Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA. Therefore, the substance is considered non-mutagenic in a bacterial reverse mutation assay.

Two further in vitro genotoxicity studies with the structural analogue substance Reaction mass of calcium 2,6-bis(3-carboxylatopropanamido)hexanoate and isomers of calcium amino-(3-carboxylatopropanamido)hexanoate are available:

In a Chromosome Aberration Assay (CA) the analogue test item did not induce structural chromosome aberrations in Chinese Hamster lung cells. Furthermore, the analogue substance tested both without and with metabolic activation, did not induce increases in mutant frequency in mammalian 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:
9 October 2017 - 23 November 2017
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:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use
Version / remarks:
June 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
TOXI-COOP ZRT. 8230 Balatonfüred, Arácsi út 97. Hungary
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, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: S.ty.mur. TA98,100,1537,1535 rfa (cell wall), uvrB (DNA-repair) mutation
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from the Phenobarbital and β-Naphthoflavone-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 following concentrations were examined: ±S9 Mix: 5000, 1600, 500, 160, 50 and 16 μg/plate in Experiment I (Plate incorporation method) and Experiment II (pre-incubation method).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ultrapure water, Dimethyl sulfoxide (DMSO)

- Justification for choice of solvent/vehicle: Test item was completely dissolved in ultrapure water. In the study two vehicle control groups were used depending on the solubility of the test item and the solubility of positive control chemicals.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: NPD (4-Nitro-1,2-phenylenediamine)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- 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.

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 E.coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
Conditions for the Validity of the Test:
The tests (initial and confirmatory mutation experiments) are considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The E.coli uvrA culture demonstrates the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titer is in the 10E9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).
A dose level is considered toxic if:
- reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs
Statistics:
none
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test item was completely dissolved in ultrapure water.
- Precipitation: No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

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 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 not observed. The colony and background lawn development was not affected in any case. All of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

HISTORICAL CONTROL DATA (Please refer to "Any other information on results incl.tables")
In the Initial Mutation Test and Confirmatory Mutation Test most of the obtained higher revertant colony numbers (higher than the revertant colony numbers of the vehicle control) remained within the corresponding historical control data ranges.

Table 1: Summary Table of the Results of the Range Finding Test

Range Finding Test (Informatory Toxicity Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester 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

18.3

1.04

24.3

0.80

101.3

1.05

108.0

1.12

DMSO Control

16.0

1.00

27.7

1.00

93.0

1.00

Ultrapure Water Control

17.7

1.00

30.3

1.00

96.3

1.00

96.7

1.00

5000

25.0

1.42

24.7

0.81

82.3

0.85

118.0

1.22

1600

23.3

1.32

24.0

0.79

79.3

0.82

106.0

1.10

500

26.0

1.47

25.7

0.85

91.3

0.95

106.3

1.10

160

20.3

1.15

53.0

1.75

79.7

0.83

117.0

1.21

50

24.3

1.38

24.3

0.80

90.3

0.94

113.0

1.17

16

19.0

1.08

26.0

0.86

88.3

0.92

111.7

1.16

5

19.0

1.08

29.0

0.96

98.0

1.02

123.7

1.28

NPD (4mg)

257.7

16.10

SAZ (2mg)

1353.3

14.05

2AA (2mg)

1341.3

48.48

1212.0

13.03

MR:Mutation Rate

NPD:4-Nitro-1,2-phenylenediamine

SAZ:Sodium azid

2AA:2-aminoanthracene

 Ultrapure water was applied as vehicle of the test item and the positive control substance: SAZ and the DMSO was applied as vehicle for positive control substances: NPD and 2AA. The mutation rate of the test item, SAZ and untreated control is given referring to the ultrapure water; the mutation rate of NPD and 2AA is given referring to DMSO.

