DMSO
Untreated negative controls validity:
other: untreated cells
Positive controls validity:
valid
Remarks:
2-aminoanthracene, 2-nitrofluorene
Remarks on result:
other: negative

Any other information on results incl. tables

Table 1: Mutagenicity Assay Results Test 1 – TA1537 strain

Strain

+/-

pre-incubation

Dose level

(µg/ml)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

TA1537

-

0

5

2.7

1.5

3, 4, 1

1580

7.7

1.5

  8, 9, 6 *

2500

8.5

0.7

  C, 9, 8 *

5000

6

2.6

  7, 3, 8 *

7500

4

1.7

2, 5, 5

10000

3.7

2.1

3, 2, 6

-

0

10

8.3

4

4, 9, 12

1580

7.3

0.6

8, 7, 7

2500

5.7

1.2

7, 5, 5

5000

5.7

0.6

6, 6, 5

7500

5.7

2.5

3, 6, 8

10000

4.3

2.9

6, 6, 1

-

0

20

4

0

4, 4, 4

1580

6

3.5

2, 8, 8

2500

3.3

1.2

2, 4, 4

5000

3.3

1.5

2, 3, 5

7500

4

2.6

3, 2, 7

10000

1.7

1.2

1, 3, 1

Untreated

0

9.7

4.9

12, 4, 13

+

0

5

4.3

2.9

1, 6, 6

1580

5.3

3.5

5, 9, 2

2500

6.3

2.1

8, 4, 7

5000

5.3

2.9

2, 7, 7

7500

1.7

1.2

1, 1, 3

10000

2.7

1.5

3, 1, 4

+

0

10

6.3

0.6

6, 6, 7

1580

6

3

6, 9, 3

2500

3.3

2.5

6, 3, 1

5000

4

1

5, 4, 3

7500

        0.3 T    

0.6

0, 0, 1

10000

   0.3 T

0.6

1, 0, 0

+

0

20

6

2.6

4, 9, 5

1580

4.7

1.5

5, 6, 3

2500

5.7

1.5

4, 6, 7

5000

         1      

1

2, 1, 0

7500

      0  T  

 

 

10000

0

 

 

Untreated

0

6.7

1.5

5, 7, 8

Positive control

+/-

pre-incubation

Concentration

(µg/plate)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

DMSO

-

 

 

10

8.3

4

4, 9, 12

2AA

2

 

554

56

497, 556, 609

DMSO

+

 

10

6.3

0.6

6, 6, 7

2AA

2

 

446.3

21.6

431, 437, 471

* = doubling of negative control values

S.D. = standard deviation

T = toxic dose level

+ = with pre-incubation

- = without pre-incubation

2AA = 2-aminoanthracene administered with DMSO as a vehicle


Table 2: Mutagenicity Assay Results Test 1 – TA1538 Strain

Strain

+/-

pre-incubation

Dose level

(µg/ml)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

TA1538

-

0

5

19.3

6.5

24, 28, 16

1580

19.7

5

24, 17, 19

2500

16.3

2.1

28, 19, 16

5000

19.7

5.7

23, 16, 25

7500

16

2.6

25, 24, ND

10000

12.7

6.4

23, 26, 16

-

0

10

22.7

6.1

24, 28, 16

1580

20

3.6

24, 17, 19

2500

21

6.2

28, 19, 16

5000

21.3

4.7

23, 16, 25

7500

24.5

0.7

25, 24, ND

10000

21.7

5.1

23, 26, 16

-

0

20

23.7

5.7

19, 22, 30

1580

 

24

5.8

C, 22, 26

2500

 

19

3

22, 16, 19

5000

 

21

2.6

20, 19, 24

7500

 

22.7

5.1

27, 17, 24

10000

 

21.3

2.1

23, 19, 22

Untreated

0

11.3

2.1

12, 13, 9

+

0

5

16

1

15, 17, 16

1580

24.7

2.1

23, 27, 24

2500

20

4.6

25, 16, 19

5000

13

2.6

16, 11, 12

7500

    6 T

6.9

14, 2, 2

10000

    0 T

 

