Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test: negative

HPRT Assay: negative

Micronucleus Assay: negative

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:
2018-09-07 to 2018-12-05
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
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use,
Version / remarks:
2012
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:
phenobarbital and beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Initial and confirmatory test I
±S9: 5000; 1600; 500; 160; 50 and 16 μg/plate

Confirmatory test II
-S9: 16, 5, 1.6, 0.5, 0.16 and 0.05 µg/plate
Vehicle / solvent:
- Vehicle/solvent used:
test item: acetone
positive controls: water or DMSO
- Justification for choice of solvent/vehicle: The test item solutions were prepared in acetone and diluted prior to treatment. This vehicle was compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary solubility test.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine (NPD) 4 µg/plate, -S9, TA98; 2-aminoanthracene (2AA) 2 or 50 µg/plate for all salmonella strains or E.coli, +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h in the dark

SELECTION AGENT: his and trp gene

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: background lawn
Evaluation criteria:
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.

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:
not examined
True negative controls validity:
not examined
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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
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
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:
not examined
True negative controls validity:
not examined
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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Table 1 summary of signs of cytotoxicity

Confirmatory Mutation Test

Concentrations

(µg/plate)

Salmonella typhimurium

Escherichia coli WP2 uvrA

TA98

TA100

TA1535

TA1537

‑S9

+S9

‑S9

+S9

‑S9

+S9

‑S9

+S9

‑S9

+S9

5000

B0

SB <<

B0

B <<

B0

B

A

B <<

B <<

1600

B0

SB <

B0

B <<

B0

SB

B0

B <<

B <<

500

B <<

SB <<

B <<

SB <<

B0

SB

B0

B

B <<

160

B <<

B <<

< *

SB <<

SB0

SB <<

50

B <<

B <<

< *

SB <<

SB0

<< 

16

B <<

B <<

< *

SB <<

SB0

<< 

Complementary Pre-Incubation Test

Concentrations

(µg/plate)

Salmonella typhimurium

Escherichia coli WP2 uvrA

TA98

TA100

TA1535

TA1537

‑S9

+S9

‑S9

+S9

‑S9

+S9

‑S9

+S9

‑S9

+S9

16

B <<

 

B <<

 

B <<

 

B0

 

SB <<

 

5

<< 

 

SB <

 

 

B <<

 

 

1.6

 

< *

 

 

 

 

0.5

 

 

 

 

 

0.16

 

 

 

 

 

0.05

 

 

 

 

 

A:           No bacterial growth: absent revertant colonies and absent background lawn;

B0:        Absent revertant colonies and reduced background lawn development;

B:           Reduced background lawn development;

SB:         Slightly reduced background lawn development;

SB0:      Absent revertant colonies and slightly reduced background lawn development;

<< :      Revertant colony numbers below the vehicle and historical control data ranges;

< :          Revertant colony numbers within of the vehicle control range but below the historical control data ranges;

< * :      Revertant colony numbers within of the vehicle control range but below the historical control data ranges; however considered as not inhibition, but being within the biological variability range of the applied test system;

:       Revertant colony numbers lower than the revertant colony number of the vehicle control but within the historical control data ranges;

‑:            No inhibition.

Cells with dark grey filling: The concentration was not tested for the corresponding strain.

 


Table 2 Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations (mg/plate)

Salmonella typhimurium tester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

