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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:
08 March 2018 - 16 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO/IEC 17025:2005 (ISO/IEC, 2005)
Version / remarks:
2005
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
Details on test material:
Storage Conditions:Stable at ambient temperature (10-30°C). Store in darkness; may be used/formulated in light.

Method

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:
S9 mix
Test concentrations with justification for top dose:
10.0, 33.3, 100, 333, 1000, 3333 and 5000 μg per plate.Doses selected for the mutagenicity assay are based on data generated in BioReliance study AF02PT.502.BTL, details was not documented in the study report
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: guideline recommended
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene: 1.0 μg/plate for TA98, TA1535, 2.0 μg/plate forTA100, TA1537 and 15 μg/plateWP2 uvrA in S9.
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicate
- Number of independent experiments: one – Plate incorporation method

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 50.0 μL of tester strain (cells seeded)
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: 50.0 μL of the test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: n/a
- Exposure duration/duration of treatment: 48 to 72 hours at 37±2°C
- Harvest time after the end of treatment (sampling/recovery times): not stated
Rationale for test conditions:
The study was based on the in vitro (plate incorporation) technique described by Ames et al (1975), Maron and Ames (1983), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence is used to complement the Salmonella strains.
Evaluation criteria:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:

1. Strains TA1535 and TA1537 – considered positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

2. Strains TA98, TA100 and WP2 uvrA - considered positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.
Statistics:
Not stated

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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:
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:
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MTEST-SPECIFIC CONFOUNDING FACTORS: 
- Data on pH:Not specified
- Data on osmolality:Not specified
- Possibility of evaporation from medium:Not specified
- Water solubility:Not specified
- Precipitation and time of the determination: Precipitate was observed at 5000 μg per plate with all conditions.
- Definition of acceptable cells for analysis: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- Other confounding effects: no



RANGE-FINDING/SCREENING STUDIES (if applicable): Was not conducted



STUDY RESULTS
- Concurrent vehicle negative and positive control data: Both with historical control range


For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: Yes in TA98, the dose dependent increases were also outside the historical control limit for this tester strain.
- Statistical analysis; not stated
- Any other criteria: not stated

Ames test:
- Signs of toxicity: Toxicity was observed beginning at 5000 μg per plate in TA1535 , 3333 in WP2 uvrA with and without S9 and 5000 μg per plate in TA1537 - S9
- Individual plate counts: yes
- Mean number of revertant colonies per plate and standard deviation: yes


Remarks on result:
other: TX16797induce reverse mutations at selected loci of Salmonella typhimurium (TA98) in the presence of metabolic activation

Any other information on results incl. tables

TABLE 1. Mutagenicity Assay without S9 activation

Strain

Substance

Dose level per plate

Mean Standard revertants

Deviation

per plate

Ratio treated / solvent

Individual revertant colony counts and background codes

TA98

TX16797

5000 μg

11

3

0.5

15A1NP,9A1NP, 9A1NP

3333 μg

12

4

0.6

14A, 7A, 14A

1000 μg

23

5

1.1

17A, 24A, 27A

333 μg

17

3

0.8

18A, 14A, 19A

100 μg

20

5

1.0

24A, 21A, 15A

33.3 μg

20

3

1.0

17A, 19A, 23A

 

10.0 μg

16

2

0.8

14A, 16A, 18A

DMSO

50.0 μL

21

4

 

22A, 17A, 24A

TA100

TX16797

5000 μg

53

12

0.6

52A2 NP, 42A2 NP, 66A2NP

3333 μg

61

3

0.7

62A, 63A, 58A

1000 μg

109

15

1.2

91A, 119A, 116A

333 μg

105

12

1.2

111A, 112A, 91A

100 μg

90

15

1.0

91A, 75A, 104A

33.3 μg

96

23

1.1

123A, 83A, 82A

10.0 μg

83

7

0.9

82A, 76A, 90A

DMSO

50.0 μL

89

10

 

83A, 101A, 84A

TA1535

TX16797

5000 μg

3

3

0.3

6A1NP,3A1NP,1A1 NP

3333 μg

7

5

0.6

7A, 11A, 2A

1000 μg

16

3

1.5

19A, 15A, 14A

333 μg

11

2

1.0

13A, 9A, 11A

100 μg

14

1

1.3

15A, 13A, 14A

33.3 μg

17

5

1.5

17A, 22A, 13A

10.0 μg

12

4

1.1

9A, 17A, 10A

DMSO

50.0 μL

11

4

 

11A, 8A, 15A

TA1537

TX16797

5000 μg

2

2

0.3

0A1NP,3A1NP,3A1 NP

3333 μg

4

3

0.6

3A, 8A, 2A

1000 μg

8

3

1.1

7A, 6A, 11A

333 μg

7

2

1.0

8A, 7A, 5A

100 μg

9

4

1.3

6A, 14A, 8A

33.3 μg

7

2

1.0

7A, 5A, 9A

10.0 μg

7

2

1.0

5A, 7A, 8A

DMSO

50.0 μL

7

4

 

3A, 9A, 10A

WP2uvrA

TX16797

5000 μg

4

1

0.1

3A1NP,5A1NP,5A1 NP

3333 μg

6

1

0.2

7A, 7A, 5A

1000 μg

15

3

0.5

13A, 18A, 15A

333 μg

23

4

0.8

26A, 19A, 24A

100 μg

24

2

0.9

23A, 27A, 23A

33.3 μg

27

4

1.0

32A, 25A, 24A

10.0 μg

24

5

0.9

21A, 29A, 21A

DMSO

50.0 μL

28

3

 

