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Toxicological information

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
Study period:
2013-12-09 to 2014-01-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2013-04-11
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Physical state: liquid, clear pale yellow
- Storage condition of test material: at room temperature

Method

Target gene:
TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
E. coli WP2 uvrA: trp-
Species / strainopen allclose all
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:
Phenobarbital/β-naphthoflavone induced rat liver S9 were used as the metabolic activation system. Protein concentration of the S9 prepartion was 37.8 mg/mL.
Test concentrations with justification for top dose:
Pre-experiment/Experiment 1 (with and without S9 mix): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Experiment II (without S9 mix): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Experiment II (with S9 mix): 1, 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control without metabolic activation: dissolved in deionised water; concentration: 10 μg/plate (strains TA 1535, TA 100)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Positive control without metabolic activation: dissolved in DMSO; concentration: 10 μg/plate (strain TA 98); 50 μg/plate (strain TA 1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Positive control without metabolic activation: dissolved in deionised water; concentration: 2 μL/plate (strain WP2 uvrA)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Positive control with metabolic activation: dissolved in DMSO; concentration: 2.5 μg/plate (strains TA 1535, TA 1537, TA 98, TA 100); 10.0 μg/plate in WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation; pre-experiment/Experiment 1) and preincubation (Experiment 2)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours at 37°C

NUMBER OF REPLICATIONS: for each strain and dose level including the controls, three plates were used.

The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager (v.1.21). Due to reduced background growth, the colonies were partly counted manually.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. Whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in the presence of metabolic activation. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate in the presence of metabolic activation in experiment I. The undissolved particles had no influence on the data recording.

CYTOTOXICITY:
- reduced background growth was observed at higher concentrations in all experimental parts with and without metabolic activation (please refer to the table 1 in the field "Any other information on results incl. tables" below).
- toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred at higher concentrations in all experimental parts with and without metabolic activation (please refer to the table 2 in the field "Any other information on results incl. tables" below).


MAIN EXPERIMENTS:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration 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 in induced revertant colonies.

Please also refer to table 3 and 4 in the field "Any other information on results incl. tables" below.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Reduced background growth was observed at the following concentration (µg/plate):

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

2500, 5000

2500, 5000

333, 5000

1000 - 5000

TA 1537

1000 - 5000

2500, 5000

333 - 5000

1000 - 5000

TA 98

2500, 5000

2500, 5000

333 - 5000

1000 - 5000

TA 100

2500, 5000

1000, 5000

333 - 5000

1000 - 5000

WP2 uvrA

5000

2500, 5000

1000 - 5000

2500 - 5000

Table 2: Toxic effects (µg/plate)

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

2500, 5000

2500, 5000

2500, 5000

1000 - 5000

TA 1537

1000 - 5000

2500, 5000

2500, 5000

1000 - 5000

TA 98

2500, 5000

2500, 5000

2500, 5000

1000 - 5000

TA 100

2500, 5000

2500, 5000

1000 - 5000

1000 - 5000

WP2 uvrA

5000

2500, 5000

5000

5000

Table 3: Summary of experiment I

Metabolic activation

Test group

Dose level (per plate)

Revertant Colony counts (Mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without activation

DMSO

 

21 ± 1

10 ± 5

24 ± 2

88 ± 5

48 ± 5

Untreated

 

15 ± 6

14 ± 3

26 ± 5

108 ± 15

44 ± 7

PI 27137

3 µg

15 ± 3

9 ± 4

24 ± 2

97 ± 13

43 ± 1

10 µg

15 ± 3

9 ± 3

26 ± 3

99 ± 10

46 ± 8

33 µg

18 ± 6

9 ± 4

23 ± 3

89 ± 11

44 ± 9

100 µg

23 ± 4

8 ± 2

22 ± 2

94 ± 6

46 ± 5

333 µg

18 ± 5

10 ± 2

25 ± 3

71 ± 7

41 ± 10

1000 µg

23 ± 1

3 ± 1MR

12 ± 4

62 ± 4

24 ± 3

2500 µg

8 ± 2MR

2 ± 1MR

5 ± 1MR

9 ± 2MR

22 ± 4

5000 µg

1 ± 1MR

1 ± 1MR

1 ± 1MR

1 ± 1MR

14 ± 4MR

NaN3

10 µg

2506 ± 413

 

 

2065 ± 15

 

4-NOPD

10 µg

 

 

271 ± 12

 

 

4-NOPD

50 µg

 

69 ± 9

 

 

 

MMS

2.0 µL

 

 

 

 

1157 ± 102

With activation

DMSO

 

15 ± 7

17 ± 4

31 ± 3

102 ± 4

50 ± 3

Untreated

 

