Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay 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.

The test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concnentrations in the absence and presence of metabolic activation.

The test item tested up to cytotoxic concentrations both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in a gene mutation assay according to OECD 476 in Chinese hamster ovary cells.

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:
2016-03-08 to 2016-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use, June 2012
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
Test concentrations with justification for top dose:
Selection of the concentrations was done on the basis of a Solubility Test and a concentration range finding test (Informatory Toxicity Test).
The highest dose was 1600 μg test item/plate in absence (-S9 Mix) and 5000 μg/plate in the presence of exogenous metabolic activation (+S9 Mix) in the final treatment mixture under the actual conditions of the test at the start of the experiments for all test strains used.
The following seven concentrations of the test item were tested in the main experiments:
-S9 Mix: 1600, 500; 160; 50; 16; 5 and 1.6 μg/plate;
+S9 Mix: 5000; 1600; 500; 160; 50; 16 and 5 μg/plate.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
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; vehicle: DMSO; strain: Salmonella TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9; vehicle: ultrapure water; strains: Salmonella TA100, TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9; vehicle: DMSO; strain: Salmonella TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9; vehicle: ultrapure water; strain: E. coli WP2 uvr A
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
with S9; vehicle: DMSO; strains: all of Salmonella strains and E. coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

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

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony numbers, background lawn development
- Any supplementary information relevant to cytotoxicity: The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.
Rationale for test conditions:
Selection of the concentrations was done on the basis of a Solubility Test and a concentration range finding test (Informatory Toxicity Test).
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 TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control.
- in strain 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 article 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 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 160 µg/plate and above
Vehicle controls validity:
valid
Untreated 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
Remarks:
at 160 µg/plate and above
Vehicle controls validity:
valid
Untreated 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
Remarks:
500 µg/plate and above
Vehicle controls validity:
valid
Untreated 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
Remarks:
500 µg/plate and above
Vehicle controls validity:
valid
Untreated 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
Remarks:
1600 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test item was dissolved, suspended in anhydrous dimethyl sulfoxide (DMSO).
- Precipitation: In the performed experiments the test item solution precipitated in a form of drops (“microdrops”) on the plates; in all examined strains at the concentration of 5000 μg/plate, with addition of metabolic activation (+S9 Mix) following the plate incorporation and pre-incubation procedures (Initial and Confirmatory Mutation Tests).

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate. In the Informatory Toxicity Test inhibitory effect of the test item was observed; that was indicated by lower revertant colony numbers (compared to that of the vehicle and historical control data ranges) and/or reduced or slightly reduced background lawn development. The inhibitory, cytotoxic effect of the test item was observed in both strains at the concentrations of 5000 and 1600 μg/plate in the absence and also in the presence of exogenous metabolic activation (±S9 Mix). The slightly lower revertant colony numbers in S. typhimurium TA98 at 500 μg/plate (-S9 Mix) as well as the slightly higher revertant colony numbers in S. typhimurium TA100 at 50 μg/plate (-S9 Mix) remained in the corresponding historical control data ranges, and were considered as reflecting the biological variability of the applied test system. Microdrops (colloid-chemical phenomenon) were noticed in both strains at the highest examined concentration of 5000 μg/plate, without and with addition of exogenous metabolic activation (±S9 Mix).

HISTORICAL CONTROL DATA
Mutation frequencies were within historical control data (see "Any other information on results" table 1).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Signs of cytotoxicity has been observed in all tested strains with and/or without metabolic activation. The 160 μg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium TA1535 and TA1537 strains, following the pre-incubation procedure, in absence of exogenous metabolic activation (-S9 Mix).

Table 1 Historical control values for revertants/plate (for the period of 2008-2015)

 

 

Bacterial strains

Historical control data of DMSO control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

20.9

101.4

10.3

7.9

24.9

SD

3.5

26.2

1.4

2.5

4.9

Minimum

10

65

3.

2

11

Maximum

39

150

23

20

44

 

 

 

 

 

 

 

+S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

27.1

114.7

12.0

8.8

34.2

SD

4.0

19.3

1.5

2.1

5.2

Minimum

15

71

4.

3

16

Maximum

48

161

24

20

56

 

 

 

 

 

 

 

 

Historical control data of water control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

22.4

150.5

10.4

7.5

26.3

SD

3.6

27.6

1.6

2.3

5.9

Minimum

12

67

3.

2

13

Maximum

36

156

24

15

47

 

 

 

 

 

 

 

+S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

28.0

117.4

11.5

8.7

35.2

SD

4.0

19.8

1.4

2.3

5.2

Minimum

15

83

4.

4.

