Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse gene mutation assay: Positive (Mutagenic); OECD 471; S. Bruce. (2018)

In vitro cytogenicity/micronucleus in mammalian cells: Negative; OECD 487; A. Dutta 2018

In vitro gene mutation in mammalian cells: Positive; OECD 490; A. Dutta 2016

Link to relevant study records

Referenceopen allclose all

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
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
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
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
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

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

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26th September - 12 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Recognised supplier.
- Suitability of cells: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line has been used successfully in in vitro experiments for many years and is guideline specified (OECD TG 490). The test system has been extensively validated.
- Cell cycle length, doubling time or proliferation index: Doubling time ca. 12 hours. Determined under normal growth conditions.
- Sex, age and number of blood donors if applicable: Not applicable.
- Whether whole blood or separated lymphocytes were used if applicable: Not applicable.
- Number of passages if applicable: Not applicable.
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: The karyotype for the cell line has been published and the modal chromosome number is 40 (ref: OECD TG 490).
- Normal (negative control) cell cycle time: Doubling time ca. 12 hr
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Fnal concentrations of the S9 mix (components) in the test system were S9 (10 μL/mL ), NADP (3 mM) and DL-isocitric acid (17.4 mM)
Test concentrations with justification for top dose:
I. Preliminary toxicity test: 0 (control), 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. Visible precipitate was observed at concentrations ≥500 μg/mL at the beginning and end of treatment.

The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%. The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).

Relative suspension growth (RSG) was 21, 16 and 10% at concentrations of 250 μg/mL (4-hour treatment with S9), 500μg/mL (4-hour treatment without S9) and 250μg/mL (24-hour treatment without S9), respectively. RSG was or approximated 0% at all higher concentrations using all treatment conditions.


II. Main Test: Based upon the results of the preliminary toxicity assay, the concentrations selected for the definitive mutagenicity assay were as indicated.

4-hour without S9: 0 (control), 35, 70, 140, 285, 375 and 500 μg/ml
4-hour with S9: 0 (control), 35, 70, 125, 250 and 285 μg/ml
24-hour without S9: 0 (control), 35, 70, 140, 188 and 250 μg/ml
Vehicle / solvent:
DMF was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMF at a concentration of approximately 500 mg/mL with sonication at 37.0°C for 5 minutes in the solubility test conducted at BioReliance.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMF
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:

- Number of cultures per concentration (single, duplicate, triplicate): Duplicate

- Number of independent experiments: Preliminary and main experiment



METHOD OF TREATMENT/ EXPOSURE:

- Cell density at seeding (if applicable):1 x 10^6 cells/100 mm plate
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk:1 x 10^6 cells/100 mm plate

TREATMENT AND HARVEST SCHEDULE:

- Preincubation period, if applicable: incubation at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air
- Exposure duration/duration of treatment:For 10 to 11 days.
- Harvest time after the end of treatment (sampling/recovery times):


 FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):10 to 11 days.
- Selection time (if incubation with a selective agent):

- Fixation time (start of exposure up to fixation or harvest of cells):

- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:

- Criteria for small (slow growing) and large (fast growing) colonies:



METHODS FOR MEASUREMENT OF CYTOTOXICITY

- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other:

- Any supplementary information relevant to cytotoxicity:



METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:
Rationale for test conditions:
The rationale for selection of concentrations was based on preliminary toxicity testing and number of cultures was in accordance with the guideline (duplicate). Optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved.
Evaluation criteria:
In evaluation of the data, increases in induced mutant frequency which occurred only at highly toxic concentrations (i.e., less than 10% total growth) were not considered biologically relevant. All conclusions were based on scientific judgment; however, the following criteria are presented as a guide to interpretation of the data (Moore et al., 2006).
•A result was considered positive if a concentration-related increase in mutant frequency was observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibited induced mutant frequencies of ≥90 mutants/106 clonable cells (based on the average mutant frequency of duplicate cultures). If the average vehicle control mutant frequency was >90 mutants/106 clonable cells, a doubling of mutant frequency over the vehicle would also be required (Mitchell et al., 1997).
•A result was considered negative if the treated cultures exhibited induced mutant frequencies of less than 90 mutants/106 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.
There are some situations in which a chemical would be considered negative when there was no culture showing between 10 to 20% survival (Office of Food Additive Safety, 2001).
•There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants/106) in a series of data points within 100 to 20% survival and there was at least one negative data point between 20 and 25% survival.
•There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants/106) in a series of data points between 100 to 25% survival and there was also a negative data point between 10 and 1% survival. In this case, it would be acceptable to count the TFT colonies of cultures exhibiting <10% total growth.
Statistics:
Not stated
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
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:
Visible precipitate was observed at concentration 500 μg/mL at the beginning and end of treatment. The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).
No increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed 4-hour treatment without S9 and 24-hour treatment without S9 treatment conditions. Increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed at concentration 285 μg/mL using a 4-hour treatment with S9.
Remarks on result:
other: Mutagenic with metabolic activation

Table 2. Summary Result for TX16797 Definitive Mutagenicity Assay

Metabolic Activation

Dose Level μg/mL

& Susp. Growth

VC Colonies Plate counts (mean)

TFT Colonies Plate counts (mean)

Total Mutant Frequency (per 106cells)

Induced Mutant Frequency (per 106cells)

Relative Total Growth

(4-Hour Exposure –S9)

 

Solvent A

100

151

42

55

N/A

100

Solvent B

169

35

41

UTC A

100

163

31

39

-10

101

UTC B

101

152

32

42

-6

96

35 A

95

197

39

40

-9

117

35 B

93

181

28

31

-17

105

70 A

91

179

32

36

-12

102

70 B

100

186

26

28

-20

116

140 A

92

156

24

30

-18

90

140 B

83

203

32

32

-16

105

285 A

70

165

26

31

-17

72

285 B

68

178

34

38

-10

75

375 A

37

****

****

****

****

****

375 B

44

****

****

****

****

****

500 A

23P

188

48

51

3

27

500 B

33P

150

36

48

0

31

Positive Control methyl methansulfonate μg/mL

 20

37

47

132

567

519

11

15

47

54

153

568

520

16

Mean Solvent Suspension growth: 32.3, Mean Solvent Cloning Efficiency: 80%, Mean Solvent Mutant Frequency: 48 (per 106cells)

(4-Hour Exposure +S9)

 

Solvent A

100

151

44

58

 

 

Solvent B

162

33

40

UTC A

101

175

26

30

-19

113

UTC B

98

164

37

45

-4

102

35 A

98

135

32

48

-1

84

35 B

99

166

42

51

1

106

70 A

87

124

37

60

11

69

70 B

86

159

48

60

11

88

125 A

67

181

68

75

26

77

125 B

74

173

48

55

6

82

250 A

28

160

103

129

80

28

250 B

29

150

77

103

54

28

285 A

19

145

116

160

111

18

285 B

26

143

97

136

87

23

Positive Control 7,12-dimethylbenz(a)anthracene (DMBA) μg/mL

1.5

41

128

259

405

356

34

1

60

139

236

340

291

53

Mean Solvent Suspension growth: 30.9, Mean Solvent Cloning Efficiency: 78%, Mean Solvent Mutant Frequency: 49 (per 106cells)

(24-Hour Exposure –S9)

 

Solvent A

100

158

37

47

N/A

100

Solvent B

161

39

48

UTC A

133

192

29

30

-17

161

UTC B

147

205

29

29

-19

188

35 A

164

157

32

41

-6

162

35 B

170

135

30

45

-3

144

70 A

144

160

40

50

2

145

70 B

134

155

45

58

10

130

140 A

71

120

44

73

25

54

140 B

65

154

47

61

13

63

188 A

48

157

49

63

15

47

188 B

44

161

46

87

10

45

250 A

21

154

63

82

34

21

250 B

27

139

47

68

20

23

Positive control: Methyl Methanesulfonate (MMS) μg/mL

7.5

46

54

222

817

770

16

5

64

96

224

466

418

39

Mean Solvent Suspension growth: 30.5, Mean Solvent Cloning Efficiency: 80%, Mean Solvent Mutant Frequency: 48 (per 106cells)