 

+

0

10

17.3

4.5

13, 17, 22

1580

 

21.7

5.5

16, 27, 22

2500

 

24

9

33, 24, 15

5000

 

10

1.7

9, 9, 12

7500

 

    0   T

 

 

10000

 

     0.3 T

0.6

0, 0, 1

+

0

20

21.3

302

25, 19, 20

1580

 

21.3

5

16, 26, 22

2500

 

24.7

308

22, 23, 29

5000

 

18

5.3

24, 14, 16

7500

 

  12.3 T

5.1

11, 8, 18

10000

 

     0  T

 

 

Untreated

0

19.3

5.1

15, 18, 25

Positive control

+/-

pre-incubation

Concentration

(µg/plate)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

DMSO

-

 

10

22.7

6.1

24, 28, 16

2AA

 

2

 

1785.3

33.8

1750, 1817, 1789

DMSO

+

 

10

17.3

4.5

13, 17, 22

2AA

 

2

 

642

30.8

607, 665, 654

S.D. = standard deviation

T = toxic dose level

+ = with pre-incubation

- = without pre-incubation

2AA = 2-aminoanthracene administered with DMSO as a vehicle


Table 3: Mutagenicity Assay Results Test 1 – TA98 strain

Strain

+/-

pre-incubation

Dose level

(µg/ml)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

TA98

-

0

5

33

4.4

38, 30, 31

1580

27.7

7.1

20, 34, 29

2500

26

10.1

15, 38, 35

5000

26.7

11

26, 38, 16

7500

27.7

6.4

35, 25, 23

10000

24.3

2.5

27, 22, 24

 

-

0

10

38.7

3.1

38, 42, 36

1580

27.7

3.2

24, 30, 29

2500

34.3

6.7

31, 42, 30

5000

28

3.5

30, 24, 30

7500

29.3

9

38, 30, 20

10000

29

5.6

28, 35, 24

 

-

0

20

31

3

34, 28, 31

1580

33

4

33, 37, 29

2500

32

4.6

33, 27, 36

5000

28.7

7.6

27, 37, 22

7500

28.3

5.5

34, 28, 23

10000

36

0

36, 36, 36

Untreated

0

34.3

6.4

39, 27, 37

 

+

0

5

32

4.4

34, 35, 27

1580

29.3

7.8

27, 38, 23

2500

29.7

3.5

26, 33, 30

5000

26

7

18, 29, 31

7500

18.3

2.1

16, 20, 19

10000

15.3

5.7

9, 20, 17

 

+

0

10

33

5.6

34, 27, 38

1580

31

3.5

29, 29, 35

2500

31.7

4

28, 31, 36

5000

25

2.6

28, 23, 24

7500

   5 T        

5

5, 0, 10

10000

     5.7 T

9

16, 1, 0

 

+

0

20

37.7

2.5

35, 40, 38

1580

36.5

2.1

38, 35, ND

2500

35.3

2.3

34, 38, 34

5000

25

2.6

27, 22, 26

7500

    6.3 T

7.6

3, 1, 15

10000

     0.7 T

1.2

0, 2, 0

Untreated

0

29.7

13.3

45, 22, 22

Positive control

+/-

pre-incubation

Concentration

(µg/plate)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

DMSO

-

 

10

38.7

3.1

38, 42, 36

2AA

 

2

 

1986.3

179.6

2096, 2084, 1779

DMSO

+

 

10

33

5.6

34, 27, 38

2AA

 

2

 

2101

178.3

2285, 2089, 1929

S.D. = standard deviation

T = toxic dose level

S.D. = standard deviation

T = toxic dose level

2-AA = 2-aminoanthracene administered with DMSO as a vehicle

 