18

Maximum

43

166

22

16

56

 

 

 

 

 

 

 

 

Historical control data of positive

control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

255.6

958.9

842.1

467.4

712.3

SD

30.7

149.9

134.0

105.7

57.5

Minimum

123

522

354

109

320

Maximum

647

1927

1871

1498

1283

 

 

 

 

 

 

 

+S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

1224.8

1431.9

165.4

148.0

264.7

SD

293.8

399.9

35.1

21.3

74.2

Minimum

409

581

85

68

141

Maximum

2587

2923

507

407

487

Table 2. Summary of the results of the Range Finding Test

Range Finding Test (Informatory Toxicity Test)

Concentrations (mg/plate)

Salmonella typhimurium tester strains

TA 98

TA 100

-S9

+S9

-S9

+S9

Mean values of revertants per plate and
Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

19.0

1.14

33.0

1.39

85.7

1.00

109.3

1.10

DMSO Control

19.3

1.00

21.3

1.00

104.0

1.00

Ultrapure Water Control

76.3

1.00

DMSO (Anhydrous) Control

16.7

1.00

23.7

1.00

85.7

1.00

99.0

1.00

5000

3.0

0.18

7.7

0.32

0.3

0.00

58.3

0.59

1600

7.7

0.46

8.0

0.34

59.0

0.69

82.3

0.83

500

13.0

0.78

21.0

0.89

91.3

1.07

90.3

0.91

160

14.0

0.84

25.7

1.08

84.0

0.98

109.0

1.10

50

14.3

0.86

25.0

1.06

107.7

1.26

101.0

1.02

16

19.0

1.14

21.7

0.92

99.7

1.16

101.0

1.02

5

17.7

1.06

20.7

0.87

94.0

1.10

109.7

1.11

NPD (4mg)

269.7

13.95

SAZ (2mg)

1337.3

17.52

2AA (2mg)

1170.7

54.88

1392.0

13.38

Table 3. Summary 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

20.7

1.22

24.0

1.16

94.0

0.96

118.7

1.03

7.7

0.92

8.7

0.87

7.0

0.88

8.0

1.00

16.0

0.94

17.7

0.88

DMSO Control

17.0

1.00

22.3

1.00

119.7

1.00

5.3

1.00

8.0

1.00

8.3

1.00

21.0

1.00

Ultrapure Water Control

98.3

1.00

8.7

1.00

18.0

1.00

DMSO (Anhydrous) Control

17.0

1.00

20.7

1.00

98.3

1.00

114.7

1.00

8.3

1.00

10.0

1.00

8.0

1.00

8.0

1.00

17.0

1.00

20.0

1.00

5000

17.0

0.82

52.0

0.45

2.7

0.27

0.7

0.08

10.3

0.52

1600

5.7

0.33

15.0

0.73

50.3

0.51

72.7

0.63

1.3

0.16

9.3

0.93

0.7

0.08

4.3

0.54

9.3

0.55

21.7

1.08

500

13.3

0.78

17.7

0.85

62.3

0.63

82.7

0.72

5.3

0.64

8.3

0.83

3.3

0.42

8.3

1.04

19.7

1.16

21.0

1.05

160

15.3

0.90

18.0

0.87

78.0

0.79

99.0

0.86

8.0

0.96

8.0

0.80

6.0

0.75

11.0

1.38

17.3

1.02

19.3

0.97

50

19.3

1.14

16.0

0.77

89.7

0.91

93.3

0.81

9.3

1.12

8.0

0.80

10.0

1.25

7.0

0.88

19.7

1.16

18.7

0.93

16

20.7

1.22

18.7

0.90

86.3

0.88

113.7

0.99

11.0

1.32

8.0

0.80

10.7

1.33

11.7

1.46

18.3

1.08

16.7

0.83

5

15.3

0.90

16.7

0.81

82.0

0.83

102.3

0.89

11.7

1.40

9.7

0.97

12.3

1.54

10.0

1.25

18.0

1.06

18.0

0.90

1.6

16.0

0.94

75.7

0.77

7.3

0.88

11.0

1.38

19.0

1.12

NPD (4mg)

279.0

16.41

SAZ (2mg)

1066.7

10.85

809.3

93.38

9AA (50mg)

804.0

100.50

MMS (2mL)

382.7

21.26

2AA (2mg)

1156.0

51.76

1474.7

12.32

162.0

30.38

177.0

21.24

2AA (50mg)

194.7

9.27

Conclusions:
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.
Executive summary:

