Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro bacterial reverse mutation assay (Ames test) according to OECD TG 471, the test item did not induce gene mutations in the tester strains used. Therefore, the test item is considered as non-mutagenic.

In an in vitro chromosome aberration assay in the mammalian cell line V79 according to OECD TG 473, the test item did not show clastogenic properties.

In an in vitro mammalian cell forward mutation assay (HPRT) according to OECD TG 476 performed with the structurally similar substance EPOTEC R 101, the test item showed no mutagenic potential.

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:
from 2020-03-10 to 2020-03-19
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:
1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
440/2008/EC, 30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998-08
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use
Version / remarks:
2011-11
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
his/trp
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:
Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
Test concentrations with justification for top dose:
16.0, 50.0, 160.0, 500.0, 1600.0, 5000.0 μg/plate, recommended maximum test concentration
Vehicle / solvent:
- Solvent used: ultrapure water (ASTM Type 1)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine (NPD)
Remarks:
without S9 mix, 4 μg/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Remarks without S9 mix, 2 μg/plate for TA100 and TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix, 50 μg/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix, 2 μL/plate for E.coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
with S9 mix, 2 μg/plate for all TA strains, 50 μg/plate for E.coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: nutrient agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: colony and background lawn development
Evaluation criteria:
Evaluation of Experimental Data
The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined (counted manually, evaluated by unaided eye), the mean values, standard deviations and the mutation rates were calculated.
Mutation Rate = (Mean revertants at the test item (or control*) treatments) / mean revertants of vehicle control
* untreated, vehicle or positive control

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces 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.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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 nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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 nor precipitates, but tested up to recommended limit concentrations
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1:Summary Table of the Results of the Initial Mutation Test (Plate Incorporation Test)

Concentrations (μg/plate)

Salmonella typhimuriumtester strains

Escherichia coli WP2 uvrA

 TA98

TA100 

TA1535 

TA1537 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean Values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

21.0

0.89

22.7

0.84

61.7

0.88

79.7

0.94

11.0

1.03

10.0

1.07

6.0

1.06

5.0

1.00

27.3

0.89

42.3

0.86

DMSO Control

26.3

1.00

23.3

1.00

99.3

1.00

10.0

1.00

5.0

1.00

4.7

1.00

41.7

1.00

Ultrapure Water Control

23.7

1.00

27.0

1.00

70.0

1.00

84.3

1.00

10.7

1.00

9.3

1.00

5.7

1.00

5.0

1.00

30.7

1.00

49.3

1.00

5000

26.3

1.11

27.0

1.00

57.0

0.81

89.0

1.06

10.7

1.00

9.0

0.96

6.7

1.18

6.0

1.20

38.3

1.25

36.3

0.74

1600

27.0

1.14

28.3

1.05

55.3

0.79

90.0

1.07

8.7

0.81

12.3

1.32

6.3

1.12

8.0

1.60

42.0

1.37

51.7

1.05

500

25.3

1.07

18.7

0.69

56.0

0.80

88.7

1.05

8.0

0.75

10.7

1.14

4.7

0.82

4.7

0.93

45.3

1.48

49.7

1.01

160

27.0

1.14

25.3

0.94

70.3

1.00

78.7

0.93

8.0

0.75

10.7

1.14

4.3

0.76

6.0

1.20

38.3

1.25

46.3

0.94

50

20.7

0.87

27.0

1.00

61.0

0.87

88.7

1.05

9.7

0.91

10.3

1.11

4.3

0.76

5.7

1.13

35.3

1.15

40.7

0.82

16

23.3

0.99

24.3

0.90

69.7

1.00

90.3

1.07

8.0

0.75

9.7

1.04

8.0

1.41

5.7

1.13

37.3

1.22

44.7

0.91

NPD (4 μg)

377.7

14.34

SAZ (2 μg)

654.3

9.35

956.0

89.63

9AA (50 μg)

204.0

40.80

MMS (2 μL)

629.7

20.53

2AA (2 μg)

1293.3

55.43

1130.7

11.38

141.7

14.17

98.3

21.07

2AA (50 μg)

167.3

4.02

Table 2: Summary Table of the Results of the Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (μg/plate)

Salmonella typhimuriumtester strains

Escherichia coli WP2 uvrA

 

 

 