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

12.7

0.97

21.0

1.21

72.3

0.84

95.0

0.97

10.0

1.07

12.3

1.28

6.3

1.06

6.7

1.18

35.0

1.14

42.3

1.15

DMSO Control

17.0

1.00

22.7

1.00

91.3

1.00

11.0

1.00

6.0

1.00

6.7

1.00

43.0

1.00

Ultrapure Water Control

74.0

1.00

16.7

1.00

39.0

1.00

Acetone Control

13.0

1.00

17.3

1.00

85.7

1.00

98.0

1.00

9.3

1.00

9.7

1.00

6.0

1.00

5.7

1.00

30.7

1.00

36.7

1.00

5000

35.7

2.74

19.3

1.12

75.3

0.88

70.7

0.72

11.7

1.25

11.7

1.21

9.0

1.50

8.0

1.41

31.7

1.03

38.7

1.05

1600

18.7

1.44

19.7

1.13

78.7

0.92

110.0

1.12

7.7

0.82

9.7

1.00

8.3

1.39

8.7

1.53

32.7

1.07

39.7

1.08

500

13.7

1.05

16.0

0.92

67.3

0.79

88.0

0.90

8.3

0.89

8.3

0.86

7.3

1.22

9.0

1.59

39.7

1.29

41.0

1.12

160

12.7

0.97

18.7

1.08

64.7

0.75

90.0

0.92

11.3

1.21

12.0

1.24

6.7

1.11

5.3

0.94

32.0

1.04

39.0

1.06

50

14.0

1.08

16.3

0.94

62.0

0.72

88.7

0.90

10.3

1.11

12.0

1.24

7.0

1.17

5.3

0.94

31.3

1.02

39.7

1.08

16

18.3

1.41

22.7

1.31

65.3

0.76

96.3

0.98

10.3

1.11

10.3

1.07

6.3

1.06

6.3

1.12

40.0

1.30

37.0

1.01

NPD (4mg/plate)

498.0

29.29

SAZ (2mg/plate)

1397.3

18.88

965.3

57.92

9AA (50mg/plate)

736.7

122.78

MMS (2mL/plate)

1157.3

29.68

2AA (2mg/plate)

1154.7

50.94

1306.7

14.31

157.0

14.27

123.7

18.55

2AA (50mg/plate)

253.0

5.88

 

 

 

Table 3 Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (mg/plate)

Salmonella typhimurium tester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

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

16.0

0.91

29.0

1.78

68.3

0.87

87.3

0.94

14.7

1.05

9.0

0.87

7.7

1.28

9.7

0.91

24.0

0.81

39.7

0.97

DMSO Control

16.7

1.00

17.0

1.00

85.7

1.00

10.0

1.00

6.3

1.00

8.7

1.00

43.3

1.00

Ultrapure Water Control

75.7

1.00

11.3

1.00

32.3

1.00

Acetone Control

17.7

1.00

16.3

1.00

78.3

1.00

93.3

1.00

14.0

1.00

10.3

1.00

6.0

1.00

10.7

1.00

29.7

1.00

41.0

1.00

5000

0.0

0.00

11.3

0.69

0.0

0.00

11.3

0.12

0.0

0.00

7.0

0.68

0.0

0.00

2.7

0.25

10.3

0.35

41.3

1.01

1600

0.0

0.00

13.7

0.84

0.0

0.00

35.3

0.38

0.0

0.00

5.3

0.52

0.0

0.00

4.3

0.41

8.0

0.27

37.7

0.92

500

1.7

0.09

12.0

0.73

7.0

0.09

69.0

0.74

0.0

0.00

8.7

0.84

0.0

0.00

5.7

0.53

8.0

0.27

42.7

1.04

160

0.7

0.04

17.0

1.04

2.0

0.03

77.3

0.83

4.0

0.29

8.7

0.84

0.0

0.00

10.3

0.97

10.0

0.34

34.7

0.85

50

0.7

0.04

25.3

1.55

1.0

0.01

88.7

0.95

2.7

0.19

9.3

0.90

0.0

0.00

8.7

0.81

12.3

0.42

40.0

0.98

16

1.3

0.08

18.3

1.12

27.0

0.34

81.3

0.87

4.3

0.31

11.3

1.10

0.0

0.00

8.7

0.81

13.7

0.46

40.7

0.99

NPD (4mg/plate)

546.7

32.80

SAZ (2mg/plate)

840.0

11.10

845.3

74.59

9AA (50mg/plate)

404.0

63.79

MMS (2mL/plate)

824.0

25.48

2AA (2mg/plate)

1253.3

73.73

1237.3

14.44

119.0

11.90

121.7

14.04

2AA (50mg/plate)

342.7

7.91

 

Table 4 Summary Table of the Results of the Complementary Pre-Incubation Test

Complementary Pre-Incubation Test

Concentrations (mg/plate)

Salmonella typhimurium tester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

