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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the conditions of this study, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used with or without metabolic activation.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 February 2001 to 09 March 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
S. typhimurium: Histidine locus
E. coli: Tryptophan locus
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
CELLS USED
- Properly maintained: Yes: regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (if applicable):
CELLS USED
- Properly maintained: Yes: regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment I: 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II: 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene and 4-nitro-o-phenylene-diamine
Details on test system and conditions:
PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test material a pre-experiment was performed with all bacterial strains to be used in the main study. Eight concentrations of the test material were tested for toxicity and mutation induction with three plates each. The experimental conditions were the same as described below for Experiment I and II.
In the pre-experiment the concentration range of the test material was 3 to 5000 µg/plate. The pre-experiment is reported as Experiment I since no relevant toxic effects were observed and 5000 µg/plate was chosen as the maximum concentration.

METHOD OF APPLICATION: Pre-incubation
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
From the thawed ampoules of the strains 0.5 ml suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µI ampicillin (25 µg/ml) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth and 5 g NaCl. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 .c;o-factor solution. The amount of S9 supernatant was 15% vlv in the cultures. The concentrated co-factor solution yields the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCI, 5 mM Glucose-6-phosphate 5 mM NADP, in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath.

NUMBER OF REPLICATIONS: test material, vehicle and positive controls were plated in triplicate.

METHOD OF APPLICATION: in agar (plate incorporation)
For each strain and dose level including the controls, three plates were used.The following materials were mixed in a test tube and incubated at 37 °C for 20 minutes 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control), 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation) and 100 µL Bacteria suspension (cf. test system, pre-culture of the strains).

After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

DURATION: 48 hours
Evaluation criteria:
A test material is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.

A test item producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic (negative) in this system.

A test item is considered mutagenic (positive) if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions is at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate (3,4).

Statistics:
No statistical evaluation of the data was required.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Additional information on results:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In the tester strain TA 1535, toxicity was observed at dose levels of 1000 µg/plate without the S9 mix and no toxic effects was observed at any of the dose levels tested with the S9 mix in experiment I. On the other hand, in experiment II, toxic effects were observed at dose levels of 2500 µg/plate with S9 mix.

In the tester strain TA 1537, toxicity was observed at dose levels of 1000-5000 µg/plate without the S9 mix and no toxic effects was observed at any of the dose levels tested with the S9 mix in experiment I. On the other hand, toxicity was observed at dose level 1250 µg/plate without S9 mix and no toxic effects was observed at any of the dose levels tested with the S9 mix.

In the tester strain TA 98, toxicity was observed at dose levels of 5000 µg/plate in experiment I with the S9 mix and no toxic effect was observed at any of the dose levels tested without the S9 mix and experiment II.

Table 1: Toxicity of the test material at different concentrations

Strain

Experiment I

Experiment II

 

Without S9 Mix

With S9 Mix

Without S9 Mix

With S9 Mix

TA 1535

1000

/

/

2500

TA 1537

1000 - 5000

/

1250

/

TA 98

/

500

/

/

TA 100

/

/

/

/

WP2uvra

/

/

/

/

/ = no toxic effect observed

Table 2: Summary of Experiment 1

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Negative

Solvent

33

100

333

1000

2500

5000

85

81

76

75

83

96

99

81

9

14

9

8

13

5

9

8

31

28

33

28

33

25

27

25

15

15

13

15

11

12

14

9

5

6

4

4

6

3

2

2

+

Negative

Solvent

33

100

333

1000

2500

5000

103

97

92

91

94

97

112

112

10

11

9

7

13

17

10

6

27

29

40

33

34

26

29

36

14

19

14

17

20

18

17

10

6

6

6

7

5

4

5

5

Positive Controls

-

Name

SA

SA

MMS

4NQO

4NQO

Concentration (µg/plate)

100

10

5

10

50

Mean no. colonies/plate

812

594

331

176

46

+

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

2.5

2.5

10

2.5

2.5

Mean no. colonies/plate

702

77

170

375

78

Table 3: Summary of Experiment 2

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Negative

Solvent

156.25

312.5

625

1250

2500

5000

111

117

119

93

101

135

135

112

9

10

10

9

11

10

10

9

45

33

41

39

49

45

42

43

25

27

27

31

26

23

28

20

7

9

7

7

8

3

6

6

+

Negative

Solvent

156.25

312.5

625

1250

2500

5000

148

135

168

136

161

193

125

120

11

11

10

9

13

13

5

10

39

44

56

55

54

56

49

47

47

25

34

37

38

29

35

27

9

12

13

14

15

12

12

12

Positive Controls

-

Name

SA

SA

MMS

4NQO

4NQO

Concentration (µg/plate)

100

10

5

10

50

Mean no. colonies/plate

1009

642

551

167

48

+

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

2.5

2.5

10

2.5

2.5

Mean no. colonies/plate

1435

137

475

802

114

Conclusions:
Interpretation of results: Negative with and without metabolic activation

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.
Executive summary:

The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted in accordance with the standardised guidelines OECD 471, EU Method B13/14 and Japanese Guidelines under GLP conditions.

In this study, the test material was assessed for its potential to induce gene mutations according to the pre-incubation test (both experiments) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. Due to a very low number of revertant colonies in strain TA 100 with metabolic activation the second experiment had to be repeated. Only the results of the repeat experiment are reported.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

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

Additional information

The test material was assessed for its potential to induce gene mutations according to the pre-incubation test (both experiments) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. Due to a very low number of revertant colonies in strain TA 100 with metabolic activation the second experiment had to be repeated. Only the results of the repeat experiment are reported.

The assay was performed in two independent experiments both with and without liver microsomal activation (S9 Mix). Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Experiment I: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 156.25; 312.5; 625; 1250; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

In the tester strain TA 1535, toxicity was observed at dose levels of 1000 µg/plate without the S9 mix and no toxic effects was observed at any of the dose levels tested with the S9 mix in experiment I. On the other hand, in experiment II, toxic effects were observed at dose levels of 2500 µg/plate with S9 mix. In the tester strain TA 1537, toxicity was observed at dose levels of 1000-5000 µg/plate without the S9 mix and no toxic effects was observed at any of the dose levels tested with the S9 mix in experiment I. On the other hand, toxicity was observed at dose level 1250 µg/plate without S9 mix and no toxic effects was observed at any of the dose levels tested with the S9 mix. In the tester strain TA 98, toxicity was observed at dose levels of 5000 µg/plate in experiment I with the S9 mix and no toxic effect was observed at any of the dose levels tested without the S9 mix and experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Under the conditions of this study, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used with or without metabolic activation.

Short description of key information:

- Non mutagenic with and without metabolic activation (Ames test); OECD 471.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to genetic toxicity.