Solvent = DMF

A & B are duplicate cultures

P= Precipitation observed at the end of treatment

**** = Discarded prior to cloning

Conclusions:
Under the conditions of the assay described in this report, TX16797 was concluded to be positive for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.
Executive summary:

OECD 490 (2016): The test substance, TX16797 α-phenyl-1H-benzimidazole-2-methanol , was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system. Dimethylformamide (DMF) was used as the vehicle with test concentration of 35, 70, 125, 250 and 285 μg/mL (4-hour treatment with S9), 35, 70, 140, 285, 375 and 500 μg/mL (4-hour treatment without S9) and 35, 70, 140, 188 and 250 μg/mL (24-hour treatment without S9).  These doses were selected based on  the preliminary toxicity assay test at concentrations range of 7.81 - 2000 μg/mL  where precipitation was observed at concentrations ≥500 μg/mL. Relative suspension growth (RSG) was 21, 16 and 10% at concentrations of 250 μg/mL (4-hour treatment with S9), 500 μg/mL (4-hour treatment without S9) and 250 μg/mL (24-hour treatment without S9), respectively. RSG was or approximated 0% at all higher concentrations using all treatment conditions.

In the main study, precipitation was observed  at concentration 500 μg/mL at the beginning and end of treatment. Cultures treated at all concentrations of 4-hour treatment with S9, 35, 70, 140, 285 and 500 μg/mL (4-hour treatment without S9) and all concentrations 24-hour treatment without S9 exhibited 19 to 101%, 23 to 101% and 21 to 170% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 18 to 113% (4-hour treatment with S9), 27 to 117% (4-hour treatment without S9) and 21 to 188% (24-hour treatment without S9). No increases in induced mutant frequency ≥90mutants/106 clonable cells were observed 4-hour treatment without S9 and 24-hour treatment without S9 treatment conditions. Increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed at concentration 285 μg/mL using a 4-hour treatment with S9.

Under the conditions of the assay described in this report, TX16797 was concluded to be positive for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Nov 2017 - 02 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
26 September 2014
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Not applicable (in vitro micronucleus test of: clastogenic and aneugenic potential).
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
The TK6 cell line was obtained from the American Type Culture Collection (respiratory number CRL-8015, lot#: 58078532), Manassas, VA. The cell line is a lymphocytic cell line of human origin with a doubling time of 12-14 hours.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Lot No. 3784, Exp. Date: 18 Apr 20189
Test concentrations with justification for top dose:
The maximum recommended dose level was 450 µg/mL, limited by cytotoxicity of the test item observed in preliminary test. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).


Micronucleus assay:
4(23)-hour without S9: 50, 200, 300, 350, 375, 400, 425, 450 and MMC
4(23)-hour with S9: 50, 150, 350, 370, 380, 390, 400, 425, 450 and CP
27-hour without S9: 5, 50, 130, 140, 150, 160, 175, 200 and VB

where:
* = dose levels selected for analysis of MN frequency in binucleate cells
CP = Cyclophosphamide
VP = Vinblastine
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMF
- Justification for choice of solvent/vehicle: DMF was the vehicle of choice based on the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, upon sonication for 5 minutes at 37.0 °C, the test substance was soluble in DMF at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.
Applicant assessment indicates: solubility test conducted at BioReliance using the suitability of DMF.
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Vinblastine Sulfate -
Evaluation criteria:
The test substance was considered to have induced a positive response if
* at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and 

* the increase was concentration-related (p ≤ 0.05), and 

* results were outside the 95% control limit of the historical negative control data. 
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met. 

