Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In two key studies of 1986, the potential of WS-23 to induce bacterial mutagenicity was investigated using the AMES assays equivalent or similar to the OECD TG 471.

In the first study (report number: CC/81/86), 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 was 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.

In the second key study (report number: 10120/5), the toxicity assay was performed with five concentrations of WS 23: 0.5 µg, 5 µg, 50 µg, 500 µg and 5 mg. These concentrations were tested against all 5 strains of S. typhimurium both with and without addition of S9. Different levels of toxicity of WS-23 towards each bacterial strain were observed, and toxicity was also dependent on the presence or absence of S9.

The mutagenicity assay (AMES test) was performed twice with five concentrations of WS-23: 50 µg, 158 µg, 500 µg, 1.58 mg and 5 mg/plate. The assay was performed with and without addition of S9. Briefly, the bacterial plates were inverted and incubated at 37 Celsius degrees for 2-3 days. The mean number of colonies and standard deviation for each set of plates were calculated.

The results of the main assay showed, that WS-23 did not induce mutations in the genomes of the organisms used under the conditions of the assay. Therefore, it was concluded, that there was no evidence to support that WS-23 is a mutagen.

 

Overall, negative results for mutagenicity were concluded for the test substance, WS-23.

Link to relevant study records

Referenceopen allclose all

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
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
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.





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 / 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.
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.
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

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

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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
27 February 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test material: N,2,3 trimethyl-2-isopropyl butanamide (WS23)
ESL Sample Number 515100 -TOl
No analyses to confirm the identity of the test material were performed as part of this study.
Target gene:
The strains of Salmonella typhimurium used were as follows:
TA 1535 hisG46, rfa-, uvrB-
TA 1537 hisC3076, rfa-, uvrB-
TA 1538 hisD3052, rfa-, uvrB-
TA 98 hisD3052, rfa-, uvrB-, pKMlOl
TA 100 hisG46, rfa-, uvrB-, pKMlOl.
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
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (derived from Aroclor-induced rat liver)
Test concentrations with justification for top dose:
The concentrations of WS-23 used in this 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. The amounts of test material used per Petri dish were 0.5 µg, 5 µg, 50 µg, 500 µg and 5 mg and these were tested against all 5 strains of S. typhimurium both with and without addition of S9. The concentrations of WS-23 used in the mutation assays were chosen to comply with OECD/EEC guidelines which recommend a top dose level of 5 mg/plate or a top dose which is within the dose range producing a toxic response.
Vehicle / solvent:
WS-23 was found to be readily soluble in dimethylsulphoxide (DMSO) and so stock solutions were prepared in this solvent at 50 mg/ml. No analyses were performed to confirm these concentrations.

Positive controls:
With 59 mix;
2-aminoanthracene, 0.63, 2 and 4 µg/plate as
appropriate.
Without 59 mix;
2-nitrofluorene 10 µg/plate, sodium azide 5
and 10 µg/plate, 9-aminoacridine 20 and 40
µg/plate.

Stock solutions of all the above mutagens were prepared at 1 mg/ml in the appropriate solvents and dilutions prepared as required. Solvent-only negative controls were used as appropriate.

Water was used as a solvent for sodium azide only and DMSO was a solvent for test material and remaining positive control materials.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO/water
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, Sodium azide, 2-nitrofluorene, 9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION and DURATION

0.1 ml of an overnight culture of bacteria of about 2 x l0 E9 cells / ml was added to 2.0 ml of soft agar containing a trace of histidine and biotin, followed by 0.1 ml of test material and 0.5 ml of S9 μM mix (for metabolism) or cofactor solution (non-metabolism). This was poured over and poured onto a 9 cm Petri dish containing Vogel-Bonner E medium. Three plates were prepared for each concentration of the tested substance. Similar numbers of plates were also prepared for appropriate positive and negative (solvent) controls. The plates were incubated at 37 degrees C for either 2 or 3 days. Colonies were counted on an Artek 880 counter.

Rationale for test conditions:
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.
Some materials are themselves non-mutagenic but may become mutagenic after metabolism. To allow for this, a postmitochondrial supernatant fraction from rat liver, together with the required cofactors (collectively known as the S9 mix) may be incorporated into the test system. Materials are routinely tested with and without exogenous metabolism.