The test item was dissolved in anhydrous dimethyl sulfoxide (DMSO). In the Initial and Confirmatory Mutation Tests the following concentrations were examined: -S9 Mix: 1600; 500; 160; 50; 16; 5 and 1.6 μg/plate; +S9 Mix: 5000; 1600; 500; 160; 50; 16 and 5 μg/plate. In the Initial and Confirmatory Mutation Tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were investigated. Five bacterial strains were used to investigate the mutagenic potential of the test item 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, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria, regarding the investigated strains, negative and positive controls, S9 activity and number of investigated analyzable 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 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values 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 performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was observed in all examined bacterial strains. The 160 μg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium TA1535 and TA1537 strains, in the absence of exogenous metabolic activation (-S9 Mix). Microdrops (as colloid-chemical phenomenon) was observed on the plates in the above bacterial strains at the concentration of 5000 μg/plate with addition of exogenous metabolic activation, following the plate incorporation and pre-incubation procedures (Initial and Confirmatory Mutation Tests).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-28 to 2017-12-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian chromosome aberration assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)
- doubling time: 12-14 h
- Modal number of chromosomes: 2n=22

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of phenobarbital and beta-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
Test concentrations were based on preliminary cytotoxicity test and highest concentration was selected to induce 50 +/-5 % cytotoxicity according to the guidelines:

Experiment A with 3/20 h treatment/sampling time
without S9 mix: 30, 50, 70 and 80 µg/mL test item
with S9 mix: 70, 80, 90 and 100 µg/mL test item

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 30, 50, 70 and 80 µg/mL test item

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 30, 50, 70 and 80 µg/mL test item

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 70, 80, 90 and 100 µg/mL test item
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours (experiment A) and 20 hours (experiment B)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h and 28 h (experiment B)

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 300 (150 per replicate)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells was scored.
- Determination of endoreplication: The number of endoreplicated cells was scored.
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,
– 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. The lower and upper 95% confidence intervals of historical control were calculated with C-chart.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
above 50 % at 80 and 100 µg/mL without or with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: the test item was well soluble in DMSO
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES: yes

HISTORICAL CONTROL DATA
Please refer to "Any other information on results incl. tables"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC

Mean percentage of cells with structural chromosome aberrations.

Table 1: Experiment A, treatment time 3 h, harvesting time 20 h

Concentration [µg/mL]

S9 mix

Mean aberrant cells / 150 cells including gaps

Mean aberrant cells / 150 cells excluding gaps

Solvent control (DMSO)

-

8

3

30

-

7

4

50

-

8

3

70

-

8

4

80

-

8

3

Ethyl methanesulfonate

[1 µL/mL]

-

44**

33**

Solvent control (DMSO)

+

8

4

70

+

9

3

80

+

8

4

90

+

9

3

100

+

8

4

Cyclophosphamide [5 µg/mL]

+

48**

40**

**: p < 0.01

Table 2: Experiment B, treatment time 20 h, harvesting time 20 h

Concentration [µg/mL]

S9 mix

Mean aberrant cells / 200 cells including gaps

Mean aberrant cells / 200 cells excluding gaps

Solvent control (DMSO)

-

8

4

30

-

7

4

50

-

8

4

70

-

7

3

80

-

9

4

Ethyl methanesulfonate

[0.4 µL/mL]

-

44**

38**

**: p < 0.01

Table 3: Experiment B, treatment time 3 h, harvesting time 28 h

Concentration [µg/mL]

S9 mix

Mean aberrant cells / 200 cells including gaps

Mean aberrant cells / 200 cells excluding gaps

Solvent control (DMSO)

-

8

3

70

+

8

3

80

+

8

3

90

+

8

3

100

+

9

4

Cyclophosphamide

[5 µL/mL]

+

42**

37**

**: p < 0.01

Table 4: Experiment B, treatment time 20 h, harvesting time 28 h

Concentration [µg/mL]

S9 mix

Mean aberrant cells / 200 cells including gaps

Mean aberrant cells / 200 cells excluding gaps

Solvent control (DMSO)

-

8

3

30

-

6

3

50

-

9

4

70

-

9

3

80

-

8

4

Ethylmethanesulfonate [0.4 µL/mL]

-

47**

40**

**: p < 0.01

Conclusions:
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.
Executive summary:

The test item, dissolved in DMSO, 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 on (without and with metabolic activation using rat S9 mix) in accordance with the current OECD Guideline 473:

 

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 30, 50, 70 and 80 µg/mL test item

with S9 mix: 70, 80, 90 and 100 µg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 30, 50, 70 and 80 µg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 30, 50, 70 and 80 µg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 70, 80, 90 and 100 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. There were no relevant changes in pH or osmolality after treatment with the test item.