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

12.0

0.86

67.0

1.03

7.7

0.88

5.7

1.06

30.7

1.00

DMSO Control

12.0

1.00

6.3

1.00

Ultrapure Water Control

 

 

73.7

1.00

8.7

1.00

29.3

1.00

Acetone Control

14.0

1.00

65.0

1.00

8.7

1.00

5.3

1.00

30.7

1.00

16

4.7

0.33

44.0

0.68

2.7

0.31

0.0

0.00

15.7

0.51

5

10.3

0.74

57.0

0.88

6.7

0.77

3.3

0.63

25.7

0.84

1.6

16.7

1.19

72.0

1.11

8.7

1.00

5.7

1.06

32.0

1.04

0.5

17.7

1.26

77.0

1.18

8.3

0.96

4.7

0.88

24.7

0.80

0.16

14.0

1.00

75.3

1.16

9.7

1.12

6.0

1.13

29.7

0.97

0.05

13.0

0.93

75.7

1.16

7.3

0.85

5.0

0.94

21.3

0.70

NPD (4mg/plate)

526.7

43.89

SAZ (2mg/plate)

846.7

11.49

1072.0

123.69

9AA (50mg/plate)

473.3

74.74

MMS (2mL/plate)

1173.3

40.00

 

 

MR: Mutation Rate;      NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarcs: Acetone was applied as vehicle for the test item, ultrapure water was applied as vehicle of the positive control substances SAZ and MMS and DMSO was applied as vehicle of NPD, 9AA and 2AA. The mutation rate of the test item and the untreated control is given referring to the acetone, the mutation rate of SAZ and MMS is given referring to ultrapure water and the mutation rate of NPD, 9AA and 2AA is given referring to the DMSO.

 

Conclusions:
Results of this mutagenicity assay show 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.
Executive summary:

An in vitro bacteria reverse mutation assay according to OECD 471 was conducted with the test item. The test item was dissolved in acetone. In the initial and confirmatory mutation tests the following concentrations were examined: ±S9: 5000, 1600, 500, 160, 50 and 16 μg/plate. Because of the noticed strong inhibition obtained in the confirmatory mutation test an additional, complementary pre-incubation test was carried out in the absence of exogenous metabolic activation and the following concentration levels were investigated: -S9: 16, 5, 1.6, 0.5, 0.16 and 0.05 µg/plate. In the initial, confirmatory mutation and complementary pre-incubation tests Salmonella typhimurium TA98, TA100, TA1535, TA1537 strains and Escherichia coli WP2 uvrA were investigated.
Five bacterial strains were used to investigate the mutagenic potential of the test item in independent experiments, in a plate incorporation test (experiment I, initial mutation test), in a pre-incubation test (experiment II, confirmatory mutation test) and because of the strong inhibitory effect obtained in the confirmatory mutation test, in an additional, complementary pre-incubation test. The initial and confirmatory mutation tests were conducted with and without metabolic activation (±S9), the complementary pre-incubation test was performed in absence of metabolic activation (-S9), only. In the performed experiments the concentrations, including the controls, were tested in triplicate (positive and negative controls were run concurrently).
In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (±S9) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

In the initial mutation test inhibitory effects of the test item were not observed. In the confirmatory mutation test and as well as in the completing complementary pre-incubation test inhibitory effect of the test item was noticed in all strains examined. The cytotoxicity was indicated by affected background lawn development (absent, reduced or slightly reduced background lawn) and/or decreased revertant colony counts (absent revertants or revertants below historical control data and/or below the corresponding vehicle data ranges).

In the initial mutation test following plate incorporation procedure microdrops (colloid-chemical phenomenon) were noticed in all strains at the highest examined concentration of 5000 µg/plate, without and with addition of exogenous metabolic activation (±S9). The obtained microdrops did not interfere with the scoring of the colonies and evaluation of background lawn development in any case.

No microdrops or precipitate of the test item were observed on the plates in the examined bacterial strains at any examined concentration level (±S9) following the pre-incubation procedures.