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean Values of

revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

24.3

0.91

28.0

0.70

80.7

0.97

90.3

0.85

6.3

0.70

10.7

1.00

6.7

1.05

5.3

1.14

19.3

0.69

27.3

0.79

DMSO Control

20.0

1.00

26.0

1.00

88.3

1.00

10.7

1.00

6.0

1.00

6.7

1.00

45.0

1.00

Ultrapure Water Control

26.7

1.00

40.0

1.00

83.3

1.00

106.0

1.00

9.0

1.00

10.7

1.00

6.3

1.00

4.7

1.00

28.0

1.00

34.7

1.00

5000

9.7

0.36

9.7

0.24

32.3

0.39

57.7

0.54

9.7

1.07

10.0

0.94

2.3

0.37

6.3

1.36

14.0

0.50

19.0

0.55

1600

7.7

0.29

32.7

0.82

36.7

0.44

114.0

1.08

8.3

0.93

9.3

0.88

2.3

0.37

6.3

1.36

27.3

0.98

45.7

1.32

500

17.7

0.66

28.0

0.70

55.0

0.66

120.3

1.14

10.7

1.19

12.0

1.13

7.7

1.21

3.7

0.79

41.3

1.48

50.0

1.44

160

23.7

0.89

29.7

0.74

83.7

1.00

113.0

1.07

7.0

0.78

8.7

0.81

8.3

1.32

3.7

0.79

42.3

1.51

53.0

1.53

50

25.3

0.95

29.0

0.73

83.3

1.00

108.3

1.02

10.7

1.19

10.7

1.00

5.3

0.84

6.7

1.43

39.0

1.39

36.3

1.05

16

27.7

1.04

23.0

0.58

85.3

1.02

117.0

1.10

8.3

0.93

11.0

1.03

5.3

0.84

6.3

1.36

45.0

1.61

61.3

1.77

NPD (4 μg)

281.3

14.07

SAZ (2 μg)

552.0

6.62

777.3

86.37

9AA (50 μg)

799.3

133.22

MMS (2 μL)

1312.0

46.86

2AA (2 μg)

2044.0

78.62

1375.3

15.57

110.0

10.31

153.3

23.00

2AA (50 μg)

211.0

4.69

Conclusions:
In an bacterial reverese mutation assay (AMES) according to OECD Guideline 471, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used.
Executive summary:

The mutagenic potential of the test item was determined in an in vitro bacterial reverse mutation assay (AMES) according to OECD Guideline 471. Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations (16, 50, 160, 500, 1600, 5000 µg/plate), including the controls, were tested in triplicate (positive and negative controls were run concurrently). Negative, vehicle and positive controls were valid. No cytotoxicity was observed up to the max. concentration. No precipitation was observed throughout the study. No substantial increases were observed in revertant colony numbers of any of the five tester strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to Section 13 for Read Across Justification.
Reason / purpose:
read-across source
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2020-03-03 to 2020-03-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29th July, 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
28 April 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Chinese hamster lung cells (male), ECACC (European Collection of Cell Cultures)
- Suitability of cells:The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations.
- Normal cell cycle time (negative control): These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h).

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 °C in a humidified atmosphere in an incubator, set at 5% CO2. The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%).
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH
- concentration or volume of S9 mix and S9 in the final culture medium : 0.3 mL/ML S9 fraction
Test concentrations with justification for top dose:
Experiment A with 3/20 h treatment/sampling time
without S9 mix: 250, 500, 1000 and 1500 μg/mL test item
with S9 mix: 500, 1000, 2000 and 2500 μg/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 62.5, 125, 250, 500 and 1000 μg/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 62.5, 125, 250, 500 and 1000 μg/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 500, 1000, 2000 and 2500 μg/mL test item
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x 10E5 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment and harvest:
Experiment A with 3/20 h treatment/sampling time
Experiment B with 20/20 h treatment/sampling time
Experiment B with 20/28 h treatment/sampling time
Experiment B with 3/28 h treatment/sampling time

- Spindle inhibitor: Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting
- Methods of slide preparation and staining technique used including the stain used:
Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes free of cytoplasm) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
All slides were independently coded before microscopic analysis and scored blind. 300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration, negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative increase in cell count (RICC)
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.
Both biological and statistical significance should be considered together.
There is no requirement for verification of a clearly positive or negative response.
Statistics:
For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH values of the treatment solutions were similar to the control.
- Data on osmolality: The osmolarity values of the treatment solutions were similar to the control.
- Precipitation and time of the determination: No precipitation of the test item was observed at any of the applied concentrations.

