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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:
21 September 2011 to 20 October 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Physical state: White to pale yellow powder with a slightly characteristic odour.
- Storage condition of test material: Room temperature (15-25 ºC) in the dark.

Method

Target gene:
Histidine and tryptophan operon.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
- Type and identity of media: Nutrient Broth No.2, 25.0 g per 1000 mL. Sterilization was performed at 121 °C in an autoclave.
- Properly maintained: Yes, all strains were stored at -80 ± 10 °C. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent. Additional strain characteristics were checked regularly.
- Periodically "cleansed" against high spontaneous background: Yes.
Additional strain / cell type characteristics:
other: rfa, uvrB, uvrA and pKM101
Metabolic activation:
with and without
Metabolic activation system:
Liver extract, S9 fraction.
Test concentrations with justification for top dose:
Main test: 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water.
- Justification for choice of solvent/vehicle: The solvent was chosen based on a preliminary solubility test. The solubility of the test material was examined in distilled water, dimethyl sulfoxide (DMSO), and acetone. The test material was soluble in distilled water at 100 mg/mL concentration, but only partial dissolution was observed using DMSO or acetone as solvent at the same concentration. Therefore, distilled water was chosen for solvent of the study. The obtained formulations (50 μL) with the solution of top agar (2 mL) and phosphate buffer (500 µL) were examined in a test tube without test bacterium suspension and found to form a clear solution at 5000 µg/tube.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water and DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene; 4-nitro-1,2-phenylene-diamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation). Before the overlaying, 50 μL of the test material solution (or solvent or reference control), 100 μL of the overnight culture of the tester strains (cultured in Nutrient Broth No.2) and 0.5 mL of the S9 mix or phosphate buffer (pH 7.4) were added into appropriate tubes to provide direct contact between bacteria and the test material. After the pre-incubation period, 2 mL of molten top agar (kept at 45°C) were added to the tubes; the content was mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activation and an activation test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated.

DURATION
- Preincubation period: 20 mins at 37 °C in a shaking incubator.
- Exposure duration: 48 hours, incubated at 37°C.

NUMBER OF REPLICATIONS: Each concentration was performed in triplicate. A second test was performed at the same dosing range to confirm the results of the first assay.

OBSERVATIONS
The colony numbers on the untreated / negative / positive control and test material treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported.

RANGE FINDING TEST:
- Test concentrations: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
- Tester strains: TA98 and TA100.
Evaluation criteria:
The study was considered valid if:
- The number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests.
- At least five analyzable (non-cytotoxic) concentrations were presented in all strains of the main tests.

Criteria for a positive (mutagenic) response:
- A dose related increase in the number of revertants occurred and/or,
- A reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- In all strains: the number of reversion more than twice higher than the reversion rate of the solvent control

Criteria for a negative (non-mutagenic) response:
The test material was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test material and for the controls using a spreadsheet.

Mutation factor (MF) = mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
MAIN TESTS
In the Initial Mutation Test and Confirmatory Mutation Tests none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed in the main tests.

RANGE-FINDING STUDIES
- Results used for determination of the concentration range for the definitive test: The observed numbers of revertant colonies were mostly in the normal range. Lower colony counts compared to controls were considered to reflect the biological variability of the test system. Inhibition was observed in tester strains TA98 and TA100 at 5000, 2500 and 1000 μg/plate concentrations without metabolic activation; and in S. typhimurium TA100 tester strain at 5000, 2500 and 1000 μg/plate concentrations with metabolic activation. No precipitation was observed in the preliminary experiment.
Based on the observed result of the preliminary experiment, 5000 μg/plate concentration was selected as the highest examined dose in the main test and lower concentrations will be evenly spaced by approximately half log intervals to cover the range from cytotoxicity to no cytotoxicity.

ADDITIONAL INFORMATION ON CYTOTOXICITY
Inhibitory, cytotoxic effects (reduced / slightly reduced background lawn development and / or reduced number of revertant colonies, in some cases pinpoint colonies were also detected) of the test material were observed in both assays in all S. typhimurium strains at concentrations of 5000 and 1581 μg/plate without metabolic activation and at 5000 μg/plate with metabolic activation. Cytotoxiciy was observed on the Initial Mutation Test with E. coli WP2 uvrA strain at 5000 μg/plate without metabolic activation. Furthermore in the Confirmatory Mutation Test, in S. typhimurium TA98, TA1535 and TA1537 at 500 μg/plate without metabolic activation, in S. typhimurium TA100 and TA1537 at 1581 μg/plate with metabolic activation, and in E. coli WP2 uvrA strain at 5000 μg/plate without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of Initial Mutation Test

Concentration (µg/plate)

