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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
First Addendum to OECD Guidelines No. 4 71, "Salmonella typhimurium, Reverse Mutation Assay", adopted May 26, 1983
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EEC Directive 84/449, L 251, B 14, p. 143-145
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Histidine locus in selected strains
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: nutrient medium: 8 g Difco Nutrient Broth, 5 g NaCl in 20 ml (for 0,5 ml bacterial suspension)
- Storage: stock cultures in ampoules with nutrient broth and 5 % DMSO in liquid nitrogen
- Properly maintained: yes

Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in C C R according to Ames et al. (1970, 1977) . In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Aroclor 1254 induced rat liver)
Test concentrations with justification for top dose:
Pre-study with strain TA 98 and TA 100: 1 – 5000 µg/plate

Main-experiments:
Exp. I : TA 1535, TA 1537, TA 98, TA 100:
10.0; 33.3; 100.0; 333.3; 1000.0 and 5000.0 µg /plate
Exp. II : TA 1535, TA 98, TA 100:
10.0; 33.3; 100.0; 333.3; 1000.0 and 5000.0 µg/plate
Exp. II: TA 1537:
10.0; 33.3; 66.6; 333.3; 100.0; 666.6 and 1000.0 µg/plate
Vehicle / solvent:
Solvent: Methanol. On the day of experiment, the test article was dissolved.
Justification: The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Concurrent untreated and solvent controls were performed.
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
sodium azide for strains: TA 1535, TA 100 without metabolic activation, 10 µg/plate, dissolved in aqua dest..
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
4-nitro-o-phenylene-diamine for strains TA 1537, TA 98 without metabolic activation, dissolved in DMSO, 50 µg/plate.
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2-aminoanthracene for all strains with metabolic activation, dissolved in DMSO, 10 µg /plate.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72h

NUMBER OF REPLICATIONS: 3/strain/dose for each of the two experiments.
Evaluation criteria:
The test material is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.

A significant response is described as follows:
The test material is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test material regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No appropriate statistical method was available.

Results and discussion

Test results
Key result
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:
cytotoxicity
Remarks:
partial or complete reduction in the number of revertants, at the higher dose levels with and without metabolic activation in experiment I and II in all strains used
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration- dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.

Table 1: Results WITHOUT S9 mix (/ = not performed)

Dose µg/plate

Strains

Experiment

TA 1535

TA 1537

TA 98

TA 100

I

II

I

II

I

II

I

II

Neg. control

9

9

10

6

24

21

107

96

Solvent control

8

7

6

7

23

16

99

94

10.0

10

13

5

6

24

19

111

98

33.3

8

6

6

7

32

16

99

99

66.6

/

/

/

10

/

/

/

/

100.0

8

7

6

9

21

15

78

76

333.3

8

8

5

7

23

17

75

44

666.6

/

/

/

2

/

/

/

/

1000.0

5

5

3

0

11

9

29

8

5000.0

3

2

0

/

0

1

24

4

Pos. contr.: Sodium azid 10 µg/plate

122

964

 

 

 

 

29

8

Pos. control:

4-Nitro-o-phenylene-diamine 50 µg/plate

 

 

193

318

1981

1913

 

 

Table 2: Results WITH S9 mix (/ = not performed)

Dose µg/plate

Strains

Experiment

TA 1535

TA 1537

TA 98

TA 100

I

II

I

II

I

II

I

II

Neg. control

11

13

5

8

43

20

95

93

Solvent control

9

13

7

11

43

32

99

115

10.0

11

13

12

11

44

27

128

120

33.3

10

10

11

14

46

27

147

102

66.6

/

/

/

9

/

/

/

/

100.0

11

12

8

10

30

28

112

122

333.3

12

15

7

10

38

21

132

126

666.6

/

/

/

6

/

/

/

/

1000.0

7

11

4

5

27

18

123

86

5000.0

4

1

0

/

1

0

27

13

Pos. contr.: 2-Amino-anthracene 10 µg/plate

126

117

111

132

772

753

783

926

Applicant's summary and conclusion

Conclusions:
Interpretation of results : negative

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test material did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.