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 considered to be a mutagenic response.
Statistics:
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 and 95% confidence limits as described by Duncan and Brookes (1973).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Remarks:
DMSO, water
Positive controls validity:
valid
Remarks:
2AA, NAAZ
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Remarks:
DMSO
Positive controls validity:
valid
Remarks:
2AA, 9AA
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Remarks:
DMSO
Positive controls validity:
valid
Remarks:
2AA, 2NF
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Remarks:
DMSO
Positive controls validity:
valid
Remarks:
2AA, 2NF
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Remarks:
DMSO, water
Positive controls validity:
valid
Remarks:
2AA, NAAZ

Table 1. Toxicity of test material – WS-23 towards different strains of Salmonella Typhimurium

Amount of material per plate (µg)

Metabolic activation

(% S9)

% Survival (+/-95% C.L.)

TA1535

TA1537

TA100

TA1538

TA98

DMSO

 

100

100

100

100

100

0.5

 

90.2 (9.2)

97.6 (14.6)

98.6 (17.3)

104.5 (7.8)

92.9 (15.2)

5

10

90.8 (9.6)

98.1 (15.4)

102.3 (16)

92.7 (7.2)

89.8 (18.1)

50

 

94.4 (9.6)

91.9 (15.9)

92.6 (3.1)

101 (9.1)

95.5 (16.2)

500

 

90.1 (9.9)

92.6 (11.3)

110.2 (14.4)

106.1 (8.3)

93.6 (15.3)

5000

 

40.6 (16.1)

45.4 (9)

95.2 (22.6)

18 (2)

48.4 (8.1)

DMSO

 

100

100

100

100

100

0.5

 

98.8 (5.1)

89.6 (13.8)

89.6 (11)

80.9 (26.6)

75.9 (14.7)

5

0

96.7 (10.4)

90.7 (9.2)

95 (10.9)

103.7 (42.4)

87.6 (14.9)

50

 

106.4 (8.6)

89.5 (9.1)

90.5 (6.1)

122 (15.4)

69.2 (14.4)

500

 

97.8 (3.3)

92.4 (13.3)

91.4 (10.7)

96.8 (32.3)

82.3 (12)

5000

 

79.8 (5)

24.5 (3.6)

51.5 (7.8)

11 (2.1)

9.7 (3.8)

 

C.L. = confidence limit                 

Figures correct to one decimal place                           

Table 2. Mutagenicity of test material – WS-23 towards Salmonella TyphimuriumTA1535

 

Amount of test material per plate (µg)

Metabolic activation

(% S9)

Revertant colony counts

Individual plates

Mean

S.D. (+/-)

DMSO

 

 

10

11, 7, 14

10.7

3.5

50

13, 13, 8

11.3

2.9

158

11, 6, 11

9.3

2.9

500

9, 15, 22

12

3

1580

12, 9, 15

12

3

5000

7, 11, 12

10

2.6

DMSO

 

 

0

8, 11, 14

11

3

50

15, 12, 14

13.7

1.5

158

9, 14, 6

9.7

4

500

9, 15, 19

14.3

5

1580

18, 11, 16

15

3.6

5000

16, 20, 20

18.7

2.3

Positive control mutagens

DMSO

 

11, 7, 14

10.7

3.5

2AA (0.63)

10

123, 78, 73

91.3

27.5*

2AA (2.0)

 

147, 151, 155

151

4*

Water

 

12, 6, 11

9.7

3.2

NAAZ (5.0)

0

619, 649, 670

646

25.6*

NAAZ (10.0)

 

889, 958, 927

924.7

34.6*

 

2AA = 2-aminoanthracene                                                           NAAZ = sodium azide

C = contaminated                                                                         N.D. = not done

S.D. = standard deviation                                                             * = doubling of negative value

Figures correct to one decimal place

Table 3. Mutagenicity of test material – WS-23 towards Salmonella Typhimurium TA1535

 

Amount of test material per plate (µg)

Metabolic activation (% S9)

Revertant colony counts

Individual plates

Mean

S.D. (+/-)

DMSO

 

 

10

12, 15, 12

13

1.7

50

15, 17, 17

16.3

1.2

158

6, 8, 9

7.7

1.5

500

9, 9, 12

10

1.7

1580

9, 9, 15

11

3.5

5000

8, 8, 15

10.3

4

DMSO

 

 

0

16, 13, 17

15.3

2.1

50

19, 15, C

17

2.8

158

16, 9, 16

13.7

4

500

12, 18, 13

14.3

3.2

1580

C, 18, 9

13.5

6.4

5000

9, 9, 5

7.7

2.3

Positive control mutagens

DMSO

 

12, 15, 12

13

1.7

2AA (0.63)

10

135, 120, 141

132

10.8*

2AA (2.0)

 

185, 171, 184

180

7.8*

Water

 