 

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

Initial Mutation Test (Plate Incorporation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-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

25.3

1.07

27.0

0.98

87.3

0.99

115.0

1.13

12.3

1.03

12.0

1.00

8.7

1.04

9.3

1.47

34.0

0.94

43.3

0.86

DMSO Control

20.3

1.00

26.0

1.00

94.7

1.00

12.0

1.00

6.0

1.00

9.3

1.00

40.0

1.00

Ultrapure Water Control

23.7

1.00

27.7

1.00

88.3

1.00

101.3

1.00

12.0

1.00

12.0

1.00

8.3

1.00

6.3

1.00

36.0

1.00

50.7

1.00

5000

31.3

1.32

30.3

1.10

95.3

1.08

101.0

1.00

14.3

1.19

10.3

0.86

10.3

1.24

7.7

1.21

45.7

1.27

59.0

1.16

1600

22.3

0.94

28.3

1.02

100.3

1.14

80.7

0.80

12.7

1.06

10.0

0.83

9.3

1.12

7.7

1.21

46.0

1.28

56.0

1.11

500

29.3

1.24

25.0

0.90

89.3

1.01

80.0

0.79

11.7

0.97

10.7

0.89

6.7

0.80

6.3

1.00

36.7

1.02

39.3

0.78

160

23.0

0.97

29.3

1.06

89.7

1.02

88.7

0.88

15.0

1.25

11.7

0.97

7.0

0.84

7.0

1.11

36.7

1.02

47.0

0.93

50

26.3

1.11

35.3

1.28

93.7

1.06

92.7

0.91

11.0

0.92

11.7

0.97

7.7

0.92

6.3

1.00

39.0

1.08

59.0

1.16

16

23.7

1.00

27.3

0.99

93.0

1.05

93.7

0.92

10.0

0.83

11.7

0.97

8.3

1.00

7.7

1.21

40.3

1.12

44.7

0.88

NPD (4mg)

464.3

22.84

SAZ (2mg)

1658.7

18.78

1717.3

143.11

9AA (50mg)

1052.7

175.44

MMS (2mL)

919.3

25.54

2AA (2mg)

2664.0

102.46

2954.7

31.21

258.0

21.50

239.0

25.61

2AA (50mg)

179.0

4.48

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

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

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

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-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

27.3

1.08

29.0

1.19

92.7

1.06

92.0

0.88

11.0

1.00

11.0

0.92

10.3

1.19

9.0

1.13

17.0

0.46

39.3

0.84

DMSO Control

23.0

1.00

22.7

1.00

94.3

1.00

13.0

1.00

7.3

1.00

6.3

1.00

38.7

1.00

Ultrapure Water Control

25.3

1.00

24.3

1.00

87.3

1.00

104.0

1.00

11.0

1.00

12.0

1.00

8.7

1.00

8.0

1.00

36.7

1.00

46.7

1.00

5000

25.3

1.00

37.3

1.53

97.7

1.12

106.0

1.02

10.7

0.97

11.7

0.97

7.3

0.85

12.0

1.50

52.0

1.42

51.3

1.10

1600

23.7

0.93

31.0

1.27

84.0

0.96

110.0

1.06

9.7

0.88

12.3

1.03

9.0

1.04

11.7

1.46

42.3

1.15

48.3

1.04

500

26.0

1.03

37.3

1.53

82.3

0.94

98.7

0.95

10.3

0.94

12.3

1.03

8.0

0.92

10.0

1.25

35.3

0.96

43.7

0.94

160

23.7

0.93

27.3

1.12

79.3

0.91

100.3

0.96

11.0

1.00

13.7

1.14

7.7

0.88

9.3

1.17

40.7

1.11

43.7

0.94

50

27.7

1.09

33.3

1.37

77.7

0.89

94.3

0.91

11.3

1.03

10.0

0.83

9.0

1.04

10.3

1.29

40.3

1.10

39.7

0.85

16

26.0

1.03

31.0

1.27

88.0

1.01

103.3

0.99

9.7

0.88

12.3

1.03

9.3

1.08

11.0

1.38

35.0

0.95

47.0

1.01

NPD (4mg)

253.3

11.01

SAZ (2mg)

797.3

9.13

509.3

46.30

9AA (50mg)

528.0

72.00

MMS (2mL)

741.3

20.22

2AA (2mg)

1532.0

67.59

2826.7

29.96

164.0

12.62

121.7

19.21

2AA (50mg)

243.7

6.30

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

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

Table 4: Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

 

Bacterial strains

Historical control data of untreated control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

21.0

105.0

10.5

8.1

25.4

SD

3.7

25.7

1.4

2.3

5.2

Minimum

9

66

3

2

11

Maximum

39

155

23

19

45

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

27.5

117.1

11.8

9.0

33.9

SD

4.3

18.1

1.4

1.9

5.2

Minimum

12

75

4

2

17

Maximum

46

166

23

20

56

 

