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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:
March 17 - April 23 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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:
equivalent or similar to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
other: unspecified EPA OPPTS harmonised guidelines
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): FLL sample 4
- Physical state: viscous liquid
- Lot/batch No.: 240210
- Expiration date of the lot/batch: not reported
- Storage condition of test material: room temperature in the dark
- Other: yellow/brown color
- All other template details: Not reported

Method

Target gene:
histidine in Salmonella strains and tryptophan in E. coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: uvrB-, and pKM101
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
in µg/plate: 0, 50, 150, 500, 1500, 5000
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: test material was immiscible in sterile distilled water, dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- All other template details: Not reported

SELECTION AGENT (mutation assays): not reported

NUMBER OF REPLICATIONS: three

NUMBER OF CELLS EVALUATED: 0.1 ml of bacterial culture, in the range of 1-9.9 x 10^9 bacteria per ml

DETERMINATION OF CYTOTOXICITY
- Method: growth, numbers of revertant colonies

OTHER EXAMINATIONS: Not reported
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used to aid evaluation, however, statistical significance will not be the only determining factor for a positive response.
Statistics:
Not reported

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitate: a very fine precipitate was noted on the 5000 µg/plate tests
- All other template details: none reported

RANGE-FINDING/SCREENING STUDIES: concentrations tested ranged from 0.15 to 5000 µg/plate, and after 48 hours was determined to be non-toxic to the strains of bacteria used.

COMPARISON WITH HISTORICAL CONTROL DATA: vehicle and positive control data presented as Appendix 1

ADDITIONAL INFORMATION ON CYTOTOXICITY: not reported

Any other information on results incl. tables

Table 2. Mean (and standard deviation of 3 replicates) numbers of revertant colonies without metabolic activation.

Test substance concentration (µg/plate)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

118 (± 15.5)

17 (± 2.1)

22 (± 1.5)

31 (± 4.4)

12 (± 2.9)

50

118 (± 3.6)

14 (± 4.0)

23 (± 5.9)

27 (± 4.5)

12 (± 1.7)

150

120 (± 14.0)

17 (± 5.9)

22 (± 3.6)

29 (± 3.0)

14 (± 3.5)

500

120 (± 18.2)

16 (± 4.0)

25 (± 4.5)

26 (± 5.2)

13 (± 2.1)

1500

111 (± 11.1)

12 (± 1.2)

23 (± 2.6)

34 (± 3.8)

14 (± 1.0)

5000

94 (± 11.4)

11 (± 1.5)

24 (± 2.6)

26 (± 4.5)

10 (± 1.0)

 

 

 

 

 

 

Positive controls

ENNG (3µg/plate)

ENNG (5µg/plate)

ENNG (2µg/plate)

4NQO (0.2µg/plate)

9AA (80µg/plate)

 

309 (± 52.0)

181 (± 28.0)

448 (± 13.6)

139 (± 15.9)

2742 (± 725.8)

Table 3. Mean (and standard deviation of 3 replicates) numbers of revertant colonies with metabolic activation

Test substance concentration (µg/plate)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

100 (± 10.4)

11 (± 3.2)

41 (± 6.0)

30 (± 1.2)

 9 (± 1.7)

50

103 (± 6.0)

8 (± 1.5)

44 (± 2.9)

30 (± 3.2)

10 (± 2.1)

150

109 (± 14.7)

11 (± 4.5)

43 (± 2.5)

28 (± 2.6)

11 (± 2.9)

500

112 (± 22.6)

11 (± 1.7)

49 (± 5.8)

33 (± 1.2)

10 (± 2.5)

1500

106 (± 5.5)

12 (± 0.6)

47 (± 2.9)

31 (± 2.0)

11 (± 2.5)

5000

100 (± 3.1)

11 (± 0.6)

43 (± 4.9)

30 (± 3.0)

11 (± 2.5)

 

 

 

 

 

 

Positive controls

2AA
(1µg/plate)

2AA
(2µg/plate)

2AA
(10µg/plate)

BP
(5µg/plate)

2AA
(2µg/plate)

 

1023 (± 199.8)

242 (± 24.2)

301 (± 6.8)

176 (± 8.7)

215 (± 21.2)

Table 4. Mean (and standard deviation of 3 replicates) numbers of revertant colonies without metabolic activation after 20 minutes of pre-incubation with the test substance.

Test substance concentration (µg/plate)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

118 (± 15.0)

18 (± 6.7)

32 (± 1.0)

15 (± 7.2)

10 (± 4.7)

50

109 (± 11.2)

19 (± 2.3)

21 (± 1.5)

22 (± 7.5)

13 (± 2.5)

150

110 (± 4.2)

22 (± 2.6)

28 (± 6.9)

19 (± 3.8)

10 (± 0.6)

500

105 (± 1.7)

24 (± 1.2)

25 (± 3.5)

18 (± 5.0)

11 (± 1.5)

1500

123 (± 8.0)

21 (± 1.7)

23 (± 2.1)

20 (± 3.6)

11 (± 4.4)

5000

114 (± 23.9)

22 (± 14.2)

25 (± 6.1)

23 (± 2.5)

12 (± 2.1)

 

 

 

 

 

 

Positive controls

ENNG (3µg/plate)

ENNG (5µg/plate)

ENNG (2µg/plate)

4NQO (0.2µg/plate)

9AA (80µg/plate)

 

398 (± 27.1)

217 (± 39.5)

391 (± 30.2)

488 (± 187.0)

2162 (± 185.7)

Table 5. Mean (and standard deviation of 3 replicates) numbers of revertant colonies with metabolic activation after 20 minutes of pre-incubation with the test substance.

Test substance concentration (µg/plate)

TA100

TA1535

WP2uvrA-

TA98

TA1537

0

112 (± 4.2)

23 (± 3.5)

22 (± 1.7)

19 (± 6.1)

18 (± 2.9)

50

101 (± 13.2)

19 (± 4.6)

23 (± 1.2)

25 (± 9.6)

13 (± 0.6)

150

112 (± 9.7)

15 (± 0.6)

26 (± 3.6)

15 (± 2.3)

16 (± 2.1)

500

113 (± 7.9)

13 (± 4.0)

28 (± 4.6)

11 (± 2.3)

11 (± 0.6)

1500

100 (± 4.0)

 9 (± 2.1)

28 (± 5.2)

20 (± 9.5)

15 (± 1.2)

5000

91 (± 8.7)

19 (± 2.5)

28 (± 3.2)

15 (± 4.7)

16 (± 3.8)

 

 

 

 

 

 

Positive controls

2AA
(1µg/plate)

2AA
(2µg/plate)

2AA
(10µg/plate)

BP
(5µg/plate)

2AA
(2µg/plate)

 

1579 (± 157.0)

331 (± 21.6)

202 (± 16.2)

235 (± 33.0)

236 (± 59.3)

 

Applicant's summary and conclusion

Conclusions:
This substance is considered to be non-mutagenic under the conditions of this test.
Executive summary:

Study Summary:

Introduction: This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Agencies including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonized guidelines.

 

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

 

Results. The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S-9 mix were validated.

 

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore tested up to the maximum recommended dose level of 5000µg/plate. A very fine precipitate was noted at 5000µg/plate, this observation did not prevent the scoring of revertant colonies.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

 

Conclusion. The test material was considered to be non-mutagenic under the conditions of this test.