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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:
24 Apr - 31 May 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1983
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
yes
Remarks:
only 4 tester strains used
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Histidin operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (rat liver supernatant from rats treated with Aroclor 1254)
Test concentrations with justification for top dose:
Without and without metabolic activation: 4, 20, 100, 500, 2500, 5000 μg/plate in both experiments.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
The test compound did not precipitate on the plates up to the highest investigated dose of 5000 μg/plate.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

SELECTION AGENT (mutation assays): histidine (in agar)

NUMBER OF REPLICATIONS:
1st experiment: Triplicates (3 plates per dose)
2nd experiment: Triplicates (3 plates per dose)

DETERMINATION OF CYTOTOXICITY
A reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.

Parallel to the 2nd experiment, precise toxicity testing was performed: 0.1 mL of the different concentrations of the test compound were thoroughly mixed with 0.1 mL of 1.0E+07 dilution of the overnight culture of TA 100 (designated TA 100D) and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control was compared with the number of colonies per plate in the presence of the test compound. Results were given as a ratio of these values (= surviving fraction).

- OTHER:
The strains of Salmonella typhimurium were obtained from Professor B.N. Ames, University of California, U.S.A..
The different bacterial strains were checked half-yearly with regard to their respective biotin, histidine requirements, membrane permeability, ampicillin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens.

Results and discussion

Test results
Key result
Species / strain:
other: TA 100, TA 1535, TA 1537, TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the toxicity test using histidine enriched agar plates and a dilution of the tester strain TA 100 (designated TA 100 D), which was performed in parallel with the second experiment, toxicity was found at concentrations of 2500 μg/plate and above in the absence of metabolic activation and 5000 μg/plate in the presence of metabolic activation.

Any other information on results incl. tables

Table 1: Results of first experiment (number of revertants)

 

 

plate 1

plate 2

plate 3

Bacterial lawn

 

 

 

 

 

 

TA 100

Solvent control

137

145

146

 

with S9

Negative control

145

170

160

 

 

4 µg/plate

143

151

147

 

 

20 µg/plate

151

145

99

 

 

100 µg/plate

136

153

145

 

 

500 µg/plate

136

138

145

 

 

2500 µg/plate

124

140

130

 

 

5000 µg/plate

153

162

139

incomplete

 

2-aminoanthracene (0.5 µg/plate)

942

914

1052

 

 

 

 

 

 

 

TA 100

Solvent control

148

133

129

 

without S9

Negative control

170

165

146

 

 

4 µg/plate

129

116

146

 

 

20 µg/plate

145

142

119

 

 

100 µg/plate

118

134

145

 

 

500 µg/plate

134

147

144

 

 

2500 µg/plate

110

126

110

 

 

5000 µg/plate

111

76

84

 

 

sodium azide (1.0 µg/plate)

634

718

652

incomplete

 

 

 

 

 

 

TA 1535

Solvent control

17

15

20

 

with S9

Negative control

9

14

12

 

 

4 µg/plate

11

10

16

 

 

20 µg/plate

17

11

11

 

 

100 µg/plate

12

11

11

 

 

500 µg/plate

16

15

12

 

 

2500 µg/plate

10

7

13

incomplete

 

5000 µg/plate

8

11

7

incomplete

 

2-aminoanthracene (1.0 µg/plate)

129

125

143

 

 

 

 

 

 

 

TA 1535

Solvent control

10

13

11

 

without S9

Negative control

9

12

12

 

 

4 µg/plate

14

12

12

 

 

20 µg/plate

10

11

14

 

 

100 µg/plate

10

10

10

 

 

500 µg/plate

11

8

14

 

 

2500 µg/plate

6

8

10

incomplete

 

5000 µg/plate

3

4

5

incomplete

 

sodium azide (1.0 µg/plate)

385

545

415

 

 

 

 

 

 

 

TA 1537

Solvent control

10

7

11

 

with S9

Negative control

8

9

12

 

 

4 µg/plate

9

9

8

 

 

20 µg/plate

7

10

10

 

 

100 µg/plate

7

6

8

 

 

500 µg/plate

8

8

6

incomplete

 

2500 µg/plate

6

2

5

incomplete

 

5000 µg/plate

3

1

2

incomplete

 

2-aminoanthracene (1.0 µg/plate)

125

137

184

 

 

 

 

 

 

 

TA 1537

Solvent control

6

7

7

 

without S9

Negative control

9

10

7

 

 

4 µg/plate

6

9

8

 

 

20 µg/plate

8

6

9

 

 

100 µg/plate

6

9

7

 

 

500 µg/plate

4

8

10

 

 

2500 µg/plate

8

3

8

incomplete

 

5000 µg/plate

8

4

4

incomplete

 

9-aminoacridine (50 µg/plate)

236

117

118

 

 

 

 

 

 

 

TA98

Solvent control

21

28

21

 

with S9

Negative control

28

29

29

 