Table 4: Mutagenicity Assay Results Test 1 – TA1535 strain

Strain

+/-

pre-incubation

Dose level

(µg/ml)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

TA1535

-

0

5

9

4

9, 5, 11

1580

6.7

3.8

5, 11, 4

2500

12.7

1.5

13, 14, 11

5000

7

2.6

4, 9, 8

7500

7.7

4.6

5, 5, 13

10000

6.3

1.5

5, 6, 8

-

0

10

8

2.6

6, 7, 11

1580

7

2

9, 7, 5

2500

8

3

11, 5, 8

5000

9.7

1.2

9, 9, 11

7500

6.7

3.8

4, 5, 11

10000

3.7

1.2

5, 3, 3

-

0

20

10.7

1.5

9, 12, 11

1580

4.7

2.1

7, 3, 4

2500

5

1.7

6, 3, 6

5000

7.3

3.2

6, 11, 5

7500

6.3

4.9

4, 12, 3

10000

8

5.3

4, 14, 6

Untreated

0

6.3

2.5

6, 4, 9

+

0

5

7.3

1.2

8, 8, 6

1580

9.3

3.8

5, 12, 11

2500

7.3

2.1

9, 8, 5

5000

8.3

3.2

12, 7, 6

7500

 3 T

1.7

1, 4, 4

10000

   3.3 T

1.5

2, 3, 5

+

0

10

10.3

2.1

8, 11, 12

1580

13.3

2.5

13, 11, 16

2500

8

1

9, 7, 8

5000

    5.3 T 

1.5

7, 4, 5

7500

    3.7 T

3.8

8, 1, 2

10000

     2.3 T

1.5

4, 2, 1

+

0

20

6.7

0.6

6, 7, 7

1580

8.7

3.8

7, 6, 13

2500

6

1

7, 6, 5

5000

8

3.6

12, 5, 7

7500

            2 T

3.5

0, 6, 0

10000

     3.3 T

0.6

3, 4, 3

Untreated

0

3.7

2.1

2, 3, 6

Positive control

+/-

pre-incubation

Concentration

(µg/plate)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

DMSO

-

 

10

38.7

3.1

38, 42, 36

2AA

 

2

 

1986.3

179.6

2096, 2084, 1779

DMSO

+

 

10

33

5.6

34, 27, 38

2AA

 

2

 

2101

178.3

2285, 2089, 1929

S.D. = standard deviation

T = toxic dose level

S.D. = standard deviation

T = toxic dose level

2-AA = 2-aminoanthracene administered with DMSO as a vehicle


Table 5: Mutagenicity Assay Results Test 1 – TA100 strain

Strain

+/-

pre-incubation

Dose level

(µg/ml)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

TA100

-

0

5

105.3

15.5

94, 123, 99

1580

98

17.3

108, 108, 78

2500

94.3

20.5

118, 82, 83

5000

83.7

1.1

85, 83, 83

7500

90

2.6

89, 88, 93

10000

82.7

12.6

96, 71, 81

 

-

0

10

105.3

15.7

100, 93, 123

1580

114.3

13.2

117, 100, 126

2500

121.7

6.7

120, 116, 129

5000

103

3

106, 100, 103

7500

107.7

29.4

121, 128, 74

10000

101.3

4

99, 106, 99

 

-

0

20

122.3

4.2

119, 121, 127

1580

120.3

18

139, 119, 103

2500

103.7

4

108, 103, 100

5000

102.3

14.5

119, 95, 93

7500

111.3

10.4

108, 103, 123

10000

127.7

13.4

143, 122, 118

Untreated

0

55.3

12.7

49, 47, 70

 

+

0

5

109.7

16.4

116, 91, 122

1580

126.7

4.2

128, 130, 122

2500

127.3

2.9

129, 129, 124

5000

103.3

2.1

101, 105, 104

7500

83

10.6

95, 75, 79

10000

    0  T

 

 

 

