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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 98, TA 100, TA 1535, TA 1537, and E. coli WP2 uvrA: negative with and without metabolic activation (according to OECD TG 471)

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:
17 Feb 2011 to 28 Feb 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon for S. typhimurium strains and trp operon for E.coli strains
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital (PB) and 5,6-Benzoflavone (BF)
Test concentrations with justification for top dose:
Dose range finding study:
1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate with and without metabolic activation - in all strains
Main study:
39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation - S. typhimurium TA1535 and TA1537 strains
156, 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation - S. typhimurium TA98, TA100 and E.coli WP2 uvr A
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was used as the vehicle in this study because the test article was soluble at 50 mg/mL and since there was no reaction such as exothermic reaction or generation of gases observed.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) and 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine.2HCl (ICR-191)
Remarks:
Without S9 mix: AF-2 in DMSO for TA100 (0.01 µg/plate), WP2uvrA (0.01 µg/plate) + TA98 (0.1 µg/plate). SAZ in water for TA1535 (0.5 µg/plate). ICR-191 in DMSO for TA1537 (1.0 µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9 mix: 2-AA in DMSO for TA1535 (2.0 µg/plate) + WP2uvrA (10.0 µg/plate). B[a]P in DMSO for TA100, TA 98 + TA1537 (5.0 µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 2 plates per test concentration in the preliminary study and the main study

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn

Evaluation criteria:
If a two-fold or more increase in the number of revertant colonies in the test substance groups were observed compared to the negative control group, dose-response and reproducibility were noted, or even if no clear dose-response was observed but there was at least two-fold increase in the number of revertant colonies in comparison with the negative control group and reproducibility was observed in the main test, the test article was judged to be positive.
Statistics:
Statistical analysis was not performed. Individual plate counts and mean values of revertant colonies are presented.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
was evident at 2500 µg/plate and above without metabolic activation and at 5000 µg/plate with metabolic activation in WP2uvrA strain
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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:
was evident at 625 µg/plate and above for TA1535 and TA1537 with metabolic activation, at 1250 µg/plate and above for TA1535 and TA1537 without metabolic activation and at 2500 µg/plate and above for TA100 and TA98 with or without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: To set the dose levels for the main test, the 50 mg/mL solution was diluted 6 times using a common ratio of 4 and a total of 7 dose levels were selected (1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate) in the dose-finding test. Precipitation on the plate and coloration by the test substance in the test systems were not observed at any dose levels with or without metabolic activation. Growth inhibition was investigated by observing the bacterial background lawn using a stereoscopic microscope. Growth inhibition was observed at 1250 µg/plate and above for TA1535 and 1537 with and without metabolic activation, and at 5000 µg/plate for TA100, TA98 and WP2uvrA with and without metabolic activation. Therefore, for the main test the maximum dose, the minimum dose at which growth inhibition was observed, was set at 1250 µg/plate in TA1535 and TA1537 with and without metabolic activation and set at 5000 µg/plate in TA100, TA98 and WP2uvrA with or without metabolic activation. A total of 6 dose levels were selected by diluting 5 times at a common ratio of 2.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Test results of main study

With or without S9-mix

Test substance concentration (µg/plate)

Mean number of revertant colonies per plate (average of 2 plates)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

0

92

12

18

14

8

-

39.1

NT

13

NT

NT

5

-

78.1

NT

12

NT

NT

7

-

156

93

13

25

16

6

-

313

89

11

21

15

5

-

625

99

6

17

11

4

-

1250

83

8*

24

12

7*

-

2500

46*

NT

9*

12*

NT

-

5000

0*

NT

0*

0*

NT

Positive controls, -S9

Name

AF-2

SAZ

AF-2

AF-2

ICR-191

Concentration (µg/plate)

0.01

0.5

0.01

0.1

1.0

Mean No. of colonies/plate (average of 2)

564

235

85

523

1063

+

0

112

9

27

39

11

+

39.1

NT

10

NT

NT

10

+

78.1

NT

6

NT

NT

8

+

156

104

9

24

29

8

+

313

111

9

20

35

8

+

625

112

11*

30

28

11*

+

1250

106

9*

23

24

7*

+

2500

93*

NT

23

25*

NT

+

5000

65*

NT

5*

13*

NT

Positive controls, +S9

Name

B[a]P

2-AA

2-AA

B[a]P

B[a]P

Concentration (µg/plate)

5.0

2.0

10.0

5.0

5.0

Mean No. of colonies/plate (average of 2 plates)

910

315

773

353

96

AF-2 =2-(furyl)-3-(5-nitro-2-furyl)acrylamide

SAZ = Sodium azide

ICR-191 = 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine.2HCl

2-AA = 2-Aminoanthracene

B[a]P=Benzo[a]pyrene

* = Growth inhibition of tester strains was observed

NT: Not tested

Table 2. Test results of dose-finding study

With or without S9-mix

Test substance concentration (µg/plate)

Mean number of revertant colonies per plate (average of 2 plates)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

0

81

10

22

15

7

-

1.22

93

7

25

16

8

-

4.88

84

8

27

15

6

-

19.5

111

7

35

12

8

-

78.1

91

5

33

17

7

-

313

109

9

26

13

8

-

1250

77

7*

21

13

6*

-

5000

0*

0*

0*

0*

0*

Positive controls, -S9

Name

AF-2

SAZ

AF-2

AF-2

ICR-191

Concentration (µg/plate)

0.01

0.5

0.01

0.1

1.0

Mean No. of colonies/plate (average of 2 plates)

695

164

90

501

1788

+

0

115

7

32

32

13

+

1.22

108

10

27

36

14

+

4.88

104

10

22

36

13

+

19.5

90

8

25

26

9

+

78.1

116

9

25

26

12

+

313

92

5

28

35

9

+

1250

84

7*

25

31

10*

+

5000

63*

0*

10*

18*

7*

Positive controls, +S9

Name

B[a]P

2-AA

2-AA

B[a]P

B[a]P

Concentration (µg/plate)

5.0

2.0

10.0

5.0

5.0

Mean No. of colonies/plate (average of 2 plates)

999

223

926

333

107

AF-2 =2-(furyl)-3-(5-nitro-2-furyl)acrylamide

SAZ = Sodium azide

ICR-191 = 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine.2HCl

2-AA = 2-Aminoanthracene

B[a]P=Benzo[a]pyrene

* = Growth inhibition of tester strains was observed

NT: Not tested

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Genetic toxicity in vitro

The test item was investigated for mutagenicity to bacteria according to the OECD TG 471 and in compliance with GLP (Oguma, 2013).

A dose-finding test was conducted initially with concentrations between 1.22 and 5000 µg/plate. The minimum dose which showed growth inhibition in the test was then selected as the maximum dose for the main test. In the main pre-incubation experiment (2 plates per concentration) the test material was tested in S. typhimurium strains TA98, TA100, TA1535, TA1537, and in E. coli WP2 uvrA up to limit concentration of 5000 µg.plate with and without a metabolic activation system, except for TA1535 and TA1537 strain which were tested up to cytotoxic concentration of 1250 µg/plate. No significant increase in the number of revertants was observed in any of the tested strains with and without metabolic activation in the dose-finding test or the main test. Appropriate solvent and positive controls were included into the test and gave the expected results. Hence, the test item was considered to be not mutagenic to bacteria under the conditions of the test.

Justification for selection of genetic toxicity endpoint

There is only one study available

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.