The reported data of this mutagenicity assay show, 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 cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-02-11 to 2019-03-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
For cell lines:
- Absence of Mycoplasma contamination: yes
- doubling time: 12-14 h
- Modal number of chromosomes: 2n = 22

MEDIA USED
- Type and composition of media:
DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2 mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin B) and heat-inactivated foetal bovine serum (final concentration 10 %)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
- concentration or volume of S9 mix and S9 in the final culture medium: The protein concentrations of the S9 batch used in the experiments were 34.8, 39.7 and 37.8 mg/mL
Test concentrations with justification for top dose:
Based on a cytotoxicity pre-experiment, the concentration ranges for the main study were chosen as follows:

4/24 h treatment/sampling time:
without S9-mix: 5, 10, 20 and 40 µg/mL test item

24/24 h treatment/sampling time
without S9-mix: 5, 10, 20 and 40 µg/mL test item

4/24 h treatment/sampling time
with S9-mix: 40, 60, 80 and 100 µg/mL test item
Vehicle / solvent:
- Vehicle/solvent used: Acetone
- Justification for choice of solvent/vehicle: solubility of the test item and suitability with test system

- Justification for percentage of solvent in the final culture medium: according to the guideline
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
colchicine
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : three

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 and 24 hours
- Harvest time after the end of treatment: 24 hours

FOR MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used: V79 cells were kept in fixative at 1-10°C before slides were prepared, but slides was not made on the day of harvest to ensure cells were adequately fixed. Cells were centrifuged and resuspended in a minimal amount of fresh fixative. Several drops of suspension were gently spread onto multiple clean, dry microscope slides. After the slides had dried the cells were stained for 25 minutes in filtered 10 % (v/v) Giemsa pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.

- Number of cells spread and analysed per concentration: 1000 cells per culture (2000 cells per concentration)

- Criteria for scoring micronucleated cells: The number of cells containing micronuclei on each slide was noted. Observations were recorded on raw data sheets. A micronucleus was only recorded if it met the following criteria:
1) The micronucleus has the same staining characteristics and a similar morphology to the main nuclei, and
2) Any micronucleus present are separate in the cytoplasm or only just touching a main nucleus, and
3) micronuclei is smooth edged and smaller than approximately one third the diameter of the main nuclei.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative increase in cell count (RICC)

Rationale for test conditions:
according to guideline
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.

When all of these criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.


Providing that all acceptability criteria are fulfilled, a 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 historical negative control data (based 95% control limit).
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
In cases when the response is neither clearly negative nor clearly positive or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment or further investigations. Performing a repeated experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions [i.e. S9 concentration or S9 origin]) could be useful.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: pH in the test item treated cultures was between 7.5 - 7.7 in the main test
- Data on osmolality: osmolality in the test item treated cultures was between 334 - 343 mmol/kg in the main test
- Possibility of evaporation from medium: no
- Water solubility: not soluble in water
- Precipitation and time of the determination: no precipitation observed in main test

RANGE-FINDING/SCREENING STUDIES: yes

STUDY RESULTS
- Concurrent vehicle negative and positive control data : valid

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : no

Table 1 Results of the micronucleus analysis: 4-hour treatment without S9-mix / 24-hour sampling time (1000 cells scored per replicate; n = 2)

Test Group

Concentration

Total MN cells scored

Frequency of MN cells (%)

Untreated control

-

13

0.7

Negative (solvent control)

0.4 µL/mL

15

0.8

Test item

5 µg/mL

15

0.8

10 µg/mL

15

0.8

20 µg/mL

13

0.7

40 µg/mL

16

0.8

Colchicine

0.15 µg/mL

162

8.1**

Mitomycin C

0.8 µg/mL

170

8.5**

** = p < 0.01 to the concurrent negative control and to the historical control

Table 2 Results of the micronucleus analysis: 24-hour treatment without S9-mix / 24-hour sampling time (1000 cells scored per replicate; n = 2)

Test Group

Concentration

Total MN cells scored

Frequency of MN cells (%)

Untreated control

-

11

0.6

Negative (solvent control)

0.4 µL/mL

14

0.7

Test item

5 µg/mL

14

0.7

10 µg/mL

12

0.6

20 µg/mL

12

0.6

40 µg/mL

13

0.7

Colchicine

0.03 µg/mL

201

10.1**

Mitomycin C

0.8 µg/mL

176

8.8**

** = p < 0.01 to the concurrent negative control and to the historical control

Table 3 Results of the micronucleus analysis: 4-hour treatment with S9-mix / 24-hour sampling time (1000 cells scored per replicate; n = 2)