+

0

10

117.3

20.1

123, 134, 95

1580

121

20.5

120, 101, 142

2500

122.7

4.1

127, 119, 122

5000

94.7

13.8

79, 105, 100

7500

63.3

24.5

35, 78, 77

10000

    52  T

20.3

70, 56, 30

 

+

0

20

130

8.9

123, 140, 127

1580

123.7

9.9

117, 119, 135

2500

114.3

10.8

119, 122, 102

5000

94.7

5.5

95, 89, 100

7500

76

14.8

69, 66, 93

10000

      0.3  T

0.6

1, 0, 0

Untreated

0

115.7

8.5

122, 106, 119

Positive control

+/-

pre-incubation

Concentration

(µg/plate)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

DMSO

-

 

 

10

38.7

3.1

38, 42, 36

2AA

 

2

 

1986.3

179.6

2096, 2084, 1779

DMSO

+

 

10

33

5.6

34, 27, 38

2AA

 

2

 

2101

178.3

2285, 2089, 1929

S.D. = standard deviation

T = toxic dose level

S.D. = standard deviation

T = toxic dose level

2AA = 2-aminoanthracene administered with DMSO as a vehicle

Table 6: Mutagenicity Assay Results Test 2 – TA1537 strain

Strain

+/-

pre-incubation

Dose level

(µg/ml)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

TA1537

-

0

5

6.3

2.5

9, 4, 6

1580

6

1

6, 5, 7

2500

9.3

4.6

12, 4, 12

5000

5

2

3, 7, 5

7500

8

1

7, 9, 8

10000

             2  T

1

3, 1, 2

-

0

10

7.7

1.2

7, 9, 7

1580

7

1

8, 7, 6

2500

6.3

1.2

7, 7, 5

5000

6

3.6

7, 2, 9

7500

5.3

1.5

5, 7, 4

10000

2.7

1.5

3, 1, 4

-

0

20

12

5.6

7, 11, 18

1580

9.3

3.2

8, 13, 7

2500

5.7

1.2

5, 7, 5

5000

7.7

1.5

8, 9, 6

7500

6

2

8, 6, 4

10000

6.7

1.5

7, 8, 5

Untreated

0

6.3

1.2

7, 5, 7

+

0

5

5.3

2.3

8, 4, 4

1580

7

1.7

5, 8, 8

2500

8.3

2.5

11, 8, 6

5000

6.3

2.9

3, 8, 8

7500

   1.3 T

1.5

1, 3, 0

10000

    3.3 T

3.2

1, 7, 2

+

0

10

6.3

2.1

8, 4, 7

1580

4.3

1.2

5, 5, 3

2500

5.3

2.9

2, 7, 7

5000

7

1

6, 7, 8

7500

4.7

1.5

6, 5, 3

10000

   2.3 T

1.2

1, 3, 3

+

0

20

10

4.6

5, 14, 11

1580

9.3

2.3

8, 8, 12

2500

8.3

3.2

7, 12, 6

5000

   1  T

1.7

3, 0, 0

7500

  3 T

3

3, 0, 6

10000

    1.7 T

1.2

3, 1, 1

Untreated

0

7.7

2.9

6, 6, 11

Positive control

+/-

pre-incubation

Concentration

(µg/plate)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

DMSO

-

 

 

10

7.7

1.2

7, 9, 7

2AA

2

 

499

18.1

480, 516, 501

DMSO

+

 

10

6.3

2.1

8, 4, 7

2AA

2

 

415

27.8

401, 447, 397

S.D. = standard deviation

T = toxic dose level

+ = with pre-incubation

- = without pre-incubation

2AA = 2-aminoanthracene administered with DMSO as a vehicle


Table 7: Mutagenicity Assay Results Test 2 – TA1538 Strain

Strain

+/-

pre-incubation

Dose level

(µg/ml)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

TA1538

-

0

5

32.3

7.4

38, 35, 24

1580

21.7

1.5

23, 20, 22

2500

28

2.6

29, 25, 30

5000

27.3

7

28, 20, 34

7500

24.3

4.7

26, 19, 28

10000

26

9.5

25, 17, 36

-

0

10

28.3

12.1

17, 27, 41

1580

24

4.6

19, 25, 28

2500

24.7

6.7

17, 28, 29

5000

30

3

33, 27, 30

7500

27.3

6.7

23, 35, 24

10000

29.7

4.7

35, 26, 28

-

0

20

23.3

6.7

25, 29, 16

1580

 