30A, 29A, 25A

TA98

2NF

1.00 μg

103

9

4.9

109A, 107A, 93A

TA100

SA

1.00 μg

656

45

7.4

678A, 686A, 604A

TA1535

SA

1.00 μg

546

8

49.6

547A, 538A, 553A

TA1537

9AA

75.0 μg

637

195

91.0

422A, 684A, 804A

WP2uvrA

MMS

1000 μg

455

35

16.3

415A, 477A, 474A

Key to Positive Controls

Key to Plate Postfix Codes

Key to Automatic Count Flags

2NF 2-nitrofluorene, SA sodium azide
9AAD 9-Aminoacridine
MMS methyl methanesulfonate

NP = Non-interfering 1= particulate Normal background

 A= Automatic count

TABLE 2. Mutagenicity Assay with S9 activation

Strain

Substance

Dose level per plate

Mean Standard revertants

Deviation

per plate

Ratio treated / solvent

Individual revertant colony counts and background codes

TA98

TX16797

5000 μg

194

57

6.5

253A1 NP, 189A1 NP, 140A1 NP

3333 μg

190

30

6.3

188A, 161A, 221A

1000 μg

274

54

9.1

317A, 292A, 214A

333 μg

95

18

3.2

79A, 92A, 115A

100 μg

46

9

1.5

35A, 52A, 50A

33.3 μg

32

4

1.1

36A, 29A, 31A

 

10.0 μg

25

5

0.8

27A, 19A, 29A

DMSO

50.0 μL

30

5

 

31A, 35A, 25A

TA100

TX16797

5000 μg

85

19

0.8

84A2 NP, 105A2 NP,67A2NP

3333 μg

81

28

0.7

93A, 49A, 101A

1000 μg

119

10

1.1

122A, 108A, 127A

333 μg

101

18

0.9

88A, 93A, 122A

100 μg

102

3

0.9

105A, 99A, 101A

33.3 μg

102

6

0.9

106A, 105A, 95A

10.0 μg

104

6

1.0

100A, 111A, 101A

DMSO

50.0 μL

109

3

 

109A, 111A, 106A

TA1535

TX16797

5000 μg

2

1

0.2

3A1NP,2A1NP,2A1 NP

3333 μg

5

4

0.4

9A, 2A, 5A

1000 μg

9

1

0.8

10A, 9A, 9A

333 μg

8

2

0.7

7A, 10A, 6A

100 μg

12

5

1.0

9A, 9A, 18A

33.3 μg

9

6

0.8

16A, 5A, 7A

10.0 μg

12

3

1.0

14A, 13A, 9A

DMSO

50.0 μL

12

2

 

14A, 10A, 13A

TA1537

TX16797

5000 μg

8

1

0.8

8A1NP,9A1NP,8A1 NP

3333 μg

7

3

0.7

6A, 10A, 5A

1000 μg

11

2

1.1

11A, 10A, 13A

333 μg

9

3

0.9

7A, 13A, 8A

100 μg

13

3

1.3

11A, 11A, 16A

33.3 μg

10

5

1.0

6A, 8A, 15A

10.0 μg

6

1

0.6

6A, 7A, 5A

DMSO

50.0 μL

10

1

 

10A, 11A, 9A

WP2uvrA

TX16797

5000 μg

8

3

0.4

7A1NP,11A1NP, 6A1 NP

3333 μg

8

4

0.4

13A, 7A, 5A

1000 μg

18

3

0.8

17A, 16A, 21A

333 μg

25

1

1.1

25A, 24A, 25A

100 μg

26

4

1.2

31A, 23A, 25A

33.3 μg

27

8

1.2

30A, 34A, 18A

10.0 μg

23

2

1.0

21A, 23A, 24A

DMSO

50.0 μL

22

5

 

27A, 17A, 22A

TA98

2AA

1.00 μg

242

28

8.1

274A, 223A, 230A

TA100

2AA

2.00 μg

799

40

7.3

845A, 776A, 776A

TA1535

2AA

1.00 μg

78

11

6.5

79A, 89A, 67A

TA1537

2AA

2.00 μg

44

11

4.4

41A, 35A, 57A

WP2uvrA

2AA

15.0 μg

350

18

15.9

332A, 352A, 367A

Key to Positive Controls

Key to Plate Postfix Codes

Key to Automatic Count Flags

2-aminoanthracene

NP = Non-interfering 1= particulate Normal background

 A= Automatic count

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test item did cause a positive mutagenic response with tester strain TA98 in the presence of S9 activation.
Executive summary:

OECD 471 ( 2018): The test substance was tested to evaluate its mutagenic potential using the plate incorporation method by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

Doses selected for the mutagenicity assay were 10.0, 33.3, 100, 333, 1000, 3333 and 5000 μg per plate, based on data generated in BioReliance study AF02PT.502.BTL (detail was not included in the report). The maximum dose of 5000 μg per plate was achieved using a concentration of 100 mg/mL and a 50.0 μL plating aliquot.  All criteria for a valid study were met as described in the protocol.

The test item  in DMSO, at concentrations of 0.188 and 92.4 mg/mL, was stable at room temperature for at least 3.0 hours. No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Precipitate was observed at 5000 μg per plate with all conditions. Toxicity was observed beginning at 5000 μg per plate in TA1535, 3333  in WP2 uvrA with and without S9 and 5000 μg per plate  in TA1537 - S9. Positive mutagenic responses were observed (9.1-fold, maximum increase) with tester strain TA98 in the presence of S9 activation. The dose dependent increases were also outside the historical control limit for this tester strain. The study was concluded to be positive without conducting a confirmatory (independent repeat) assay because the results were clearly positive; hence, no further testing was warranted.