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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:
1990-01-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Trilon D flussig
- Lot/batch No.: B110
- Storage condition of test material: 4 - 8 degrees C
- All other template details: Not reported

Method

Target gene:
his+ revertants
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
- Periodically checked for deep rough character: yes
- Periodically checked for UV sensitivity: yes
- Periodically checked for ampicillin resistance (R factor plasmid): yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500, 500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua dest.
- Justification for choice of solvent/vehicle: complete solubility of the test substance in aqua dest.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S-9 mix: 2-aminoanthracine (in DMSO); without S-9 mix: N-methyl-N'-nitro-N-nitrosoguianidine (in DMSO) for strains TA 100 and TA 1535; 4-nitro-o-phenylendiamine (in DMSO) for strain TA 98; 9-aminoacridine chloride monohydrate (in DMSO) for TA 1537
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation), test tubes containing 2 mL portions of soft agar (100 ml agar - 0.6% agar + 0.6% NaCl) and 10 mL amino acid solution are kept in a water bath at 45 degrees C. The remaining ingredients were added: 0.1 mL test solution, 0.1 mL bacterial suspension, 0.5 mL S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation)

DURATION
- Preincubation period: 20 minutes for that study
- Exposure duration: 48 hours at 37 degrees C

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: >10^7 cells

DETERMINATION OF CYTOTOXICITY
- Method: reduced his- background growth

OTHER EXAMINATIONS: No

Other: Tissue preparation for the S-9 fraction (according to Ames et al.)
- 5 male Sprague-Dawley rats (200-300 g) receive a single IP injection of 500 mg Aroclor 1254 per kg BW 5 days before sacrifice
- Rats are sacrificed, livers removed, weighted and washed in a 150 mM KCl solution
- Livers then are cut into small pieces and homogenized in three volumes of KCl solution and centrifuged at 9000 x g for 10 minutes at 4 degrees C
- 5 ml portions of the supernatant are quickly deep-frozen in dry ice and stored at -70 to -80 degrees C for up to 2 months
- S9 mix is prepared freshly prior to each experiment with: MgCl2 - 8 mM, KCl - 33 mM, glucose-S-phosphate - 5 mM, NADP - 4 mM, phosphate buffer (pH 7.4) - 100 mM
Evaluation criteria:
1) doubling of the spontaneous mutation rate (control), 2) dose-response relationship, 3) reproducibility of the results
Statistics:
Not reported

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Full results provided on Tables 1-4 showing the number of revertants/plate for each strain.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Results for Strain TA1535 – Number of Revertants/Plate.

Dose

without S9

Ave.

SD

with S9

Ave.

SD

Negative control Aqua Dest.

12

16

4

14

13

1

18

 

 

12

 

 

19

 

 

13

 

 

20

16

15

1

13

15

3

14

 

 

18

 

 

14

 

 

13

 

 

100

15

18

3

13

15

4

20

 

 

19

 

 

18

 

 

12

 

 

500

19

16

3

10

14

5

15

 

 

20

 

 

14

 

 

12

 

 

2500

15

16

2

14

12

4

19

 

 

15

 

 

15

 

 

8

 

 

5000

13

14

1

12

14

3

15

 

 

17

 

 

15

 

 

13

 

 

Pos. Control 2-AA

 

 

 

130

114

17

 

 

 

96

 

 

 

 

 

115

 

 

Pos. Control MNNG

1880

1927

64

 

 

 

1900

 

 

 

 

 

2000

 

 

 

 

 

 

Table 2. Results for Strain TA100 – Number of Revertants/Plate.

Dose

without S9

Ave.

SD

with S9

Ave.

SD

Negative control Aqua Dest.

100

105

6

119

112

13

102

 

 

97

 

 

112

 

 

121

 

 

20

124

131

13

137

126

10

123

 

 

123

 

 

146

 

 

117

 

 

100

134

119

14

127

123

9

117

 

 

129

 

 

106

 

 

113

 

 

500

85

85

1

108

111

7

84

 

 

119

 

 

86

 

 

106

 

 

2500

100

114

12

111

115

4

124

 

 

116

 

 

117

 

 

118

 

 

5000

124

144

19

133

127

7

146

 

 

130

 

 

162

 

 

119

 

 

Pos. Control 2-AA

 

 

 

1880

1913

42

 

 

 

1900

 

 

 

 

 

1960

 

 

Pos. Control MNNG

1900

2083

161

 

 

 

2150

 

 

 

 

 

2200

 

 

 

 

 

 

Table 3. Results for Strain TA1537 – Number of Revertants/Plate.

Dose

without S9

Ave.

SD

with S9

Ave.

SD

Negative control Aqua Dest.

5

8

3

10

12

2

10

 

 

12

 

 

9

 

 

13

 

 

20

12

10

2

22

18

4

10

 

 

15

 

 

9

 

 

18

 

 

100

11

11

1

20

15

6

12

 

 

9

 

 

10

 

 

15

 

 

500

12

11

4

9

11

2

14

 

 

11

 

 

6

 

 

13

 

 

2500

10

12

6

3

7

5

8

 

 

12

 

 

19

 

 

5

 

 

5000

7

10

4

8

8

1

14

 

 

7

 

 

10

 

 

8

 

 

Pos. Control 2-AA

 

 

 

204

185

19

 

 

 

184

 

 

 

 

 

167

 

 

Pos. Control MNNG

558

527

34

 

 

 

532

 

 

 

 

 

491

 

 

 

 

 

 

Table 4. Results for Strain TA98 – Number of Revertants/Plate.

Dose

without S9

Ave.

SD

with S9

Ave.

SD

Negative control Aqua Dest.

29

30

3

44

44

1

28

 

 

43

 

 

33

 

 

45

 

 

20

34

31

3

44

44

6

31

 

 

39

 

 

29

 

 

50

 

 

100

29

31

4

45

39

5

36

 

 

36

 

 

29

 

 

37

 

 

500

29

31

3

51

42

8

35

 

 

36

 

 

29

 

 

38

 

 

2500

42

31

12

41

48

6

18

 

 

52

 

 

34

 

 

52

 

 

5000

28

35

8

45

40

10

34

 

 

29

 

 

44

 

 

47

 

 

Pos. Control 2-AA

 

 

 

1460

1447

160

 

 

 

1280

 

 

 

 

 

1600

 

 

Pos. Control MNNG

800

815

19

 

 

 

836

 

 

 

 

 

810

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
There was no increase in the number of mutations (his+ revertants) in bacterial strains TA 1535, TA 100, TA 1537, and TA 98 exposed to 20-5000 ug/plate of the test material in agar for 48 hours. The test material did not appear to be cytotoxic. There was an appropriate response from the positive control materials.
Executive summary:

An increase in the number of his+ revertants was not observed both in the standard plate test and in the pre-incubation test either without S-9 mix or after the addition of a metabolizing system to strains TA 1535, TA 100, TA 1537, and TA 98 from 20 -5000 ug/plate of Trilon D. According to the results of the present study, the test substance Trilon D flussig is not mutagenic in the Ames test under the experimental conditions chosen.