C, 12, 15

13.5

2.1

NAAZ (5.0)

0

600, 645, 637

627.3

24

NAAZ (10.0)

 

804, 907, 902

871

58.1

 

2AA = 2-aminoanthracene                                                           NAAZ = sodium azide

C = contaminated                                                                         N.D. = not done

S.D. = standard deviation                                                             * = doubling of negative value

Figures correct to one decimal place

Table 4. Mutagenicity of test material – WS-23 towards Salmonella Typhimurium TA1537

 

Amount of test material per plate (µg)

Metabolic activation

(% S9)

Revertant colony counts

 

 

Individual plates

Mean

S.D. (+/-)

DMSO

 

 

10

7, 8, 9

8

1

50

 

5, 12, 6

7.7

3.8

158

 

14, 9, 4

9

5

500

 

7, 6, 12

8.3

3.2

1580

 

13, 9, 4

8.7

4.5

5000

 

8, 4, 3

5

2.6

DMSO

 

 

0

7, 9, 5

7

2

50

 

9, 8, 7

8

1

158

 

12, 7, 11

10

2.6

500

 

7, 7, 3

5.7

2.3

1580

 

6, 9, 6

7

1.7

5000

 

2, 4, 5

3.7

1.5

Positive control mutagens

DMSO

 

7, 8, 9

8

1

2AA (0.63)

10

236, 222, 219

225.7

9.1*

2AA (2.0)

 

537, 562, 532

543.7

16*

Water

 

7, 9, 5

7

2

9AA (20.0)

0

38, 31, 48

39

8.5*

9AA (40.0)

 

541, 841, 591

657.7

160.7*

 

2AA = 2-aminoanthracene                                                           9AA = 9-aminoacridine

C = contaminated                                                                         N.D. = not done

S.D. = standard deviation                                                             * = doubling of negative value

Figures correct to one decimal place

Table 5. Mutagenicity of test material – WS-23 towards Salmonella Typhimurium TA1537

Amount of test material per plate (µg)

Metabolic activation (% S9)

Revertant colony counts

 

 

Individual plates

Mean

S.D. (+/-)

DMSO

 

 

10

9, 9, 9

9

0

50

 

7, 5, 5

5.7

1.2

158

 

7, 7, 5

6.3

1.2

500

 

5, 13, 8

8.7

4

1580

 

8, 6, 6

6.7

1.2

5000

 

8, 6, 7

7

1

DMSO

 

 

0

8, 5, 9

7.3

2.1

50

 

5, 4, 4

4.3

0.6

158

 

6, 5, 3

4.7

1.5

500

 

13, 8, 3

8

5

1580

 

9, 6, 8

7.7

1.5

5000

 

9, 6, 7

7.3

1.5

Positive control mutagens

DMSO

 

9, 9, 9

9

0

2AA (0.63)

10

234, 180, 228

214

29.6*

2AA (2.0)

 

733, 714, 714

720.3

11*

Water

 

8, 5, 9

7.3

2.1

9AA (20.0)

0

29, 29, 25

27.7

2.3*

9AA (40.0)

 

497, 377, 422

432

60.6*

 

2AA = 2-aminoanthracene                                                           9AA = 9-aminoacridine

C = contaminated                                                                         N.D. = not done

S.D. = standard deviation                                                             * = doubling of negative value

Figures correct to one decimal place

Table 6. Mutagenicity of test material – WS-23 towards Salmonella Typhimurium TA100

 

Amount of test material per plate (µg)

Metabolic activation

(% S9)

Revertant colony counts

 

 

Individual plates

Mean

S.D. (+/-)

DMSO

 

 

10

107, 106, 123

112

9.5

50

 

88, 135, 135

119.3

27.1

158

 

86, 101, 107

98

10.8

500

 

101, 107, 110

106

4.6

1580

 

86, 120, 119

108.3

19.3

5000

 

93, 103, 90

95.3

6.8

DMSO

 

 

0

126, 91, 86

101

21.8

50

 

85, 91, 91

89

3.5

158

 

100, 108, 118

108.7

9

500

 

118, 95, C

106.5

16.3

1580

 

129, 93, 99

107

19.3

5000

 

119, 122, 120

120.3

1.5

Positive control mutagens

DMSO

 

107, 106, 123

112

9.5

2AA (2.0)

10

2605, 2619, 2610

2611.3

6.5*

2AA (4.0)

 

2000, 2040, 2123

2054.3

62.9*

Water

 

100, 143, 126

123

21.7

NAAZ (5.0)