Test Group

Concentration

Total MN cells scored

Frequency of MN cells (%)

Untreated control

-

13

0.7

Negative (solvent control)

0.4 µL/mL

15

0.8

Test item

5 µg/mL

13

0.7

 

10 µg/mL

15

0.8

 

20 µg/mL

17

0.9

 

40 µg/mL

12

0.6

Cyclophosphamide

6.25 µg/mL

296

14.8**

** = p < 0.01 to the concurrent negative control and to the historical control

 

Conclusions:
The test item is considered not genotoxic based on the results of the OECD 487 compliant micronucleus assay in vitro.
Executive summary:

This study was performed to evaluate the clastogenic and aneugenic potential of the test item by its effects on the frequency of micronuclei in cultured V79 cell lines treated in the absence and presence of a rat liver metabolizing system according to OECD TG 487. The test item was dissolved in Acetone and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In this experiment (both run in duplicate with concurrent negative and positive controls) at least 2000 cells were analyzed for micronucleus at concentrations and treatment (exposure)/sampling (expression) intervals given below, ranging from no or little to maximum (55 ± 5%survival) toxicity.

 

4/24 h treatment/sampling time, without S9 -mix: 5, 10, 20 and 40 µg/mL test item

24/24 h treatment/sampling time, without S9 -mix: 5, 10, 20 and 40 µg/mL test item

4/24 h treatment/sampling time, with S9 -mix: 40, 60, 80 and 100 µg/mL test item

In this In Vitro Mammalian Cell Micronucleus Test, the frequency of the cells with micronuclei did not show biologically and statistically significant increases compared to the concurrent and historical controls when the test item was examined in the absence and in the presence of metabolic activation, up to the cytotoxic concentrations. 

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

 

In the concurrent negative and positive control groups the percentage of cells with micronuclei were in the historical control range. The positive controls Mitomycin C (0.8 µg/mL), Colchicine (0.15 and 0.03 µg/mL) and Cyclophosphamide (6.25 µg/mL) caused expected biologically relevant increases of cells with micronuclei compared to solvent and historical controls. Thus, the study is considered valid.

The test item, both with and without metabolic activation, did not induce breakage and /or chromosomal loss in Chinese Hamster lung cells under the test conditions. Therefore, the test item is considered as non-genotoxic with the micronocucleus test.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-09-06 to 2018-10-16
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)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
Subline KI
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Ham's F12 medium containing 10 % fetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced male rat liver S9
Test concentrations with justification for top dose:
Test item concentrations without S9: 40, 50, 60, 80, 90, 100, 110 µg/mL
Test item concentrations with S9: 60, 70, 80, 90, 100, 110,120 µg/mL
Test item concentrations were chosen based on pre-test results on cytotoxicity.
Vehicle / solvent:
- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: recommended vehicle by the guideline.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: 5x10^6

DURATION
- Exposure duration: 5 h
- Expression time (cells in growth medium): 19 h
- Selection time (if incubation with a selection agent): 8 days

SELECTION AGENT: hypoxanthine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Assay acceptance criteria
The test is considered valid, because:
• With the exception of a single culture in the absence of metabolic activation, all concurrent negative controls are within the 95% control limits of the distribution of the laboratory historical solvent control database.
• The mutant frequency in the negative (solvent) control cultures is within the range (min-max) of historical laboratory control data.
• The positive control chemicals induce a statistically significant and biologically relevant increase in mutant frequency. These increases are compatible with the historical control data base.
• Adequate number of cells and concentrations were analysable.
• Two experimental conditions with and without metabolic activation were tested.
• The highest concentration was adequate.
• The cloning efficiency of the negative controls is between the range of 60% to 140% on Day 1 and 70% to 130% on Day 8.

Evaluation of Results
the 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.

Providing that all acceptability criteria were fulfilled, the test item is considered clearly negative if:
• 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).
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
• mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
• mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.