21 ± 6

21 ± 9

38 ± 6

113 ± 2

47 ± 6

PI 27137

3 µg

14 ± 2

17 ± 3

38 ± 12

96 ± 11

44 ± 7

10 µg

16 ± 4

16 ± 4

30 ± 4

94 ± 2

49 ± 9

33 µg

14 ± 2

13 ± 3

25 ± 3

95 ± 3

48 ± 4

100 µg

14 ± 5

12 ± 5

26 ± 3

105 ± 1

45 ±4

333 µg

11 ± 3

16 ± 8

36 ± 10

93 ± 2

45 ± 7

1000 µg

15 ± 6

14 ± 4

29 ± 7

63 ± 11R

26 ± 5

2500 µg

1 ± 1MR

1 ± 1R

3 ± 1MR

0 ± 0MR

11 ± 3MR

5000 µg

0 ± 1MRP

0 ± 0MRP

0 ± 0PMR

0 ± 0PMR

0 ± 0PMR

2-AA

2.5 µg

407 ± 35

369 ± 3

2325 ± 578

3608 ± 319

 

2-AA

10.0 µg

 

 

 

 

287 ±58

Key to positive Controls          

NaN3: sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phylene-diamine

MMS: methyl methane sulfonate

 

Key to Plate Postfix Codes

M: Manual count

R: Reduced background growth

P: Precipitate

Table 4: Summary of experiment II

Metabolic activation

Test group

Dose level (per plate)

Revertant Colony counts (Mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without activation

DMSO

 

10 ± 2

9 ± 1

21 ± 4

88 ± 12

43 ± 2

Untreated

 

11 ± 3

10 ± 4

23 ± 5

115 ± 16

42 ± 5

PI 27137

3 µg

10 ± 4

9 ± 1

33 ± 7

101 ± 12

51 ± 3

10 µg

13 ± 7

10 ± 2

25 ± 4

90 ± 6

49 ± 6

33 µg

12 ± 2

8 ± 2

16 ± 3

95 ± 18

51 ± 12

100 µg

11 ± 1

13 ± 3

23 ± 3

107 ± 12

43 ± 5

333 µg

7 ± 1MR

3 ± 1MR

13 ± 3MR

63 ± 13R

29 ± 6

1000 µg

6 ± 1MR

13 ± 2RM

23 ± 6MR

21 ± 2MR

24 ± 4R

2500 µg

4 ± 1MR

0 ± 0MR

4 ± 1MR

0 ± 0MR

23 ± 7R

5000 µg

2 ± 1MR

0 ± 0MR

0 ± 0MR

0 ± 0MR

5 ± 1MR

NaN3

10 µg

2968 ± 19

 

 

2435 ± 30

 

4-NOPD

10 µg

 

 

306 ± 20

 

 

4-NOPD

50 µg

 

59 ± 2

 

 

 

MMS

2.0 µL

 

 

 

 

669 ± 40

With activation

DMSO

 

14 ± 3

21 ± 6

45 ± 4

109 ± 16

46 ± 5

Untreated

 

12 ± 5

18 ± 3

59 ± 10

124 ± 6

56 ± 12

PI 27137

 

6 ± 1

18 ± 4

35 ± 1

123 ± 14

70 ± 13

3 µg

11 ± 5

23 ± 4

51 ± 4

101 ± 21

55 ± 12

10 µg

10 ± 4

15 ± 1

48 ± 7

123 ± 4

58 ± 12

33 µg

9 ± 4

16 ± 3

35 ± 7

115 ± 8

56 ± 9

100 µg

14 ± 6

14 ± 1

26 ± 3

116 ± 15

39 ± 9

333 µg

14 ± 5

22 ± 6

48 ± 3

83 ± 20

41 ± 5

1000 µg

0 ± 0MR

3 ± 1MR

4 ± 1MR

25 ± 2MR

31 ± 2

2500 µg

0 ± 0R

0 ± 0MR

0 ± 0MR

0 ± 0MR

23 ± 4R

5000 µg

0 ± 0R

0 ± 0MR

0 ± 0MR

0 ± 0MR

0 ± 0R

2-AA

2.5 µg

436 ± 34

342 ± 14

3217 ± 173

3801 ± 192

 

2-AA

10.0 µg

 

 

 

 

265 ± 17

Key to positive Controls          

NaN3: sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phylene-diamine

MMS: methyl methane sulfonate

 

Key to Plate Postfix Codes

M: Manual count

R: Reduced background growth

For further results please also refer to the field "Attached background material" below.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
According to Directive 67/548 /EEC and its subsequent amendments and according to Regulation (EC) No 1272/2008 and subsequent regulations, the test substance should not be considered to have a mutagenic potential, and hence no classification or labelling is required.
Executive summary:

A bacterial reverse mutation assay was performed with the test item according to the OECD guideline 471 (1997) using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 as well as E. coli WP2 uvr A. The test was carried out using the plate incorporation method and the preincubation method with plating in triplicate with and without metabolic activation. The test item was tested at the following concentrations in both experiments: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate. Additionally, a negative control and positive controls were run concurrently.

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in the presence of metabolic activation. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate in the presence of metabolic activation in the plate incorporation test. The undissolved particles had no influence on the data recording.

Reduced background growth was observed at higher concentrations in all experimental parts with and without metabolic activation.

Toxic effects, evident as a reduction of the number of revertants (below an induction factor of 0.5), occurred at higher concentrations in all experimental parts with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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.

In conclusion, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.