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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:
26 Feb - 18 Mar 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

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:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted in 1984
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
yes
Remarks:
only four S. typhimurium strains (additional E. coli WP2 strains or S. typhimurium TA 102 is missing)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch No.: 791

Method

Target gene:
his 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:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
First experiment: 8, 40, 200, 1000 and 5000 µg/plate with and without metabolic activation
Second experiment: 30, 60, 120, 240, 480 and 960 µg/plate with and without metabolic activation

The highest dose of the first experiment was chosen based on the standard protocol. Based on the bacteriotoxic effect of the test substance as determined in the first experiment 960 µg/plate was chosen as the highest concentration for the repeat test.
Vehicle / solvent:
- Vehicle/solvent used: ethanol
- Justification for choice of solvent/vehicle: The used solvent was chosen out of the following solvents, in the order given: water, methanol, ethanol, acetone, DMSO, DMF, and ethylene glycol dimethylether according to information given by the internal sponsor.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: sodium azide (10 µg/plate, -S9, TA 1535), nitrofurantoin (0.2 µg/plate, -S9, TA 100), 4-nitro-1,2-phenylene diamine (10 µg/plate, -S9, TA 1537 and 0.5 µg/plate, -S9, TA 98), 2-aminoanthracene (3 µg/plate, +S9, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 4 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other: titer
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement. In case of questionable results, investigation should continue, possibly with modifications, until a final evaluation is possible.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 480 µg/plate and above
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 480 µg/plate and above
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 480 µg/plate and above
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 240 µg/plate and above
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
HISTORICAL CONTROL DATA
Summaries of historical negative and positive controls of experiments performed within 6 month each from January 1988 to December 1990 are provided. Please refer to Table 1 under "Any other informations on results incl. tables" for the summarized historical control data of July to December 1990.

Any other information on results incl. tables

Table 1: Summary of historical negative and positive controls of experiments performed from July to December 1990

Compound

S9 mix

Strain

TA 1535

TA 100

TA 1537

TA 98

median

semi-Q range

median

semi-Q range

median

semi-Q range

median

semi-Q range

water

-

13

2

105

16

9

1

21

4

ethanol

-

12

3

93

14

9

1

22

3

Na azide

-

882

114

 

 

 

 

 

 

NF

-

 

 

380

60

 

 

 

 

4-NPDA

-

 

 

 

 

48

9

71

15

30% water

+

18

3

143

15

11

2

29

3

30% ethanol

+

19

3

118

18

10

1

39

7

2-AA

+

175

41

800

243

84

17

485

93

Na azide = sodium azide, NF = nitrofurantoin, 4-NPDA = 4-nitro-1,2-phenylenediamine

 

Table 2: First experiment - Average revertants per plate in tester strains TA98, TA 100, TA 1535 and TA 1537 without S9-mix

Concentration [µg/plate]

Revertants/plate [mean]

TA 1535

TA 100

TA 1535

TA 98

0

13

89

8

32

8

13

94

9

34

40

15

99

6

37

200

14

89

8

30

1000

5

18

4

7

5000

0

0

0

0

Na-azide1

603

 

 

 

NF2

 

477

 

 

4-NPDA3

 

 

70

167

Positive controls:1Sodium-azide, 10 µg/plate (only TA 1535)

                                2Nitrofurantoin, 0.2 µg/plate (only TA 100)

                                34-nitro-1,2-phenylenediamine, 10 µg/plate (only TA 1537), 0.5 µg/plate (only TA 98)

 

Table 3: First experiment - Average revertants per plate in tester strains TA98, TA 100, TA 1535 and TA 1537 with S9-mix

Concentration [µg/plate]

Revertants/plate [mean]

TA 1535

TA 100

TA 1535

TA 98

0

20

111

9

38

8

21

124

10

46

40

20

120

10

46

200

11

100

6

51

1000

7

35

3

13

5000

0

0

0

0

2-AA4

164

1211

74

446

Positive controls:42-aminoanthracene, 3 µg/plate

 

Table 4: Second experiment - Average revertants per plate in tester strains TA98, TA 100, TA 1535 and TA 1537 without S9-mix

Concentration [µg/plate]

Revertants/plate [mean]

TA 1535

TA 100

TA 1535

TA 98

0

14

78

9

25

30

13

86

9

28

60

13

89

9

28

120

12

91

12

29

240

12

89

7

29

480

14

66

5

23

960

5

35

2

17

Na-azide1

732

 

 

 

NF2

 

364

 

 

4-NPDA3

 

 

58

67

Positive controls:1Sodium-azide, 10 µg/plate (only TA 1535)

                                2Nitrofurantoin, 0.2 µg/plate (only TA 100)

                                34-nitro-1,2-phenylenediamine, 10 µg/plate (only TA 1537), 0.5 µg/plate (only TA 98)

 

Table 5: Second experiment - Average revertants per plate in tester strains TA98, TA 100, TA 1535 and TA 1537 with S9-mix

Concentration [µg/plate]

Revertants/plate [mean]

TA 1535

TA 100

TA 1537

TA 98

0

24

143

12

45

30

30

135

12

48

60

29

94

8

43

120

29

117

10

50

240

27

138

7

44

480

19

75

6

24

960

21

47

3

7

2-AA4

188

756

77

372

Positive controls:42-aminoanthracene, 3 µg/plate

Applicant's summary and conclusion

Executive summary:

The mutagenic potential of the test substance was tested in the Salmonella/Microsome test according to OECD Guideline 471 (1983) and in compliance with GLP. Tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA 1535 and TA 1537. Liver microsomal enzymes were prepared from at least 6 male Sprague-Dawley or male Wistar rats which had received a peritoneal injection of Aroclor 1254 at 500 mg/kg bw (S9 mix). The test substance was solved in ethanol. In a first experiment, 6 dose levels from 0 - 5000 µg/plate were plated with overnight cultures of TA 98, TA 100, TA 1535 and TA 1537 in the presence or absence of rat S9 mix. As a result, the appropriate maximum dose to be plated in the second experiment was determined to be 960 µg/plate with and without metabolic activation, respectively. In the second experiment the test substance was tested at 5 dose levels (30, 60, 120, 240, 480, 960 µg/plate) along with negative control and appropriate positive controls in the absence or presence of rat microsomal enzymes. DMSO was used as vehicle for positive controls. 0.1 mL of test-substance in ethanol or positive control in DMSO were added to 0.1 mL of bacteria solution (grown for 17 hours at 37 °C at 90 rpm) and 0.5 mL S9 mix or buffer (for non-activating tests) were added to 2.0 mL molten top-agar. After incubation for a maximum of 30 s at 45 °C in a water bath and mixing, this solution was poured onto solid agar plates. Four replicates were plated for all dose levels and controls. The plates were incubated at 37 °C for 48 h, before the colonies were counted. A result was evaluated as positive when it caused a doubling in the mean number of revertants per plate in at least one tester strain. This increase must be accompanied by a positive dose response. There was no indication of bacteriotoxic effects of the test substances at doses of up to and including 200 µg/plate. Total bacteria counts were comparable to or only slightly different from the negative controls. No inhibition of growth was noted. Doses at and above 240 µg/plate caused bacteriotoxic effects. None of the four strains tested showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls, with and without metabolic activation. The respective positive controls caused an increase in the number of revertants which proved the sensitivity of the test. Therefore, under the conditions of this study, no indications of mutagenic effects of the test substance could be found at doses up to 960 µg/plate in any of the S. typhimurium strains tested.