The data was checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software. 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: yes

Table 1 CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d (5-hour Treatment without S9-Mix)

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

Untreated control a

200.0

±

3.00

102

100

6

100

6.00

Solvent control a

196.0

±

2.65

100

100

6

100

6.00

Pos. control
(EMS 1.0 µL/mL) a

48.3

±

1.53

25

62

894

62

1441.94**

TEST ITEM

 

40g/mL a

180.7

±

1.15

92

97

5

97

5.15

50g/mL a

165.0

±

2.00

84

97

6

97

6.19

60g/mL a

157.7

±

1.53

80

98

5

98

5.10

80g/mL a

133.0

±

1.00

68

97

5

96

5.21

90g/mL a

116.0

±

3.00

59

97

5

97

5.15

100g/mL a

48.0

±

1.00

24

97

7

97

7.22

110g/mL a

36.0

±

1.00

18

97

7

96

7.29

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

Untreated control b

201.0

±

1.00

102

100

6

100

6.00

Solvent control b

198.0

±

1.73

100

100

6

100

6.00

Pos. control
(EMS 1.0 µL/mL) b

49.0

±

1.00

25

62

904

62

1458.06**

TEST ITEM

 

40g/mL b

183.0

±

2.00

92

97

5

97

5.15

50g/mL b

164.7

±

1.15

83

98

5

98

5.10

60g/mL b

156.3

±

2.08

79

98

5

98

5.10

80g/mL b

134.0

±

1.00

68

97

6

98

6.12

90g/mL b

115.7

±

2.08

58

97

5

97

5.15

100g/mL b

45.7

±

1.53

23

97

6

98

6.12

110g/mL b

34.0

±

1.00

17

97

7

97

7.22

  

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

Untreated control c

202.7

±

2.31

101

100

6

100

6.00

Solvent control c

200.7

±

2.08

100

100

7

99

7.07

Pos. control
(EMS 1.0 µL/mL) c

50.3

±

2.08

25

64

898

64

1403.13**

TEST ITEM

 

40g/mL c

181.7

±

1.53

91

98

6

98

6.12

50g/mL c

170.3

±

1.53

85

99

5

98

5.10

60g/mL c

158.3

±

2.08

79

97

6

96

6.25

80g/mL c

132.0

±

1.00

66

97

5

97

5.15

90g/mL c

120.3

±

0.58

60

97

5

97

5.15

100g/mL c

49.3

±

1.15

25

97

5

96

5.21

110g/mL c

34.3

±

0.58

17

97

7

96

7.29

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

Untreated control d

202.0

±

1.00

101

101

5

100

5.00

Solvent control d

200.3

±

1.53

100

100

7

100

7.00

Pos. control
(EMS 1.0 µL/mL) d

50.7

±

2.08

25

65

926

64

1446.88**

TEST ITEM

 

40g/mL d

184.0

±

1.00

92

98

5

98

5.10

50g/mL d

169.0

±

1.00

84

98

6

98

6.12

60g/mL d

160.0

±

1.00

80

97

5

97

5.15

80g/mL d

132.3

±

0.58

66

98

5

97

5.15

90g/mL d

120.7

±

1.53

60

97

5

97

5.15

100g/mL d

48.0

±

1.00

24

97

5

97

5.15

110g/mL d

33.3

±

0.58

17

97

8

97

8.25

 

a = parallel of first culture 
b = parallel of first culture.
c = parallel of second culture
d = parallel of second culture.

 

abs.C.E. = Absolute Cloning Efficiency

EMS= Ethyl methanesulfonate

** = p < 0.01 to the concurrent negative control and to the historical control

 

Table 2 CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d (5-hour Treatment with S9-Mix)

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

Untreated control a

201.3

±

2.08

106

103

5

99

5.05

Solvent control a

190.3

±

1.53

100

100

7

97

7.22

Pos. control
(DMBA 20 µg/mL) a

122.7

±

2.08

64

80

559

77

725.97**

TEST ITEM

 