21.3

5.1

27, 20, 17

2500

 

26

8.5

17, 27, 34

5000

 

25

12.2

39, 19, 17

7500

 

25.3

3.5

29, 22, 25

10000

 

24.3

3.8

27, 20, 26

Untreated

0

15.7

2.5

13, 18, 16

+

0

5

22.7

5.5

29, 20, 19

1580

25.7

7

33, 19, 25

2500

23

9.2

13, 25, 31

5000

23.3

4.2

20, 28, 22

7500

20.7

4.2

16, 24, 22

10000

18.3

1.2

19, 17, 19

+

0

 

32

3.6

35, 28, 33

1580

 

28.7

4

25, 28, 33

2500

10

27.7

5

27, 23, 33

5000

 

24.7

4.9

28, 19, 27

7500

 

21.7

4.7

20, 18, 27

10000

 

 14 T

13.1

26, 16, 0

+

0

 

25

3

22, 28, 25

1580

 

28.7

5.5

25, 35, 26

2500

20

27.7

4.2

29, 23, 31

5000

 

23.3

4.2

22, 20, 28

7500

 

  2  T   

2.6

5, 0, 1

10000

 

  0  T

 

 

Untreated

0

26.3

9.1

16, 33, 30

Positive control

+/-

pre-incubation

Concentration

(µg/plate)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

DMSO

-

 

10

28.3

12.1

17, 27, 41

2AA

 

2

 

1930.7

73.7

1929, 1858, 2005

DMSO

+

 

10

32

3.6

35, 28, 33

2AA

 

2

 

2176.7

98.3

2063, 2235, 2232

S.D. = standard deviation

T = toxic dose level

+ = with pre-incubation

- = without pre-incubation

2AA = 2-aminoanthracene administered with DMSO as a vehicle


Table 8: Mutagenicity Assay Results Test 2 – TA98 strain

Strain

+/-

pre-incubation

Dose level

(µg/ml)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

TA98

-

0

5

36.7

8.6

46, 29, 35

1580

25.3

4.7

29, 20, 27

2500

30.7

2.1

33, 30, 29

5000

32.7

4.5

28, 33, 37

7500

32.3

5

27, 33, 37

10000

31

11.5

44, 27, 22

 

-

0

10

38.7

2.1

37, 38, 41

1580

43.3

5.9

39, 41, 50

2500

33.7

5.8

27, 37, 37

5000

36.7

7.4

31, 34, 45

7500

29

5.6

30, 23, 34

10000

39.3

3.8

42, 35, 41

 

-

0

20

44.7

11.1

33, 46, 55

1580

37

5.3

41, 39, 31

2500

42

7.2

44, 48, 34

5000

41.3

7.1

49, 35, 40

7500

41

5.2

38, 47, 38

10000

34.3

5.7

28, 36, 39

Untreated

0

42.3

7.5

35, 50, 42

 

+

0

5

37.7

11

27, 37, 49

1580

36.7

4

41, 36, 33

2500

35

5.3

33, 41, 31

5000

26.3

2.9

28, 28, 23

7500

28

3

31, 28, 25

10000

14.7

10.8

27, 7, 10

 

+

0

10

34.7

4

37, 37, 30

1580

31.3

3.8

34, 27, 33

2500

35.3

2.5

35, 38, 33

5000

26.3

9

35, 17, 27

7500

   8.7 T

8.1

10, 16, 0

10000

    2.7  T

3.8

0, 1, 7

 