 

Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts at the concentration levels of 80 µg/mL and above.

There were no relevant increases in cells carrying structural chromosomal aberrations observed, neither in the absence nor in the presence of metabolic activation.

 

There were no biologically relevant increases in the rate of 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 historical laboratory control data. The concurrent positive controls ethylmethanesulphonate (0.4 and 1.0 L/mL) and cyclophosphamide (5 g/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.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-28 to 2017-10-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro gene mutation assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
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: 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 %), at 37 °C and 5% CO2 in the air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat livers
Test concentrations with justification for top dose:
5-hour treatment period without S9-mix:
50, 60, 65, 70, 75, 80, 85 and 90 μg/mL

5-hour treatment period with S9-mix:
50, 60, 65, 70, 75, 80, 85 and 90 μg/mL
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 h
- Expression time (cells in growth medium): 8 days
- Fixation time: after selection period

SELECTION AGENT: The selection medium for TG resistant mutants contained 10 μM/mL of 6-thioguanine (6-TG) (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL).

STAIN: Giemsa

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, 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.

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 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).

The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
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 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
Remarks:
relative survival < 50% at 80 µg/mL and above with and without S9 mix and
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Water solubility: the test item was well soluble in DMSO
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES: yes

 

Table 1 Day 8 Cloning efficiencies -Main Mutation Assay (5-hour treatment without and With S9-Mix)

 

Test Group a and b

Concentration
µg/mL

S9-mix

Mean number of colonies/200 cells/dish

 

Cloning
efficiency (%)

% of Control

Negative (Solvent Control)

200,3

100

100

Test item - Group a

50

199,7

100

100

60

195,7

98

98

65

200,0

100

100

70

203,7

102

102

75

200,7

100

100

80

197,7

99

99

85

198,7

99

99

90

0,0

0

0

EMS (1µL/mL)

144,7

72

72

Negative (Solvent Control)

200,7

100

100

 

50

199,3

100

99

60

198,0

99

99

65

199,0

100

99

Test item- Group b

70

201,3

101

100

75

199,3

100

99

80

197,7

99

99

85

199,0

100

99

90

0,0

0

0

EMS (1µL/mL)

145,7

73

73

 

 

 

 

Test Group c and d

Concentration
µg/mL

S9-mix

Mean number of colonies/200 cells/dish

Cloning
efficiency (%)

% of Control

Negative (Solvent Control)

203,3

102

100

 

Test item- Group c

50

201,3

101

99

60

200,7

100

99

65

202,0

101

99

70

200,0

100

98

75

202,3

101

100

80

200,3

100

99

85

199,0

100

98

90

0,0

0

0

EMS (1µL/mL)

141,7

71

70

Negative (Solvent Control)

203,0

102

100

Test item- Group d

50

201,0

101

99

60

200,0

100

99

65

201,3

101

99

70

201,0

101

99

75

202,0

101

100

80

201,7

101

99

85

201,0

101

99

90

0,0

0

0

EMS (1µL/mL)

142,7

71

70

 

 

Test group a and b

Concentration
µg/mL

S9-mix

Mean number of colonies/200 cells/dish

Cloning
efficiency (%)

% of Control

Negative (Solvent Control)

-

+

200,3

100

100

 

Test item- Group a

50

+

200,7

100

100

60

+

198,0

99

99

65

+

197,7

99

99

70

+

195,0

98

97

75

+

197,7

99

99

80

+

194,0

97

97

85

+

196,0

98

98

90

+

0,0

0

0

DMBA (20µg/mL)

+

156,3

78

78

Negative (Solvent Control)

-

+

201,3

101

100

Test item-Group b

50

+

199,3

100

99

60

+

198,3

99

99

65

+

198,0

99

98

70

+

197,0

99

98

75

+

198,7

99

99

80

+

196,7

98

98

85

+

196,3

98

98

90

+

0,0

0

0

DMBA (20µg/mL)

+

157,0

79

78

 

 

Test group c and d

Concentration
µg/mL

S9-mix

Mean number of colonies/200 cells/dish

Cloning
efficiency (%)

% of Control

Negative (Solvent Control)

-

+

200,3

100

100

 

Test item- Group c

50

+

198,7

99

99

60

+

196,0

98

98

65

+

196,3

98

98

70

+

195,0

98

97

75

+

196,3

98

98

80

+

194,7

97

97

85

+

194,0

97

97

90

+

0,0

0

0

DMBA (20µg/mL)

+

153,7

77

77

Negative (Solvent Control)