Bacterial strains

Historical control data of DMSO

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

20.4

100.1

10.3

7.9

24.7

SD

3.6

24.8

1.3

2.4

4.6

Minimum

10

64

3

2

11

Maximum

38

147

23

20

45

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

26.5

113.8

11.8

8.8

33.7

SD

4.1

18.3

1.5

1.9

5.0

Minimum

15

71

3

3

16

Maximum

47

162

25

20

57

 

Bacterial strains

Historical control data of Water

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

21.9

104.7

10.5

7.6

26.1

SD

3.7

25.9

1.5

2.2

5.5

Minimum

12

68

3

2

12

Maximum

35

154

24

16

48

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

27.4

117.3

11.4

8.7

34.9

SD

4.0

18.5

1.3

2.2

4.9

Minimum

15

83

4

3

18

Maximum

43

167

22

16

57

 

Bacterial strains

Historical control data of positive controls

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

260.1

977.2

847.3

478.6

724.5

SD

31.8

150.6

126.3

104.5

65.0

Minimum

123

521

359

110

320

Maximum

664

1970

1855

1601

1313

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

1222.7

1436.4

164.1

147.0

257.7

SD

274.9

318.3

33.1

20.1

72.5

Minimum

386

583

85

69

140

Maximum

2676

2988

498

399

477

                           SD: Standard deviation

 

 

Conclusions:
The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the substance is considered non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

A bacterial reverse mutation assay according OECD TG 471, EU method B.13/14 and EPA OTS 789.5100 was performed to investigate the mutagenic potential in two independent experiments, in a plate incorporation test (Initial Mutation Test) and in a pre-incubation test (Confirmatory Mutation Test).

In the Initial and Confirmatory Mutation Tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were investigated. The test item was dissolved in ultrapure water. In the Initial and Confirmatory Mutation Tests the following concentrations were examined: ±S9 Mix: 5000, 1600, 500, 160, 50 and 16 μg/plate.

Five bacterial strains were used to investigate the mutagenic potential in two independent experiments. 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.

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.

The positive controls showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the substance is considered non-mutagenic in this bacterial reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 28, 2016 - April 5, 2017
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:
29 July, 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
30 May 2008
Deviations:
yes
Remarks:
There is a deviation from the guidelines regarding the maximum concentration. The maximum concentration was 2000 μg/ml, according to current Guideline (OECD 473, 29 July 2016) instead of 5000 μg/ml.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
August 1998
Deviations:
yes
Remarks:
There is a deviation from the guidelines regarding the maximum concentration. The maximum concentration was 2000 μg/ml, according to current Guideline (OECD 473, 29 July 2016) instead of 5000 μg/ml.
GLP compliance:
yes (incl. QA statement)
Remarks:
TOXI-COOP ZRT. 8230 Balatonfüred, Arácsi út 97. Hungary
Type of assay:
other: 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)
- Suitability of cells: Stability of karyotype and morphology makes them suitable for gene toxicity assays with low background aberrations.
- Cell cycle length, doubling time or proliferation index: doubling time 12-14 h
- Modal number of chromosomes: 2n=22
MEDIA USED
- Type and identity of media including CO2 concentration: DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine and 1 % of Antibiotic-antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated fetal bovine serum (final concentration 10 %), cultivated at 37°C in a humidified atmosphere containing 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Cytokinesis block (if used):
Colchicine
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:
The concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for soluble, lower-cytotoxic substances is 2000 μg/mL (based on the updated OECD Guideline 473 (2016)).
Experiment A with 3/20 h treatment/sampling time
without and with S9 mix: 500, 1000 and 2000 μg/mL
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 250, 500, 1000 and 2000 μg/mL
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 250, 500, 1000 and 2000 μg/mL
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 500, 1000 and 2000 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: medium (DME)
- Justification for choice of solvent/vehicle: This vehicle is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test, and its suitability is confirmed with the available laboratory’s historical database.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DME (Dulbecco’s Modified Eagle’s) medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
(EMS), without metabolic activation, 0.4 and 1.0 μL/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DME (Dulbecco’s Modified Eagle’s) medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation, 5.0 μg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 5x10E5