RANGE-FINDING/SCREENING STUDIES:
A pre-test on cytotoxicity was performed as part of this study to establish an appropriate concentration range for the main chromosome aberration assays (experiment A and B), both in the absence and in the presence of a metabolic activation (rodent S9 mix).
See attached results.

STUDY RESULTS

- Results from cytotoxicity measurements:
In both experiments, clear cytotoxicity of 53% and 54 % was observed at the highest concentrations of 1500 and 2500 μg/mL after treatment with the test item for 3 hours in the absence and presence of metabolic activation. After 20-hours treatment with the test item 53 % of cytotoxicity was recorded at concentration of 1000 μg/mL without S9 mix.

- Genotoxicity results
No increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed, neither in the absence nor in the presence of metabolic activation. No polyploid and endoreduplicated metaphases were found after treatment with the different concentrations of the test item. The number of aberrations found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

HISTORICAL CONTROL DATA
- see attached results
Conclusions:
The substance tested up to the maximum recommended concentration without and with mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. Thus, the test item is considered as not clastogenic in this system.
Executive summary:

The test item dissolved in DMSO medium was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:

Experiment Awith 3/20 h treatment/sampling time

without S9 mix:250, 500, 1000 and 1500 μg/mL test item

with S9 mix:500, 1000, 2000 and 2500 μg/mL test item

Experiment Bwith 20/20 h treatment/sampling time

without S9 mix:62.5, 125, 250, 500 and 1000 μg/mL test item

Experiment Bwith 20/28 h treatment/sampling time

without S9 mix:62.5, 125, 250, 500 and 1000 μg/mL test item

Experiment Bwith 3/28 h treatment/sampling time

with S9 mix:500, 1000, 2000 and 2500 μg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).

No precipitation of the test item was observed at any of the applied concentrations. The pH and the osmolality values of the treatment solutions were similar compared to the control.

Clear cytotoxicity of 53% and 54 % were observed at the highest concentrations in all experimental parts.

No relevant increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed, neither in the absence nor in the presence of metabolic activation.

There were no polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The number of aberrations found in the solvent controls was in the range of the laboratory historical control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0μL/mL) and cyclophosphamide (5μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2020-02-11 to 2020-02-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
440/2008/EC, 30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial forward mutation assay
Target gene:
hprt locus located on the X chromosome
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Sub-line (KI) of Chinese hamster ovary cell line CHO, ECACC (European Collection of Cell Cultures)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
The CHO KI cells for this study were grown in Ham's F12 medium (F12-10) supplemented with 1 % of Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 5 hour treatment with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (F12-5). The selection medium for TG resistant mutants contained 3.4 μg/mL of 6-thioguanine (6-TG) (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL).
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH
- concentration or volume of S9 mix and S9 in the final culture medium : The concentration of S9 was 1.5% in medium. The protein concentrations of the S9 batch used in the experiments were 33.8 and 35.5 mg/mL.
Test concentrations with justification for top dose:
5-hour treatment period without S9-mix: 125, 250, 500, 1000 and 2000 μg/mL
5-hour treatment period with S9-mix: 125, 250, 500, 1000 and 2000 μg/mL
In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for lower -cytotoxic substances is 2000 μg/mL (based on the updated OECD Guideline 476 (2016)).
Vehicle / solvent:
- Vehicle used: culture medium

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ham's F12 medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ham's F12 medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x10E6 cells/concentration
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Following the exposure period of 5 hours the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours. After the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution and counted using a Barker chamber. Throughout the expression period; cells were transferred to dishes for growth or diluted to be plated for survival.
- Fixation time (start of exposure up to fixation or harvest of cells): During the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2x10E6 cells were taken on days 1, 3 and 6 and evaluated on day 8.
- If a selective agent is used: At the end of the expression period, cultures from each dose level were adjusted to 2 x 10E5 cells / dish ( 4 x five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 μg/mL of thioguanine (6-TG).
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: After the selection period, the colonies were fixed, stained with Giemsa and counted for mutant selection and cloning efficiency determination.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
• the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Test item is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria were fulfilled, the test item is considered clearly negative because:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are compatible the distribution of the historical negative control data (based 95% control limit).
• The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No relevant changes in pH was found after treatment with the test item.
- Data on osmolality: No relevant changes in osmolality was found after treatment with the test item.
- Precipitation and time of the determination: There was no precipitation observed in the medium at the end of treatment at any concentration tested.