0

714, 662, 693

689.7

26.1*

NAAZ (10.0)

 

941, 964, 867

924

50.7*

 

2AA = 2-aminoanthracene                                                           NAAZ = sodium azide

C = contaminated                                                                         N.D. = not done

S.D. = standard deviation                                                             * = doubling of negative value

Figures correct to one decimal place

Table 7. Mutagenicity of test material – WS-23 towards Salmonella Typhimurium TA100

 

Amount of test material per plate (µg)

Metabolic activation

(% S9)

Revertant colony counts

 

 

Individual plates

Mean

S.D. (+/-)

DMSO

 

 

10

129, 128, 137

131.3

4.9

50

 

133, 113, C

123

14.1

158

 

132, 125, C

128.5

5

500

 

116, 100, 125

113.7

12.7

1580

 

152, 125, 126

134.3

15.3

5000

 

102, C, 112

107

7.1

DMSO

 

 

0

112, 97, C

104.5

10.6

50

 

117, 126, 98

113.7

14.3

158

 

101, 111, 91

101

10

500

 

110, 111, 115

112

2.7

1580

 

108, 105, 115

109.3

5.1

5000

 

104, 101, 81

95.3

12.5

Positive control mutagens

DMSO

 

129, 128, 137

131.3

4.9

2AA (2.0)

10

2064, 2144, 2162

2123.3

51.9*

2AA (4.0)

 

1644, 1578, 1409

1543.7

121.2*

Water

 

C, 129, 120

124.5

6.4

NAAZ (5.0)

0

577, 572, 519

556

32.1*

NAAZ (10.0)

 

770, 762, 829

787

36.6*

 

2AA = 2-aminoanthracene                                                           NAAZ = sodium azide

C = contaminated                                                                         N.D. = not done

S.D. = standard deviation                                                             * = doubling of negative value

Figures correct to one decimal place

Table 8. Mutagenicity of test material – WS-23 towards Salmonella Typhimurium TA1538

 

Amount of test material per plate (µg)

Metabolic activation

(% S9)

Revertant colony counts

 

 

Individual plates

Mean

S.D. (+/-)

DMSO

 

 

10

24, 31, 19

24.7

6

50

 

25, 29, 27

27

2

158

 

15, 25, 28

22.7

6.8

500

 

28, 20, 25

24.3

4

1580

 

26, 22, 30

22.7

3.1

5000

 

20, 24, 30

24.7

5

DMSO

 

 

0

13, 9, 14

12

2.6

50

 

16, 13, 11

13.3

2.5

158

 

17, 15, 22

18

3.6

500

 

14, 13, 13

13.3

0.6

1580

 

13, 9, 11

11

2

5000

 

11, 17, 16

14.7

3.2

Positive control mutagens

DMSO

 

24, 31, 9

24.7

6

2AA (2.0)

10

2132, 2301, 2206

2213

84.6*

2AA (4.0)

 

194, 165, 157

172

19.5*

DMSO

 

13, 9, 14

12

2.6

2NF (10.0)

0

1199, 1197, 1086

1160.7

64.6*

 

2AA = 2-aminoanthracene                                                           2NF= 2-nitrofluorene

C = contaminated                                                                         N.D. = not done

S.D. = standard deviation                                                             * = doubling of negative value

Figures correct to one decimal place

Table 9. Mutagenicity of test material – WS-23 towards Salmonella Typhimurium TA1538

 

Amount of test material per plate (µg)

Metabolic activation

(% S9)

Revertant colony counts

Individual plates

Mean

S.D. (+/-)

DMSO

 

 

10

28, 27, 18

24.3

5.5

50

24, 31, 32

29

4.4

158

19, 27, 21

22.3

4.2

500

17, 14, 25

18.7

5.7

1580

24, 17, 21

20.7

3.5

5000

19, 24, 25

22.7

3.2

DMSO

 

 

0

14, 5, 16

11.7

5.9

50

17, 14, 12

14.3

2.5

158

15, 9, 16

13.3

3.8

500

14, 5, 13

10.7

4.9

1580

12, 17, 16

15

2.6

5000

13, 8, 12

11

2.6

Positive control mutagens

DMSO

 

28, 27, 18

24.3

5.5

2AA (2.0)

10

1800, 1884, 1926

1870

64.2*

2AA (4.0)

 

115, 122, 129

122

7*

DMSO

 

14, 5, 16

11.7

5.9

2NF (10.0)

0

1327, 1268, 1370

1321.7

51

 

2AA = 2-aminoanthracene                                                           2NF= 2-nitrofluorene