DURATION
- Exposure duration: 3 hours (Experiment A and Experiment B with S9 Mix) and 20 hours (Experiment B without S9 Mix)
- Expression time (cells in growth medium): 20 hours (Experiment A) and 28 hours (Experiment B)

SPINDLE INHIBITOR: colchicine (0.2 μg/mL)

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation became plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies. For control of bias, all slides were coded and scored blind.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE
300 metaphase cells containing 2 N ± 2 centromeres were evaluated for structural aberrations from each experimental group.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was determined by cell counting, based on the cell counts Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative because:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.
Statistics:
For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity assay a 3-hour treatment in the absence of S9 Mix was performed with the concentration levels of: 500, 1000 and 2000 μg/mL. The obtained toxicity range was 3.33-22.82 %. At the 3-hour treatment (20 h sampling time) in the presence of S9 Mix the observed range of toxicity was -1.00- 14.54 % at concentration levels of 250, 500, 1000 and 2000 μg/mL. In case of 20/20, 28 h treatment/sampling times examined concentrations were: 125, 250, 500, 1000 and 2000 μg/mL. The observed toxicity range was -0.51-57.42 %. At the 3-hour treatment (28 h sampling time) in the presence of S9 Mix the obtained range of toxicity was 0.21-15.91% at concentration levels of 250, 500, 1000 and 2000 μg/mL.

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


Table 1: Mean Percentage of cells with structural chromosome aberrations (Experiment A)

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Mean aberrant cells/
150 cells

incl. gaps

excl. gaps

Solvent control
(DME)

-

3 h

20 h

5

3

Retardan 200 P

500µg/mL

-

3 h

20 h

5

2

1000µg/mL

-

3 h

20 h

7

4

2000µg/mL

-

3 h

20 h

6

3

Pos. Control (EMS)

-

3 h

20 h

48**

35**

Solvent control
(DME)

+

3 h

20 h

6

3

Retardan 200 P

500µg/mL

+

3 h

20 h

8

3

1000µg/mL

+

3 h

20 h

9

4

2000µg/mL

+

3 h

20 h

7

3

Pos. Control (Cycl.)

+

3 h

20 h

47**

40**

Positive control (-S9): Ethyl methanesulfonate (1.0 µL/mL),Positive control (+S9): Cyclophosphamide (5.0 µg/mL);** = p < 0.01 to the concurrent negative control and to the historical control

Table 2: Mean Percentage of cells with structural chromosome aberrations (Experiment B)

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Mean aberrant cells/
150cells

incl. gaps

excl. gaps

Solvent control
(DME)

-

20 h

20 h

5

3

Retardan 200 P

250 µg/mL

-

20 h

20 h

6

3

500µg/mL

-

20 h

20 h

7

3

1000µg/mL

-

20 h

20 h

8

4

2000µg/mL

-

20 h

20 h

7

3

Pos. Control (EMS)

-

20 h

20 h

50**

43**

Solvent control
(DME)

-

20 h

28 h

5

3

Retardan 200 P

250 µg/mL

-

20 h

28 h

7

4

500µg/mL

-

20 h

28 h

7

3

1000µg/mL

-

20 h

28 h

7

4

2000µg/mL

-

20 h

28 h

7

4

Pos. Control (EMS)

-

20 h

28 h

45**

39**

Positive control (-S9): Ethyl methanesulfonate (0.4 µL/mL);** = p < 0.01 to the concurrent negative control and to the historical control

Table 3: Mean Percentage of cells with structural chromosome aberrations (Experiment B)- continued

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Mean aberrant cells/
150 cells

incl. gaps

excl. gaps

Solvent control
(DME)

+

3 h

28 h

5

2

Retardan 200 P

500µg/mL

+

3 h

28 h

6

4

1000µg/mL

+

3 h

28 h

6

3

2000µg/mL

+

3 h

28 h

7

3

Pos. Control (Cycl.)