RANGE-FINDING/SCREENING STUDIES
A Pre-test on Toxicity was performed to establish an appropriate concentration range for the main mutation assay, both in the absence and in the presence of metabolic activation (rodent S9-mix). Based upon the toxicity data of the pre-test the following concentrations were chosen for the main mutation assay:
5-hour treatment period without S9-mix:
125, 250, 500, 1000 and 2000 μg/mL
5-hour treatment period with S9-mix:
125, 250, 500, 1000 and 2000 μg/mL

STUDY RESULTS
- see attached results
There were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no significant differences between treatment and control groups and no dose-response relationships were noted. The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by distinct and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with ethyl methanesulfonate (1.0 μL/mL) and 7,12-dimethyl benz[a]anthracene (20 μg/mL).

HISTORICAL CONTROL DATA
- see attached results
All values were within the range of the laboratory historical solvent control data and no dose-related increase was observed in any of the cultures. The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory.
Conclusions:
The substance tested up to the maximum recommended concentration (2000 μg/mL) without and with metabolic activation system over a 5 hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control). It is concluded that the test item, was not mutagenic in this in vitro mammalian cell gene mutation test performed in Chinese hamster ovary cells.
Executive summary:

The test item was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according OECD TG 476. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital andβ-naphthoflavone induced rat liver and solubility of test item.

 

5-hour treatment period without S9-mix:

125, 250, 500, 1000 and 2000μg/mL

5-hour treatment period with S9-mix:

125, 250, 500, 1000 and 2000μg/mL

 

In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for lower -cytotoxic substances is 2000 μg/mL (based on the updated OECD Guideline 476 (2016)).

Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, neither in the absence nor in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups compared to the concurrent and historical control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical control data.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in the osmolality of the test system were noted at the different dose levels tested. The measured pH of treatment solution was similar compared to the control values.

The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0μL/mL) and 7,12-Dimethyl benzanthracene (20μg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.The substance tested up to the maximum recommended concentration (2000μg/mL) without and with metabolic activation system over a 5 hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control). It is concluded that the test item, was not mutagenic in this in vitro mammalian cell gene mutation test performed in Chinese hamster ovary cells.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames Test

The mutagenic potential of the test item was determined in an in vitro bacterial reverse mutation assay (AMES) according to OECD Guideline 471. Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations (16, 50, 160, 500, 1600, 5000 µg/plate), including the controls, were tested in triplicate (positive and negative controls were run concurrently). Negative, vehicle and positive controls were valid. No cytotoxicity was observed up to the max. concentration. No precipitation was observed throughout the study. No substantial increases were observed in revertant colony numbers of any of the five tester strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

HPRT Test

For the target substance no Mammalian Gene Mutation Test is available. However, a study according OECD 476 with the structurally similar source substance EPOTEC R 101 is available. In this study the following concentrations have been selected for the main test:

5-hour treatment period without S9-mix:

125, 250, 500, 1000 and 2000 μg/mL

5-hour treatment period with S9-mix:

125, 250, 500, 1000 and 2000 μg/mL

In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for lower -cytotoxic substances is 2000 μg/mL. Phenotypic expression was evaluated up to 8 days following exposure. In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, neither in the absence nor in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups compared to the concurrent and historical control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical control data. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in the osmolality of the test system were noted at the different dose levels tested. The measured pH of treatment solution was similar compared to the control values. The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 μL/mL) and 7,12-Dimethyl benzanthracene (20 μg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid. The substance tested up to the maximum recommended concentration (2000 μg/mL) without and with metabolic activation system over a 5 hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control). It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed in Chinese hamster ovary cells.

Chromosome Aberration Test

The test item dissolved in DMSO medium was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 250, 500, 1000 and 1500 μg/mL test item

with S9 mix:500, 1000, 2000 and 2500 μg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 62.5, 125, 250, 500 and 1000 μg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 62.5, 125, 250, 500 and 1000 μg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 500, 1000, 2000 and 2500 μg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).

No precipitation of the test item was observed at any of the applied concentrations. The pH and the osmolality values of the treatment solutions were similar compared to the control.

Clear cytotoxicity of 53% and 54 % were observed at the highest concentrations in all experimental parts.

No relevant increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed, neither in the absence nor in the presence of metabolic activation.

There were no polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The number of aberrations found in the solvent controls was in the range of the laboratory historical control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.