60g/mL a

179.0

±

1.00

94

99

5

96

5.21

70g/mL a

175.0

±

2.00

92

98

7

95

7.37

80g/mL a

167.7

±

1.53

88

97

8

94

8.51

90g/mL a

157.7

±

3.21

83

98

6

95

6.32

100g/mL a

126.7

±

1.15

67

97

5

94

5.32

110g/mL a

77.0

±

2.65

40

97

6

94

6.38

120g/mL a

24.0

±

1.00

13

97

5

94

5.32

  

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

Untreated control b

201.3

±

2.08

105

101

5

100

5.00

Solvent control b

191.3

±

1.53

100

100

6

99

6.06

Pos. control
(DMBB 20 µg/mL) b

122.3

±

2.08

64

79

561

78

719.23**

TEST ITEM

 

60g/mL b

180.3

±

0.58

94

97

5

96

5.21

70g/mL b

176.7

±

1.15

92

97

5

95

5.26

80g/mL b

167.0

±

2.00

87

97

8

96

8.33

90g/mL b

155.7

±

3.06

81

97

6

96

6.25

100g/mL b

126.7

±

1.53

66

97

5

95

5.26

110g/mL b

75.0

±

1.00

39

96

5

95

5.26

120g/mL b

26.7

±

1.53

14

97

7

96

7.29

  

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

Untreated control c

201.0

±

1.73

106

100

6

99

6.06

Solvent control c

189.7

±

0.58

100

100

6

99

6.06

Pos. control
(DMBC 20 µg/mL) c

120.3

±

1.53

63

78

566

77

735.06**

TEST ITEM

 

60g/mL c

178.0

±

2.00

94

98

5

97

5.15

70g/mL c

171.0

±

1.00

90

100

5

99

5.05

80g/mL c

169.0

±

1.00

89

99

7

98

7.14

90g/mL c

158.3

±

1.15

83

97

5

96

5.21

100g/mL c

125.0

±

1.00

66

97

5

96

5.21

110g/mL c

71.7

±

1.53

38

97

5

96

5.21

120g/mL c

24.3

±

1.15

13

97

5

96

5.21

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBERS.D.

PERCENT
VEH. CONTROL

Untreated control d

201.3

±

0.58

106

100

5

100

5.00

Solvent control d

190.3

±

2.08

100

100

7

99

7.07

Pos. control
(DMBD 20 µg/mL) d

121.3

±

1.53

64

78

542

78

694.87**

TEST ITEM

 

60g/mL d

179.0

±

2.00

94

98

5

97

5.15

70g/mL d

172.0

±

1.00

90

99

5

99

5.05

80g/mL d

170.3

±

0.58

89

98

7

97

7.22

90g/mL d

158.7

±

1.15

83

97

5

96

5.21

100g/mL d

121.7

±

1.53

64

97

5

96

5.21

110g/mL d

71.0

±

1.00

37

97

5

97

5.15

120g/mL d

24.7

±

1.15

13

97

6

97

6.19

 

 

a = parallel of first culture 
b = parallel of first culture.
c = parallel of second culture
d = parallel of second culture.

 

abs.C.E. = Absolute Cloning Efficiency

EMS= Ethyl methanesulfonate

** = p < 0.01 to the concurrent negative control and to the historical control

 

Conclusions:
The test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells according to OECD 476 TG.
Executive summary:

The test item, dissolved in Acetone, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according to OECD 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 and β-naphthoflavone induced rat liver and solubility of test item.

5-hour treatment period without S9-mix: 40, 50, 60, 80, 90,100 and 110 μg/mL

5-hour treatment period with S9-mix: 60, 70, 80, 90, 100, 110 and 120 μg/mL

In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity. 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 test item tested up to cytotoxic concentrations both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells, when tested up to cytotoxic concentrations. It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with 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

In vitro bacterial reverse mutation assay (Ames), OECD 471

An in vitro bacteria reverse mutation assay according to OECD 471 was conducted with the test item. The test item was dissolved in acetone. In the initial and confirmatory mutation tests the following concentrations were examined: ±S9: 5000, 1600, 500, 160, 50 and 16 μg/plate. Because of the noticed strong inhibition obtained in the confirmatory mutation test an additional, complementary pre-incubation test was carried out in the absence of exogenous metabolic activation and the following concentration levels were investigated: -S9: 16, 5, 1.6, 0.5, 0.16 and 0.05 µg/plate.