+

0

20

34.3

0.6

35, 34, 34

1580

37.7

1.2

39, 37, 37

2500

36.3

3.1

33, 39, 37

5000

23.7

1.2

23, 23, 25

7500

21.3

4

22, 25, 17

10000

    0   T

 

 

Untreated

0

42.7

3.8

47, 40, 41

Positive control

+/-

pre-incubation

Concentration

(µg/plate)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

DMSO

-

 

10

38.7

2.1

37, 38, 41

2AA

 

2

 

1645

54.8

1704, 1635, 1596

DMSO

+

 

10

34.7

4

37, 37, 30

2AA

 

2

 

1599.3

77.9

1511, 1629, 1658

S.D. = standard deviation

T = toxic dose level

+ = with pre-incubation

- = without pre-incubation

2AA = 2-aminoanthracene administered with DMSO as a vehicle


Table 9: Mutagenicity Assay Results Test 2 – TA1535 strain

Strain

+/-

pre-incubation

Dose level

(µg/ml)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

TA1535

-

0

5

8.3

4

4, 12, 9

1580

11

5.3

17, 9, 7

2500

11

2.6

8, 12, 13

5000

9.7

2.1

9, 8, 12

7500

10

2.6

9, 8, 13

10000

6.3

0.6

6, 6, 7

-

0

10

8.7

3.8

6, 7, 13

1580

7

0

7, 7, 7

2500

8.7

6.7

3, 7, 16

5000

8.7

4

11, 4, 11

7500

5.3

1.5

7, 5, 4

10000

7.3

2.1

9, 8, 5

-

0

20

14.3

2.5

14, 17, 12

1580

8.7

2.1

7, 8, 11

2500

9.3

3.5

9, 6, 13

5000

11.3

2.1

9, 13, 12

7500

7.7

4.2

9, 3, 11

10000

9

2

11, 9, 7

Untreated

0

9.3

1.5

9, 8, 11

+

0

5

7.7

2.1

6, 10, 7

1580

12.7

2.9

16, 11, 11

2500

6.3

1.5

5, 6, 8

5000

7.3

1.2

8, 8, 6

7500

6.7

1.2

6, 6, 8

10000

     1.7  T

2.9

0, 5, 0

+

0

10

6.3

1.2

7, 5, 7

1580

11

5

11, 16, 6

2500

8

3.5

12, 6, 6

5000

7

2

9, 7, 5

7500

      2   T

2

4, 0, 2

10000

      1.7 T

1.2

1, 1, 3

+

0

20

11.3

3.8

7, 13, 14

1580

8.3

2.5

6, 11, 8

2500

10

2.6

8, 13, 9

5000

10.7

3.2

7, 12, 13

7500

    1.3   T

2.3

0, 0, 4

10000

     3.7   T

3.2

0, 5, 6

Untreated

0

11

2.6

8, 13, 12

Positive control

+/-

pre-incubation

Concentration

(µg/plate)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

DMSO

-

 

10

8.7

3.8

6, 7, 13

2AA

 

2

 

75.7

10.4

64, 84, 79

DMSO

+

 

10

6.3

1.2

7, 5, 7

2AA

 

2

 

137.3

11.8

130, 131, 151

S.D. = standard deviation

T = toxic dose level

+ = with pre-incubation

- = without pre-incubation

2AA = 2-aminoanthracene administered with DMSO as a vehicle


Table 10: Mutagenicity Assay Results Test 2 – TA100 strain

Strain

+/-

pre-incubation

Dose level

(µg/ml)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

TA100

-

0

5

130.3

3.1

131, 133, 127

1580

140

14.1

142, 153, 125

2500

126.7

14.2

119, 143, 118

5000

111.7

11.9

125, 108, 102

7500

111

15.1

106, 99, 128

10000

112.7

10.7

125, 107, 106

 

-

0

10

139

6.6

132, 140, 145

1580

130

10.6

118, 138, 134

2500

121.7

18.1

105, 141, 119

5000

111.3

10.3

114, 120, 100

7500

123.7

9.1

114, 132, 125

10000

116.7

3.8

121, 114, 115

 