C = contaminated                                                                         N.D. = not done

S.D. = standard deviation                                                             * = doubling of negative value

Figures correct to one decimal place

Table 10. Mutagenicity of test material – WS-23 towards Salmonella Typhimurium TA98

 

Amount of test material per plate (µg)

Metabolic activation (% S9)

Revertant colony counts

 

 

Individual plates

Mean

S.D. (+/-)

DMSO

 

 

10

48, 38, 33

39.7

7.6

50

 

35, 38, 34

35.7

2.1

158

 

40, 29, 37

35.3

5.7

500

 

30, 31, 34

31.7

2.1

1580

 

39, 41, 36

38.7

2.5

5000

 

C, 45, 35

40

7.1

DMSO

 

 

0

22, 23, 27

24

2.6

50

 

30, 29, 20

26.3

5.5

158

 

23, 29, 30

27.3

3.8

500

 

25, 20, 28

24.3

4

1580

 

34, 22, 20

25.3

7.6

5000

 

C, 19, 22

20.5

2.1

Positive control mutagens

DMSO

 

48, 38, 33

39.7

7.6

2AA (2.0)

10

2512, 2592, 2370

2491.3

112.2*

2AA (4.0)

 

2037, 2007, 2007

2017

17.8

DMSO

 

22, 23, 27

24

2.6

2NF (10.0)

0

718, 726, 729

724.3

5.8*

 

2AA = 2-aminoanthracene                                                           2NF = 2-nitrofluorene

C = contaminated                                                                         N.D. = not done

S.D. = standard deviation                                                             * = doubling of negative value

Figures correct to one decimal place

Table 11. Mutagenicity of test material – WS-23 towards Salmonella Typhimurium TA98

 

Amount of test material per plate (µg)

Metabolic activation (% S9)

Revertant colony counts

 

 

Individual plates

Mean

S.D. (+/-)

DMSO

 

 

10

45, C, 33

39

8.5

50

 

32, 39, 31

34

4.4

158

 

38, 38, 38

38

0

500

 

41, 48, 31

40

8.5

1580

 

25, 29, 17

23.7

6.1

5000

 

25, 27, 28

26.7

1.5

DMSO

 

 

0

24, 33, 20

25.7

6.7

50

 

25, 20, 18

21

3.6

158

 

19, 20, 37

25.3

10.1

500

 

29, 17, 32

26

7.9

1580

 

15, 25, 20

20

5

5000

 

21, 9, 14

14.7

6

Positive control mutagens

DMSO

 

45, C, 33

39

8.5

2AA (2.0)

10

1876, 1724, 1911

1870.3

130.9*

2AA (4.0)

 

1804, 1591, 1673

1689.3

107.5*

DMSO

 

24, 33, 20

25.7

6.7

2NF (10.0)

0

744, 684, 793

740.3

54.6*

2AA = 2-aminoanthracene                                                           2NF= 2-nitrofluorene

C = contaminated                                                                         N.D. = not done

S.D. = standard deviation                                                             * = doubling of negative value

Figures correct to one decimal place

Conclusions:
WS-23 did not cause a dose-related reproducible increase in reversion to occur in any of the five S.typhimurium strains of bacteria up to 5 mg/plate.

Executive summary:

The objective of this study equivalent or similar to OECD TG 471, was to provide information on the mutagenic potential of WS-23 in a bacterial mutation assay (Ames Test).

 

In the Toxicity (Survival) Assay, the toxicity assay was performed with five concentrations of WS 23: 0.5 µg, 5 µg, 50 µg, 500 µg and 5 mg. These concentrations were tested against all 5 strains of S. typhimurium both with and without addition of S9. Different levels of toxicity of WS-23 towards each bacterial strain were observed, and toxicity was also dependent on the presence or absence of S9.

 

The mutagenicity assay (AMES test) was performed twice with five concentrations of WS-23: 50 µg, 158 µg, 500 µg, 1.58 mg and 5 mg/plate. The assay was performed with and without addition of S9. Briefly, the bacterial plates were inverted and incubated at 37 Celsius degrees for 2-3 days. The mean number of colonies and standard deviation for each set of plates were calculated.

 

The results of the main assay showed, that WS-23 did not induce mutations in the genomes of the organisms used under the conditions of the assay. Therefore, it was concluded, that there was no evidence to support that WS-23 is a mutagen.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Based on the negative results of two in vitro AMES assays (equivalent or similar to OECD TG 471) and in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, it is concluded that WS-23 does not have a mutagenic potential.