In the initial, confirmatory mutation and complementary pre-incubation tests Salmonella typhimurium TA98, TA100, TA1535, TA1537 strains and Escherichia coli WP2 uvrA were investigated.
Five bacterial strains were used to investigate the mutagenic potential of the test item in independent experiments, in a plate incorporation test (experiment I, initial mutation test), in a pre-incubation test (experiment II, confirmatory mutation test) and because of the strong inhibitory effect obtained in the confirmatory mutation test, in an additional, complementary pre-incubation test. The initial and confirmatory mutation tests were conducted with and without metabolic activation (±S9), the complementary pre-incubation test was performed in absence of metabolic activation (-S9), only. In the performed experiments the concentrations, including the controls, were tested in triplicate (positive and negative controls were run concurrently).

In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled .

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (±S9) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

In the initial mutation test inhibitory effects of the test item were not observed. In the confirmatory mutation test and as well as in the completing complementary pre-incubation test inhibitory effect of the test item was noticed in all strains examined. The cytotoxicity was indicated by affected background lawn development (absent, reduced or slightly reduced background lawn) and/or decreased revertant colony counts (absent revertants or revertants below historical control data and/or below the corresponding vehicle data ranges).

In the initial mutation test following plate incorporation procedure microdrops (colloid-chemical phenomenon) were noticed in all strains at the highest examined concentration of 5000 µg/plate, without and with addition of exogenous metabolic activation (±S9). The obtained microdrops did not interfere with the scoring of the colonies and evaluation of background lawn development in any case. No microdrops or precipitate of the test item were observed on the plates in the examined bacterial strains at any examined concentration level (±S9) following the pre-incubation procedures. The reported data of this mutagenicity assay show, 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.

In vitro gene mutation assay in mammalian cells (HPRT), OECD 476

The test item, dissolved in Acetone, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according to OECD 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 and β-naphthoflavone induced rat liver and solubility of test item.

5-hour treatment period without S9-mix: 40, 50, 60, 80, 90,100 and 110 μg/mL

5-hour treatment period with S9-mix: 60, 70, 80, 90, 100, 110 and 120 μg/mL

In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity. 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 test item tested up to cytotoxic concentrations both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells, when tested up to cytotoxic concentrations. It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.

In vitro micronucleus assay in mammalian cells, OECD 487

This study was performed to evaluate the clastogenic and aneugenic potential of the test item by its effects on the frequency of micronuclei in cultured V79 cell lines treated in the absence and presence of a rat liver metabolizing system according to OECD TG 487.The test item was dissolved in Acetone and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In this experiment (both run in duplicate with concurrent negative and positive controls) at least 2000 cells were analyzed for micronucleus at concentrations and treatment (exposure)/sampling (expression) intervals given below, ranging from no or little to maximum (55 ± 5%survival) toxicity.

 

4/24 h treatment/sampling time,without S9 -mix: 5, 10, 20 and 40 µg/mL test item

24/24 h treatment/sampling time,without S9 -mix: 5, 10, 20 and 40 µg/mL test item

4/24 h treatment/sampling time,with S9 -mix: 40, 60, 80 and 100 µg/mL test item

In this In Vitro Mammalian Cell Micronucleus Test, the frequency of the cells with micronuclei did not show biologically and statistically significant increases compared to the concurrent and historical controls when the test item was examined in the absence and in the presence of metabolic activation, up to the cytotoxic concentrations. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

In the concurrent negative and positive control groups the percentage of cells with micronuclei were in the historical control range. The positive controls Mitomycin C (0.8 µg/mL),Colchicine (0.15 and 0.03 µg/mL) and Cyclophosphamide (6.25 µg/mL) caused expected biologically relevant increases of cells with micronuclei compared to solvent and historical controls. Thus, the study is considered valid.

The test item, both with and without metabolic activation, did not induce breakage and /or chromosomal loss in Chinese Hamster lung cells under the test conditions. Therefore, the test item is considered as non-genotoxic with the micronocucleus test.

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity, the test item does not require classification as mutagenic according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.