-

0

20

131.3

6

132, 137, 125

1580

131.3

23.9

148, 104, 142

2500

120.7

16.7

102, 134, 126

5000

131.3

10.3

140, 134, 120

7500

133.3

4.5

133, 138, 129

10000

117.3

15.5

117, 133, 102

Untreated

0

145.3

11.8

138, 159, 139

 

+

0

5

118.7

10.7

121, 107, 128

1580

130

21.6

106, 148, 136

2500

129

15

114, 144, 129

5000

93.3

14.6

95, 78, 107

7500

82.3

2.5

82, 85, 80

10000

   46.7 T

34

60, 8, 72

 

+

0

10

109.7

8.4

115, 100, 114

1580

129

9

129, 138, 120

2500

124

19.3

145, 120, 107

5000

94

21.7

81, 119, 82

7500

       62.7 T

19.6

41, 79, 68

10000

     26 T

22.7

36, 42, 0

 

+

0

20

130

10.1

139, 132, 119

1580

138

6.6

137, 145, 132

2500

122

1.8

120, 123, 123

5000

105.3

24.5

77, 119, 120

7500

       51.3      T

40.3

71, 5, 78

10000

       21.7      T

31.7

0, 58, 7

Untreated

0

139.7

18.6

138, 122, 159

Positive control

+/-

pre-incubation

Concentration

(µg/plate)

% S9

Mean Revertant Colony Counts

S.D.

Individual Revertant Colony Counts

DMSO

-

 

10

139

6.6

132, 140, 145

2AA

 

2

 

1850

125.7

1728, 1979, 1843

DMSO

+

 

10

34.7

4

37, 37, 30

2AA

 

2

 

1599.3

77.9

1511, 1629, 1658

S.D. = standard deviation

T = toxic dose level

+ = with pre-incubation

- = without pre-incubation

2AA = 2-aminoanthracene administered with DMSO as a vehicle

Applicant's summary and conclusion

Conclusions:
Based on this testing, it is concluded, that under conditions used in the assay, WS-23 was not mutagenic towards five strains of S. typhimurium.
Executive summary:

The objective of this study was to investigate the potential of WS-23 to be a bacterial mutagen.

 

For this reason, a standard Plate Incorporation Assay (Ames test) was performed twice on WS-23 using five concentrations of WS-23 spaced at log10 intervals. The concentrations of the test material used per Petri dish were, as follows: 1.58, 2.5, 5, 7.5 and 10 mg/plate. The assay was performed with three levels of S9 (5, 10 and 20 %) with and without pre-incubation.

 

Three plates were prepared for each concentration of material tested. Similar numbers of plates were also prepared for appropriate positive and negative (solvent, DMSO) controls. Untreated controls were also included. The plates were incubated at 37 degrees C for either 2 or 3 days. Colonies were counted on an Artek 880 counter.

 

A reduction in the number of revertants and thinning or complete absence of background lawn were observed on several plates treated with the higher concentration (10 mg/plate) of WS 23. The toxicity was dependent on the concentration of S9 used and was more evident with pre-incubation (observed toxicity at 7.5 and 10 mg/plate).

 

No increase in the number of the revertant colonies occurred with any of the five strains of bacteria at test concentrations of WS-23 up to 10 mg/plate at any of the three levels of S9 mix, either with of without pre-incubation.

 

In one test only, with TA 1537 in the presence of 5% S9, there was an increase in the number of revertants three concentrations of twice the spontaneous control value. These increases were neither dose related nor reproducible. The spontaneous revertant values on the control plates were unusually low in this test and the observed increase was therefore considered spurious.

 

In the absence of any evidence of mutagenic potential it is concluded that WS-23 is not mutagenic towards five strains of S. typhimurium up to 5 mg/plate.

 

Interpretation of the data described in this study suggests that WS-23 is unlikely to present a genotoxic hazard.