Statistics:
Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.
Key result
Species / strain:
human lymphoblastoid cells (TK6)
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:
The results of the analysis indicate that the actual mean concentrations of the analyzed formulation samples, 0.5 and 45 mg/mL, were 100.4 and 98.3% of target, respectively, with S/L ratios of > 0.925. This indicates that the formulations were accurately prepared. No test substance was detected in the vehicle control sample. Additionally, TX16797 in DMF, at concentrations of 0.502 and 44.3 mg/mL, was stable at room temperature for at least 3.0 hours.
The test substance was soluble in the treatment medium at all doses tested at the beginning and conclusion of the treatment period.
The osmolality of the test substance dose in treatment medium was considered acceptable. The pH of the highest dose of test substance in treatment medium was 7.5.
The results of cytotoxicity and micronucleus data for the untreated controls were comparable to that of the vehicle control. Therefore, use of DMF as the vehicle control in this assay was justified.
The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.

Table 1: Micronucleus analysis of TK6 Cells Treated with TX16797 in absence and abseces of metabolic activation.

Metabolic activation

Treatment Condition (μg/mL)

 

Replicate Culture

Cell Density (105/mL)

 

Total cells (106/Tube)

PD1

 

RPD2

 

Cytotoxicity (%)

 

Total# of cells Counted

% Micronucleated Cell/Culture

Average % Micronucleated Cell/Culture

1

2

4-hr

Without

S9

Untreated

A

4.39

4.43

2.21

1.7

100

-

1000

0.80

0.70

B

3.95

3.99

1.99

1000

0.60

DMF

A

3.78

3.58

1.84

1.5

89

113

1000

0.70

0.75

B

3.61

3.62

1.81

1000

0.80

50

A

3.67

3.55

1.81

1.5

98

24 

1000

0.70

0.75

B

3.47

3.52

1.75

1000

0.80

200

A

2.79

2.69

1.37

1.1

72

284

1000

0.60

0.75

B

2.69

2.66

1.34

1000

0.90

300

A

2.33

2.31

1.16

0.9

59

414

 

B

2.43

2.32

1.19

350

A

2.39

2.31

1.18

0.9

58

424

B

2.29

2.34

1.16

375

A

2.47

2.33

1.20

0.9

58

424

B

2.30

2.21

1.13

400

A

2.21

2.10

1.08

0.7

42

584

1000

0.90

0.8

B

1.78

1.77

0.89

1000

0.70

425

A

1.69

1.73

0.86

0.6

36

644

 

B

1.97

1.95

0.98

450

A

1.62

1.59

0.80

0.4

23

774

B

1.59

1.62

0.80

4-hr

With

S9

Untreated

A

3.79

3.69

1.87

1.6

100

-

1000

0.70

0.65

B

3.67

3.75

1.86

1000

0.60

DMF

A

3.60

3.49

1.77

1.6

100

03

1000

0.70

0.75

B

3.91

3.85

1.94

1000

0.80

50

A

3.60

3.64

1.81

1.5

96

44

1000

0.60

0.65

B

3.45

3.47

1.72

1000

0.70

150

A

3.25

3.26

1.63

1.3

84

164 

1000

0.70

0.65

B

2.98

2.96

1.49

1000

0.60

350

A

2.40

2.34

1.19

0.9

60

404

 

B

2.45

2.44

1.22

370

A

2.24

2.23

1.12

1.0

61

394

B

2.75

2.49

1.31

380

A

2.32

2.33

1.16

0.9

56

444

B

2.30

2.29

1.15

390

A

2.21

2.19

1.10

0.8

53

474

B

2.30

2.24

1.14

400

A

2.31

2.09

1.10

0.8

49

514

B

2.04

2.05

1.02

425

A

2.19

2.09

1.07

0.7

47

534

1000

0.80

0.75

B

2.01

2.01

1.01

1000

0.70

450

A

1.86

1.77

0.91

0.5

32

684

 