S. typhimurium Tester Strains

E. Coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Untreated control

Mean

19.0

32.3

84.7

101.0

10.7

10.3

5.7

9.3

31.0

51.7

MF

0.98

1.13

1.01

0.96

1.03

0.89

0.74

1.65

0.99

1.22

DMSO control

Mean

19.3

27.0

-

94.3

-

12.3

7.0

9.7

-

44.0

MF

1.00

0.94

-

0.90

-

1.06

0.91

1.71

-

1.04

Distilled water control

Mean

19.3

28.7

83.7

104.7

10.3

11.7

7.7

5.7

31.3

42.3

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

5000

Mean

0.0*#

10.3 #

0.0*#

39.0*#

0.0*#

5.7*#

1.7*#

1.3 #

14.7 #

34.3

MF

0.00 #

0.36 #

0.00 #

0.37 #

0.00 #

0.49 #

0.22 #

0.24 #

0.47 #

0.81

1581

Mean

7.7*#

17.0

49.3*#

47.3 #

3.7*#

11.7

2.0*#

6.3

21.3

41.3

MF

0.40 #

0.59

0.59 #

0.45 #

0.35 #

1.00

0.26 #

1.12

0.68

0.98

500

Mean

13.0

21.3

57.3

69.0

6.7

10.7

2.0

8.0

25.3

40.7

MF

0.67

0.74

0.69

0.66

0.65

0.91

0.26

1.41

0.81

0.96

158.1

Mean

16.7

26.0

80.0

90.7

7.7

12.0

6.0

5.7

30.3

44.3

MF

0.86

0.91

0.96

0.87

0.74

1.03

0.78

1.00

0.97

1.05

50

Mean

21.3

26.3

83.7

98.0

8.0

10.7

4.7

7.3

29.7

38.3

MF

1.10

0.92

1.00

0.94

0.77

0.91

0.61

1.29

0.95

0.91

15.81

Mean

22.7

26.0

90.7

104.7

12.7

14.0

5.0

10.3

31.0

41.7

MF

1.17

0.91

1.08

1.00

1.23

1.20

0.65

1.82

0.99

0.98

5

Mean

21.3

28.7

81.0

96.3

9.3

10.7

4.3

9.3

28.7

36.3

MF

1.10

1.00

0.97

0.92

0.90

0.91

0.57

1.65

0.91

0.86

1.581

Mean

21.3

27.7

94.3

89.3

9.3

9.3

6.3

10.3

29.0

35.0

MF

1.10

0.97

1.13

0.85

0.90

0.80

0.83

1.82

0.93

0.83

* = growth inhibition; # = Cytotoxic concentrations; MF = mutation factor (mean number of revertants on the test material plate divided by the mean number of revertants on the vehicle control plate).

 

Table 2: Summary of Confirmatory Mutation Test

Concentration (µg/plate)

S. typhimurium Tester Strains

E. Coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Untreated control

Mean

28.7

28.0

91.0

109.3

9.7

8.7

8.3

8.7

23.3

27.7

MF

1.34

0.79

1.04

1.10

0.91

0.70

1.04

1.04

1.13

0.95

DMSO control

Mean

27.3

29.0

-

111.3

-

9.7

4.0

8.7

-

24.7

MF

1.28

0.81

-

1.12

-

0.78

0.50

1.04

-

0.85

Distilled water control

Mean

21.3

35.7

87.3

99.0

10.7

12.3

8.0

8.3

20.7

29.0

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

5000

Mean

3.3*#

9.0*#

27.0*#

38.7*#

1.0*#

5.0*#

0.0*#

1.7*#

10.7*#

30.3

MF

0.16 #

0.25 #

0.31 #

0.39#

0.09 #

0.41 #

0.00 #

0.20 #

0.52 #

1.05

1581

Mean

4.7*#

29.7 #

53.7*#

35.3*#

4.0*#

10.3

0.0*#

3.7 #

22.7

33.0

MF

0.22#

0.83

0.61 #

0.36 #

0.38 #

0.84

0.00 #

0.44 #

1.10

1.14

500

Mean

15.0*#

35.0

66.3

56.0

6.0*#

9.7 #

2.7*#

8.0

21.3

33.7

MF

0.70 #

0.98

0.76

0.57

0.56 #

0.78 #

0.33 #

0.96

1.03

1.16

158.1

Mean

24.0

29.7

88.7

71.7

8.7

6.7

8.0

9.0

22.7

40.0

MF

1.13

0.83

1.02

0.72

0.81

0.54

1.00

1.08

1.10

1.38

50

Mean

26.3

33.3

84.7

85.3

7.7

9.7

3.7

9.7

24.3

38.0

MF

1.23

0.93

0.97

0.86

0.72

0.78

0.46

1.16

1.18

1.31

15.81

Mean

26.3

31.7

84.7

89.7

7.7

9.7

5.3

8.7

24.0

39.7

MF

1.23

0.89

0.97

0.91

0.72

0.78

0.67

1.04

1.16

1.37

5

Mean

27.0

39.0

86.0

102.0

8.0

14.0

10.3

10.0

25.3

32.3

MF

1.27

1.09

0.98

1.03

0.75

1.14

1.29

1.20

1.23

1.11

1.581

Mean

21.3

39.0

92.0

93.0

4.7

8.3

8.7

5.7

26.3

35.7

MF

1.00

1.09

1.05

0.94

0.44

0.68

1.08

0.68

1.27

1.23

* = growth inhibition; # = Cytotoxic concentrations; MF = mutation factor (mean number of revertants on the test material plate divided by the mean number of revertants on the vehicle control plate).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of the study, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used, therefore the test material is considered to be non-mutagenic. Cytotoxicity was observed in both S. typhimurium and E. coli at certain concentrations at or above 500 µg/plate, however, this was dependant on the strain and the inclusion of metabolic activation.
Executive summary:

The mutagenic potential of the test material was assessed in an Ames test conducted under GLP conditions and in line with the standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100. During the study S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA were exposed to the test material at concentrations of 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate using the plate incorporation method. Positive and solvent controls were run concurrently for comparison.

Under the conditions of the study, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used, therefore the test material is considered to be non-mutagenic. Cytotoxicity was observed in both S. typhimurium and E. coli at certain concentrations at or above 500 µg/plate, however this was dependant on the strain and the presence or absence of metabolic activation.