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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 March 2012 to 30 March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
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.
Justification for type of information:
Read-across from a structurally similar substance.
Cross-reference
Reason / purpose:
other: read-across target
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
RAAF report to be provided.
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Test material information:
Composition 1
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes

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.
- Storage condition of test material: Controlled room temperature (15-25 °C, below 70 RH %), protected from humidity.

Method

Target gene:
Histidine and tryptophan operon.
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (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 ºC. 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. Growing cultures were prepared form the frozen bacterial cultures, thawed at room temperature. 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a water bath shaker.
- Periodically "cleansed" against high spontaneous background: Yes.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (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 ºC. 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. Growing cultures were prepared form the frozen bacterial cultures, thawed at room temperature. 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a water bath shaker.
- Periodically "cleansed" against high spontaneous background: Yes.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Test concentrations with justification for top dose:
Main test: 5000, 2500, 1250, 625, 312.5, 156.25 and 78.125 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The solvent was chosen based on a preliminary solubility test. The test material was soluble in distilled water at 100 mg/mL concentration. Therefore, distilled water was chosen as the solvent for this 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
Negative controls:
no
Solvent controls:
yes
Remarks:
distilled water and DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylene-diamine; 2-aminoanthracene
Details on test system and conditions:
METHOD OF APPLICATION: Preincubation method.
Before the overlaying, 50 μL of the test 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. These tubes were then gently mixed and then incubated for 20 minutes at 37 ºC in a shaking incubator. 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. After preparation, the plates were incubated at 37 °C.

DURATION
- Exposure duration: 48 hours.

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 item 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, with and without metabolic activation.
- 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.

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

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 resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
MAIN TESTS
Higher numbers of revertant colonies compared to the solvent control were detected in the Initial Mutation Test and Confirmatory Mutation Test in some sporadic cases. However, no dose-dependence was observed and they were below the biologically relevant threshold value and were non-reproducible. The numbers of revertant colonies were within the historical control range in all cases, so they were considered as reflecting the biological variability of the test.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None observed in either test with or without metabolic activation.

RANGE-FINDING/SCREENING 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. Slightly higher or slightly lower colony counts compared to the solvent control were observed in some cases; however, they were without biological significance and in the historical control range in all cases. Therefore, they were considered to reflect the biological variability of the test system. No precipitate or sign of cytotoxicity were 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 a factor of two.

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

28.0

33.7

98.3

110.3

10.3

11.3

9.7

11.3

29.3

41.3

MF

1.29

0.90

0.99

0.94

1.00

0.92

1.00

0.83

0.77

0.82

Distilled Water Control

Mean

21.7

37.3

99.3

117.3

10.3

12.3

9.7

13.7

38.0

50.7

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

DMSO Control

Mean

23.3

32.7

-

121.3

-

16.3

11.3

13.7

-

35.3

MF

1.08

0.88

-

1.03

-

1.32

1.17

1.00

-

0.70

5000

Mean

29.7

32.3

93.7

112.7

8.0

11.0

13.0

11.7

33.3

44.3

MF

1.37

0.87

0.94

0.96

0.77

0.89

1.34

0.85

0.88

0.88

2500

Mean

24.7

34.3

108.7

118.0

12.7

13.0

10.7

12.3

30.0

42.3

MF

1.14

0.92

1.09

1.01

1.23

1.05

1.10

0.90

0.79

0.84

1250

Mean

26.7

32.7

106.0

130.7

9.7

10.0

11.0

12.0

37.3

46.3

MF

1.23

0.88

1.07

1.11

0.94

0.81

1.14

0.88

0.98

0.91

625

Mean

24.3

32.7

99.7

129.0

11.3

11.0

9.0

14.7

38.7

44.7

MF

1.12

0.88

1.00

1.10

1.10

0.89

0.93

1.07

1.02

0.88

312.5

Mean

26.0

23.0

114.3

123.0

11.3

15.0

9.7

12.0

37.7

48.0

MF

1.20

0.62

1.15

1.05

1.10

1.22

1.00

0.88

0.99

0.95

156.25

Mean

33.0

28.3

100.0

134.7

6.7

11.7

8.0

13.3

36.0

41.3

MF

1.52

0.76

1.01

1.15

0.65

0.95

0.83

0.98

0.95

0.82

78.125

Mean

22.7

33.0

103.0

123.7

4.3

14.0

8.0

13.3

22.3

45.7

MF

1.05

0.88

1.04

1.05

0.42

1.14

0.83

0.98

0.59

0.90

 

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

22.0

32.3

110.3

123.3

7.0

9.7

8.3

5.7

27.3

37.3

MF

0.65

0.96

0.96

1.00

0.81

1.07

1.14

0.61

1.11

0.97

Distilled Water Control

Mean

33.7

33.7

115.3

123.3

8.7

9.0

7.3

9.3

24.7

38.7

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

DMSO Control

Mean

31.0

32.7

-

123.7

-

10.0

7.0

6.7

-

37.7

MF

0.92

0.97

-

1.00

-

1.11

0.95

0.71

-

0.97

5000

Mean

28.0

28.3

97.7

114.0

6.3

9.0

7.7

9.3

31.7

42.0

MF

0.83

0.84

0.85

0.92

0.73

1.00

1.05

1.00

1.28

1.09

2500

Mean

34.7

35.3

102.7

130.3

7.0

16.0

7.0

8.3

33.0

43.7

MF

1.03

1.05

0.89

1.06

0.81

1.78

0.95

0.89

1.34

1.13

1250

Mean

28.7

29.0

108.0

119.7

5.3

8.7

6.0

9.3

27.7

47.7

MF

0.85

0.86

0.94

0.97

0.62

0.96

0.82

1.00

1.12

1.23

625

Mean

29.3

33.3

116.3

101.0

6.0

11.3

7.7

7.7

32.7

36.3

MF

0.87

0.99

1.01

0.82

0.69

1.26

1.05

0.82

1.32

0.94

312.5

Mean

24.7

39.3

101.0

133.7

6.7

10.7

5.7

6.7

31.0

43.7

MF

0.73

1.17

0.88

1.08

0.77

1.19

0.77

0.71

1.26

1.13

156.25

Mean

25.3

33.3

114.3

124.0

6.3

13.3

6.0

5.7

34.0

40.7

MF

0.75

0.99

0.99

1.01

0.73

1.48

0.82

0.61

1.38

1.05

78.125

Mean

24.0

34.7

104.7

111.0

7.0

10.7

8.0

7.3

29.0

39.3

MF

0.71

1.03

0.91

0.90

0.81

1.19

1.09

0.79

1.18

1.02

Applicant's summary and conclusion

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

Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), response in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2 uvrA both in the presence and in the absence 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. S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2uvrA were exposed to the test material at concentrations of 5 000, 2500, 1 250, 625, 312.5, 156.25 and 78.125 µg/plate using the preincubation method. Positive and solvent controls were run concurrently for comparison.

Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), response in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2uvrA both in the presence and in the absence of metabolic activation.