B

1.75

1.74

0.87

CP, 2.5

A

2.38

2.31

1.17

1.0

62

384

B

2.57

2.56

1.28

CP, 3

A

2.13

2.14

1.07

0.9

57

434

B

2.47

2.53

1.25

CP, 4

A

2.22

2.25

1.12

0.8

50

504

1000

2.70

2.50**

B

2.06

2.07

1.03

1000

2.30

27-hr

Without

S9

Untreated

A

7.62

7.53

3.79

1.7

100

-

1000

0.70

0.70

B

8.30

8.34

4.16

1000

0.70

DMF

A

7.73

7.58

3.83

1.6

96

43

1000

0.80

0.75

B

7.56

7.59

3.79

1000

0.70

5

A

7.01

7.10

3.53

1.5

94

64

1000

0.90

0.85

B

7.25

7.23

3.62

1000

0.80

50

A

5.77

5.75

2.88

1.2

76

244

1000

1.00

0.85

B

5.84

5.88

2.93

1000

0.70

130

A

3.98

4.06

2.01

0.8

47

534

1000

0.90

0.75

B

4.42

4.41

2.21

1000

0.60

140

A

3.89

3.76

1.91

0.6

36

644

 

B

3.75

3.60

1.84

150

A

3.28

3.23

1.63

0.5

30

704 

B

3.78

3.69

1.87

160

A

3.30

3.28

1.65

0.4

27

734

B

3.49

3.41

1.73

175

A

3.20

3.19

1.60

0.3

21

794

B

3.10

3.15

1.56

200

A

3.05

3.01

1.52

0.3

16

844 

B

2.98

2.89

1.47

VB, 10 ng/mL

A

7.65

7.57

3.81

1.6

100

04

B

7.69

7.71

3.85

VB, 12 ng/mL

A

4.89

4.84

2.43

1.0

61

394

1000

7.50

7.90**

B

5.07

4.99

2.52

1000

8.30

1 PD= Population Doubling


2 Relative PD to vehicle control

3 Relative to untreated control


4 Relative to vehicle control

** p ≤ 0.01, Fisher's exact test, relative to the solvent control.

VB = Vinblastine Sulfate


CP = Cyclophosphamide

Conclusions:
Under the conditions of this study the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in the presence and absence of S9 activation.
Executive summary:

OECD TG 487- 2016: The study was conducted under GLP conditions to assess the detection of the clastogenic and aneugenic potential of the test item on the nuclei of TK6 cells. The target cells were treated for 4 hours in the absence and presence of S9, and for 27 hours in the absence of S9. N,N-Dimethylformamide (DMF) was used as the vehicle.

Following preliminary study, the doses tested in main test ranged from 50 to 450 μg per plate in the non-activated and S9-activated 4-hour exposure groups and from 5 to 200 μg/mL in the non-activated 27-hour exposure group. Cytotoxicity was observed at doses ≥ 400 μg/mL in the non-activated and S9-activated 4-hour exposure groups, and at doses ≥ 130 μg/mL in the non-activated 27-hour exposure group.

The doses selected for microscopic evaluation were 50, 200, and 400 μg/mL in the non-activated 4-hour exposure group, 50, 150, and 425 μg/mL in the S9-activated 4-hour exposure group, and 5, 50, and 130 μg/mL in the non-activated 27-hour exposure group.

No significant or dose-dependent increases in micronuclei induction were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

Under the conditions of the assay, the test tem was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using TK6 cells.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo Mammalian Alkaline Comet Assay: Negative; OECD 489; A. Morris 2018

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
In vivo Mammalian Alkaline Comet Assay - incorporated into the study design of OECD 422 Combined 28-day Repeated Dose Toxicity Study and Reproductive/Developmental Screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June 2018 to TBC
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29th July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals No 422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test and genotoxicity screening"
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Remarks:
RccHan™:WIST
Details on species / strain selection:
Rat - Wistar Han™
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Ltd., Oxon, UK
- Age at study initiation: Male 8 - 11 weeks Female: 13-14 weeks
- Weight at study initiation: Not reported.
- Assigned to test groups randomly: assigned dose groups using a randomization procedure based on body weight at approximately five days prior to the start of treatment
- Fasting period before study: No
- Housing: Individually in wooden chew blocks and cardboard fun tunnels
- Diet (e.g. ad libitum): Rodent 2018C Teklad Global Certified Pelleted Diet
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least fourteen days to allow exclusion of females not showing appropriate estrous cycling.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C)
- Humidity (%): 30-70%
- Air changes (per hr): At least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light)