-

+

202,0

101

100

Test item-Group d

50

+

199,3

100

99

60

+

196,7

98

97

65

+

198,7

99

98

70

+

197,3

99

98

75

+

196,3

98

97

80

+

197,0

99

98

85

+

197,0

99

98

90

+

0,0

0

0

DMBA (20µg/mL)

+

153,0

77

76

 

 

 

Table 2 Historical Background for Solvent Control Mutant Frequency (2015-2016)

 

 

Without S9 mix

With S9 mix

5-hour treatment

5-hour treatment

Mean

6.37

6.66

SD

0.57

0.87

Range

4.90 – 8.82

4.12 – 11.76

Lower confidence interval

5.11

4.73

Upper confidence interval

7.63

8.59

n

11

11

 

SD      =         Standard deviation

n         =         number of experiments

 

Table 3 Historical Background for Solvent Control Mutant Frequency (2015-2016)

 

 

Without S9 mix

EMS

With S9 mix

DMBA

5-hour treatment

5-hour treatment

Mean

1516.10

750.93

SD

26.13

14.53

Range

1357.81 – 1636.92

690.00-810.29b

Lower confidence interval

1457.87

718.55

Upper confidence interval

1574.32

783.50

n

11

11

EMS   =         Ethyl methanesulphonate

DMBA           =         7,12-Dimethylbenzanthracene

SD      =         Standard deviation

n         =         number of experiments

Conclusions:
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. Thus, the test item was not mutagenic under the conditions of this study.


Executive summary:

The test item, dissolved in DMSO, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. 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:

50, 60, 65, 70, 75, 80, 85 and 901µg/mL

5-hour treatment period with S9-mix:

50, 60, 65, 70, 75, 80, 85 and 901µg/mL

1:This concentration was tested but was very toxic and there were not enough cells to start the phenotypic expression period after the treatment.

 

In the performed Mutation Assay the concentration levels were chosen 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 compatible of the laboratory historical control data.

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.

 

The validity of the test 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). The mutation frequency found in the positive controls was within the range of historical laboratory control data. 

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.

Thus, the test item was not mutagenic under the conditions of this study.

 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay:

The test item was dissolved in anhydrous dimethyl sulfoxide (DMSO). In the Initial and Confirmatory Mutation Tests the following concentrations were examined: -S9 Mix: 1600; 500; 160; 50; 16; 5 and 1.6 μg/plate; +S9 Mix: 5000; 1600; 500; 160; 50; 16 and 5 μg/plate. In the Initial and Confirmatory Mutation Tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were investigated. Five bacterial strains were used to investigate the mutagenic potential of the test item 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, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria, regarding the investigated strains, negative and positive controls, S9 activity and number of investigated analyzable 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 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values 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 performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was observed in all examined bacterial strains. The 160 μg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium TA1535 and TA1537 strains, in the absence of exogenous metabolic activation (-S9 Mix). Microdrops (as colloid-chemical phenomenon) was observed on the plates in the above bacterial strains at the concentration of 5000 μg/plate with addition of exogenous metabolic activation, following the plate incorporation and pre-incubation procedures (Initial and Confirmatory Mutation Tests).

In vitro chromosome aberration assay in mammalian cells

The test item Oligomeric Silane Adduct, dissolved in DMSO, 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 on (without and with metabolic activation using rat S9 mix) in accordance with the current OECD Guideline 473:

 

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 30, 50, 70 and 80 µg/mL test item

with S9 mix: 70, 80, 90 and 100 µg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 30, 50, 70 and 80 µg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 30, 50, 70 and 80 µg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 70, 80, 90 and 100 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. There were no relevant changes in pH or osmolality after treatment with the test item.

Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts at the concentration levels of 80 µg/mL and above.

There were no relevant increases in cells carrying structural chromosomal aberrations observed, neither in the absence nor in the presence of metabolic activation.

 

There were no biologically relevant increases in the rate of 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 historical laboratory control data. The concurrent positive controls ethylmethanesulphonate (0.4 and 1.0 L/mL) and cyclophosphamide (5 g/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.

 

HPRT Assay in vitro

The test item, dissolved in DMSO, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. 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:

50, 60, 65, 70, 75, 80, 85 and 901µg/mL

5-hour treatment period with S9-mix:

50, 60, 65, 70, 75, 80, 85 and 901µg/mL

1:This concentration was tested but was very toxic and there were not enough cells to start the phenotypic expression period after the treatment.

 

In the performed Mutation Assay the concentration levels were chosen 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 compatible of the laboratory historical control data.

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.

The validity of the test 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). The mutation frequency found in the positive controls was within the range of historical laboratory control data.  

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.

Thus, the test item was not mutagenic under the conditions of this study.

 

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 is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.