 

4 µg/plate

25

33

30

 

 

20 µg/plate

27

31

30

 

 

100 µg/plate

32

25

32

 

 

500 µg/plate

29

32

28

 

 

2500 µg/plate

22

24

34

incomplete

 

5000 µg/plate

28

13

13

incomplete

 

2-aminoanthracene (0.5 µg/plate)

1212

1213

1292

 

 

 

 

 

 

 

TA98

Solvent control

26

21

22

 

without S9

Negative control

27

23

24

 

 

4 µg/plate

21

19

21

 

 

20 µg/plate

28

22

18

 

 

100 µg/plate

18

17

24

 

 

500 µg/plate

24

22

19

 

 

2500 µg/plate

18

9

18

incomplete

 

5000 µg/plate

7

5

7

incomplete

 

2-nitrofluorene (2.5 µg/plate)

220

336

265

 

Table 2: Results of second experiment (number of revertants)

 

 

plate 1

plate 2

plate 3

Bacterial lawn

 

 

 

 

 

 

TA 100

Solvent control

141

150

144

 

with S9

Negative control

134

144

145

 

 

4 µg/plate

140

123

133

 

 

20 µg/plate

123

139

130

 

 

100 µg/plate

121

150

145

 

 

500 µg/plate

137

158

155

 

 

2500 µg/plate

130

130

120

 

 

5000 µg/plate

119

143

155

 

 

2-aminoanthracene (0.5 µg/plate)

1550

1514

1562

 

 

 

 

 

 

 

TA 100

Solvent control

127

122

138

 

without S9

Negative control

173

162

141

 

 

4 µg/plate

117

139

146

 

 

20 µg/plate

135

136

132

 

 

100 µg/plate

124

130

141

 

 

500 µg/plate

116

130

120

 

 

2500 µg/plate

110

112

117

 

 

5000 µg/plate

87

80

76

 

 

sodium azide (1.0 µg/plate)

682

704

685

 

 

 

 

 

 

 

TA 1535

Solvent control

15

9

10

 

with S9

Negative control

11

14

15

 

 

4 µg/plate

14

12

9

 

 

20 µg/plate

11

14

11

 

 

100 µg/plate

8

10

12

 

 

500 µg/plate

11

14

12

 

 

2500 µg/plate

14

12

16

 

 

5000 µg/plate

12

10

9

 

 

2-aminoanthracene (1.0 µg/plate)

160

145

193

 

 

 

 

 

 

 

TA 1535

Solvent control

14

14

10

 

without S9

Negative control

14

8

8

 

 

4 µg/plate

9

10

10

 

 

20 µg/plate

9

15

11

 

 

100 µg/plate

11

14

12

 

 

500 µg/plate

11

14

14

 

 

2500 µg/plate

12

14

9

 

 

5000 µg/plate

3

2

4

incomplete

 

sodium azide (1.0 µg/plate)

464

426

424

 

 

 

 

 

 

 

TA 1537

Solvent control

7

11

10

 

with S9

Negative control

7

9

8

 

 

4 µg/plate

8

7

10

 

 

20 µg/plate

10

11

8

 

 

100 µg/plate

11

6

6

 

 

500 µg/plate

8

7

8

 

 

2500 µg/plate

10

10

8

 

 

5000 µg/plate

5

3

3

incomplete

 

2-aminoanthracene (1.0 µg/plate)

207

253

256

 

 

 

 

 

 

 

TA 1537

Solvent control

8

8

6

 

without S9

Negative control

10

6

9

 

 

4 µg/plate

10

6

10

 

 

20 µg/plate

8

9

6

 

 

100 µg/plate

9

8

14

 

 

500 µg/plate

7

10

9

 

 

2500 µg/plate

4

6

9

incomplete

 

5000 µg/plate

3

2

1

incomplete

 

9-aminoacridine (50 µg/plate)

149

16

139

 

 

 

 

 

 

 

TA98

Solvent control

44

31

33

 

with S9

Negative control

45

26

38

 

 

4 µg/plate

22

29

30

 

 

20 µg/plate

29

31

35

 

 

100 µg/plate

30

24

38

 

 

500 µg/plate

37

30

41

 

 

2500 µg/plate

20

23

30

 

 

5000 µg/plate

14

20

19

incomplete

 

2-aminoanthracene (0.5 µg/plate)

2007

1917

1933

 

 

 

 

 

 

 

TA98

Solvent control

24

27

27

 

without S9

Negative control

22

23

26

 

 

4 µg/plate

28

21

34

 

 

20 µg/plate

36

34

34

 

 

100 µg/plate

26

28

25

 

 

500 µg/plate

32

33

24

 

 

2500 µg/plate

19

11

16

incomplete

 

5000 µg/plate

5

6

8

incomplete

 

2-nitrofluorene (2.5 µg/plate)

777

756

765

 

Applicant's summary and conclusion

Conclusions:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.