IN-LIFE DATES: tbc
Route of administration:
oral: gavage
Vehicle:
PEG 400 - more details to follow

- Vehicle(s)/solvent(s) used: PEG 400
- Justification for choice of solvent/vehicle: 

- Concentration of test material in vehicle: 

- Amount of vehicle (if gavage or dermal): 

- Type and concentration of dispersant aid (if powder):

 - Lot/batch no. (if required):

 - Purity:
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Oral gavage
Five males from each dose group (Groups 1 to 4) were dosed once daily with the test item for 45 consecutive days and killed approximately 3 hours after the last dose. Another set of three males were dosed with N-nitroso-N-methylurea (NMU) on Days 44 and 45 only (approximately 27 and 3 hours before euthanasia) and used as positive controls.
Duration of treatment / exposure:
45 days
Frequency of treatment:
Daily
Post exposure period:
N/A
Tissues and cell types examined:
Liver, glandular stomach and jejunum were assessed
Details of tissue and slide preparation:
-CRITERIA FOR DOSE SELECTION:
Dose selection was based on the dose range finder study where the animals were exposed to dose range of 500, 750 and 1000 mg/kg bw/day at a treatment volume of 4 mL/kg. Based on the observations results of the study, it was considered that dose levels of 0 (control), 100, 350 and 750 mg/kg bw/day, utilizing a dose volume of 6 mL/kg, would be appropriate for further investigation of effects of the Test Item on reproduction or toxicity.

- TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The tissue samples were processed under subdued lighting and over ice to provide single cell suspensions, providing sufficient cells for scoring for the comet assay.

A small section of the glandular stomach was immersed in stomach buffer and incubated for approximately 15 minutes on ice. The mucosal layer of the glandular stomach was removed by gentle scraping and then a single cell suspension was obtained by scraping the remaining tissue into a small volume of stomach buffer

Approximately a 2 cm piece of Jejunum was processed and immersed briefly in stomach buffer and then scraped gently to remove any contents, incubated in stomach buffer (approximately 10 mL), on ice for 5 to 10 minutes. A single cell suspension was obtained by gentle scraping into approximately 1 mL of fresh liver buffer.

A small piece of liver (approximately 1 cm3) was washed in liver buffer, before being minced and filtered to provide a single cell suspension.

- DETAILS OF SLIDE PREPARATION:
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. 
Approximately 30 µL of the cell suspension was added to 270 µL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 µL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension mix was immediately covered with a glass coverslip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes in order to solidify. Once the LMP agarose had set, the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. Following lysis, the slides were removed from the solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis bath filled with chilled electrophoresis buffer (pH>13) at low temperature (2-10 °C). The slides were then left for 20 minutes after which they were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The slides were removed and placed on to a draining surface and drop wise coated with a neutralization buffer and left for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer was performed twice. The slides were further drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry, after which they were stored prior to staining and scoring.
For each tissue, two of the four processed slide gels were scored (A and B replicates) and the remaining two slides were stored as backup slides (C and D replicates).

METHOD OF ANALYSIS:
The processed Comet slides were coded to allow “blind” scoring using a computer generated code and stained just prior to analysis for comets. To each dry slide gel, 75 µL of propidium iodide (20 µg/mL) was placed on top of the slide and overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA, the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PC-based image analysis program (Comet IV version 4.3.1). Two slide gels for each tissue for each animal were scored with a maximum of 75 cells per slide gel giving an accumulative total of 150 cells per tissue per animal. The slide score data for each tissue was processed using the Excel macro program provided in Comet IV version 4.3.1

 OTHER: N/A
Evaluation criteria:
Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly
Negative if:
•None of the test concentrations exhibits a significant increase compared with the concurrent negative control.
•There is no evidence of a dose-related response.
•The results are within the laboratory historical vehicle control range.
•There is evidence, direct or indirect, to demonstrate exposure or toxicity to the target tissue has been achieved.

The test item is then considered unable to induce DNA strand breakage in the tissues studied in the test system.

Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly
Positive if:
•At least one of the test doses exhibits a statistically significant increase compared to the concurrent negative control.
•The response is considered to be dose-related.
•The results are substantially outside the laboratory historical vehicle control range.

The test item can be considered to induce DNA strand breakage in a particular tissue if all three conditions are met.

There is no requirement for verification of a clearly positive or negative response
Statistics:
Where considered appropriate, statistical analysis was performed on the mean % Tail Intensity and median % Tail Intensity data using a Students t-test on transformed data using a √(x+1) transformation.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Table 2. Comet Assay - Summary of Group Data on Glandular stomach

Dose Level

Group Mean % Hedgehogs

Group Mean % Tail Intensity

Group Mean of Median % Tail Intensity

Group Mean Standard Deviation of % Tail Intensity

Vehicle

0

1.44

0.47

2.25

Low

0

1.30

0.40

2.09

Intermediate

0.09

2.81

1.86

2.97

High

0

1.45

0.55

2.00

Positive (MNU)

20.54

38.26***

35.93***

14.39

Table 2b. Comet Assay - Summary of Group Data on Jejunum

Dose Level

Group Mean % Hedgehogs

Group Mean % Tail Intensity

Group Mean of Median % Tail Intensity

Group Mean Standard Deviation of % Tail Intensity

Vehicle

0

2.30

1.31

2.64

Low

0.10

2.17

1.21

5.69

Intermediate

0

2.19

1.24

2.56

High

0

1.52

0.62

2.15

Positive (MNU)

13.78

34.71***

32.42***

14.99

Table 2c. Comet Assay - Summary of Group Data on Liver

Dose Level

Group Mean % Hedgehogs

Group Mean % Tail Intensity

Group Mean of Median % Tail Intensity

Group Mean Standard Deviation of % Tail Intensity

Vehicle

0

0.24

0.01

0.72

Low

0

0.29

0.01

0.88

Intermediate

0

0.40

0.02

1.04

High

0

0.34

0.02

0.97

Positive (MNU)

2.80

45.30***

46.07***

10.09

MNU = N-nitroso-N-methylurea

 *** = P<0.001

Conclusions:
Under the condition of the study, the test item did not demonstrate any significant increases in the percentage tail intensity or median percentage tail intensity of the jejunum, glandular stomach and liver and was considered to not induce DNA damage in these tissues. The test item does not meet the criteria for classfication for mutagenicity in accordance with the Regulation (EC) 1272/2008.
Executive summary:

In comet assay assessment OECD 489 - 2019: Five males from each dose group (Groups 1 to 4) were dosed once daily with the test item at concentration of 0, 100, 350 & 750 mg/kg bw/day for 45 consecutive days and killed approximately 3 hours after the last dose.  Another set of three males were dosed with N-nitroso-N-methylurea (NMU) on Days 44 and 45 only (approximately 27 and 3 hours before euthanasia) and used as positive controls.

Samples of liver, glandular stomach and jejunum were taken at necropsy from these males and transferred to the Cell and Molecular Sciences Department and processed to provide slides for the comet assay.  

The vehicle control group induced percentage tail intensities which were consistent with the current laboratory historical control data ranges.  The positive control item (MNU) produced a statistically significant increase in the percentage tail intensity and median percentage tail intensity in the liver, glandular stomach and jejunum indicating that the test method itself was operating as expected and was considered to be valid under the conditions of the test.

The glandular stomach did not demonstrate any marked increases in the percentage tail intensity or the median percentage tail intensity over the vehicle control. One individual slide, the ‘B’ replicate slide for animal 56 in the intermediate dose group had a  percentage tail intensity which was much greater than the remaining slides in the group and it exceeded the upper limit of the laboratory historical control range for a vehicle.  The reason for this is unclear since the ‘A’ replicate of the same animal and tissue had percentage tail intensities which were consistent with the rest of the group.  However, since the group as a whole had no statistically significant increases in percentage tail intensity, the increase observed in one slide is considered to be artefactual and of no significance in the overall result.  Therefore, it is considered that the test item did not induce DNA damage in the glandular stomach tissue investigated under the conditions of the test.  There were no dose-related increases in the percentage hedgehog frequencies in the dose groups of the glandular stomach.  There were no significant increases in the percentage mean tail intensity or median tail intensity in the jejunum or liver for any of the test item dose groups when compared to the vehicle control.  The incidence of hedgehog cells in both tissues, however, in the jejunum was lower than minimum of the historical control range for a vehicle but this was seen in both the vehicle and the test item dose groups and was therefore considered to be of no consequence.

Under the condition of the study, the test item did not demonstrate any significant increases in the percentage tail intensity or median percentage tail intensity of the jejunum, glandular stomach and liver and was considered to not induce DNA damage in these tissues. The item does not meet the criteria for classification for mutagenicity in accordance with the Regulation (EC) 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No mode of action analysis is required since the susbtance was not mutagenic in vivo.

Additional information

OECD 471 (2018): The test substance was tested at doses of 10.0, 33.3, 100, 333, 1000, 3333 and 5000 μg per plate in Dimethyl sulfoxide (DMSO)  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. Positive mutagenic responses were observed (9.1-fold, maximum increase) with tester strain TA98 in the presence of S9 activation.

OECD 490 (2016): The test substance was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system. Dimethylformamide (DMF) was used as the vehicle with test concentration of 35, 70, 125, 250 and 285 μg/mL (4-hour treatment with S9), 35, 70, 140, 285, 375 and 500 μg/mL (4-hour treatment without S9) and 35, 70, 140, 188 and 250 μg/mL (24-hour treatment without S9). Increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed at concentration 285 μg/mL using a 4-hour treatment with S9.  Under the conditions of the assay described in this report, TX16797 was concluded to be positive for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

OECD TG 487 (2016): The study was conducted under GLP conditions to assess the detection of the clastogenic and aneugenic potential of the test item on the nuclei of TK6 cells. The target cells were treated for 4 hours in the absence and presence of S9, and for 27 hours in the absence of S9. Dimethylformamide (DMF) was used as the vehicle. Based on cytotoxicity the doses selected for microscopic evaluation were 50, 200, and 400 μg/mL in the non-activated 4-hour exposure group, 50, 150, and 425 μg/mL in the S9-activated 4-hour exposure group, and 5, 50, and 130 μg/mL in the non-activated 27-hour exposure group. No significant or dose-dependent increases in micronuclei induction were observed in all treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). Under the conditions of the assay was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using TK6 cells.

In comet assay assessment OECD 489 (2019): Five males from each dose group (Groups 1 to 4) were dosed once daily with the test item at concentration of 0, 100, 350 & 750 mg/kg bw/day for 45 consecutive days and killed approximately 3 hours after the last dose.  Another set of three males were dosed with N-nitroso-N-methylurea (NMU) on Days 44 and 45 only (approximately 27 and 3 hours before euthanasia) and used as positive controls. Samples of liver, glandular stomach and jejunum were taken at necropsy from these males and transferred to the Cell and Molecular Sciences Department and processed to provide slides for the comet assay.   There were no significant increases in the percentage mean tail intensity or median tail intensity in all tissues assessed in all of the tested groups. There were no dose-related increases in the percentage hedgehog frequencies in all dose groups, however, in the jejunum it exceeded the upper end of the historical control range for a vehicle but this was seen in both the vehicle and the test item dose groups and was therefore considered to be of no consequence.  Under the condition of the study, there were no significant increases in the percentage tail intensity or median percentage tail intensity for all tested tissues in all treatment groups. Furthermore, there were not effects on germ cell tissues such as ovaries, testis and there was no effect on post-implantation observed in this comet assay combined with the OECD 422. It can be concluded that the substance is not mutagenic on both somatic and germ cells. The item does not meet the criteria for classification for mutagenicity in accordance with the Regulation (EC) 1272/2008.

Justification for classification or non-classification

The item does not meet the criteria for classification for mutagenicity in accordance with the Regulation (EC) 1272/2008.