+

3 h

28 h

48**

39**

Positive control (+S9): Cyclophosphamide (5.0 µg/mL); ** = p < 0.01 to the concurrent negative control and to the historical control

Table 4: Number of Polyploid cells and Endoreduplicated cells (Experiment A)

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Polyploid Cells (mean)

Endoredup-lication (mean)

Solvent control
(DME)

-

3 h

20 h

0.0

0.0

Retardan 200 P

500µg/mL

-

3 h

20 h

0.0

0.0

1000µg/mL

-

3 h

20 h

0.0

0.0

2000µg/mL

-

3 h

20 h

0.0

0.0

Pos. Control (EMS)*

-

3 h

20 h

0.0

0.0

Solvent control
(DME)

+

3 h

20 h

0.0

0.0

Retardan 200 P

500µg/mL

+

3 h

20 h

0.0

0.0

1000µg/mL

+

3 h

20 h

0.0

0.0

2000µg/mL

+

3 h

20 h

0.0

0.0

Pos. Control (Cycl.)**

+

3 h

20 h

0.0

0.0

* Ethyl methanesulfonate (1.0 µL/mL); ** Cyclophosphamide (5.0 µg/mL); The number of polyploid and endoreduplicated cells was determined in 150 cells of each test group.

Table 5: Number of Polyploid cells and Endoreduplicated cells (Experiment B)

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Polyploid Cells (mean)

Endoredup-lication (mean)

Solvent control
(DME)

-

20 h

20 h

0.0

0.0

Retardan 200 P

250 µg/mL

-

20 h

20 h

0.0

0.0

500µg/mL

-

20 h

20 h

0.0

0.0

1000µg/mL

-

20 h

20 h

0.0

0.0

2000µg/mL

-

20 h

20 h

0.0

0.0

Pos. Control (EMS)

-

20 h

20 h

0.0

0.0

Solvent control
(DME)

-

20 h

28 h

0.0

0.0

Retardan 200 P

250 µg/mL

-

20 h

28 h

0.0

0.0

500µg/mL

-

20 h

28 h

0.0

0.0

1000µg/mL

-

20 h

28 h

0.0

0.0

2000µg/mL

-

20 h

28 h

0.0

0.0

Pos. Control (EMS)

-

20 h

28 h

0.0

0.0

Ethyl methanesulfonate (0.4 µL/mL)

Table 6: Number of Polyploid cells and Endoreduplicated cells (Experiment B)- continued

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Polyploid Cells (mean)

Endoredup-lication (mean)

Solvent control
(DME)

+

3 h

28 h

0.0

0.0

Retardan 200 P

500µg/mL

+

3 h

28 h

0.0

0.0

1000µg/mL

+

3 h

28 h

0.0

0.0

2000µg/mL

+

3 h

28 h

0.0

0.0

Pos. Control (Cycl.)**

+

3 h

28 h

0.0

0.0

**Cyclophosphamide (5.0 µg/mL); The number of polyploid and endoreduplicated cells was determined in 150 cells of each test group.

Table 7: Historical Control Data

A. 3h/20h treatment/sampling time without S9-mix

 

number of aberrant cells/ 150 cells

negative control

positive control
(Ethyl methanesulfonate)

incl. Gaps

excl. Gaps

incl. Gaps

excl. Gaps

Mean

5.70

2.65

40.20

30.85

SD

0.63

0.59

3.83

3.41

Lower confidence interval

4.27

1.31

31.53

23.13

Upper confidence interval

7.13

3.99

48.87

38.57

n

10

10

10

10

B. 3h/20h treatment/sampling time with S9-mix

 

number of aberrant cells/150cells

negative control

positive control
(
Cyclophosphamide)

incl. Gaps

excl. Gaps

incl. Gaps

excl. Gaps

Mean

5.75

2.80

46.40

39.65

SD

0.74

0.55

2.10

1.83

Lower confidence interval

4.07

1.56

41.66

35.51

Upper confidence interval

7.43

4.04

51.14

43.79

n

10

10

10

10

C. 20h/20h treatment/sampling time without S9-mix

 

number of aberrant cells/150cells

negative control

positive control
(Ethyl methanesulfonate)

incl. Gaps

excl. Gaps

incl. Gaps

excl. Gaps

Mean

5.70

2.85

45.20

37.85

SD

1.00

0.59

2.32

2.13

Lower confidence interval

3.44

1.51

39.94

33.02

Upper confidence interval

7.96

4.19

50.46

42.68

n

10

10

10

10

D. 20h/28h treatment/sampling time without S9-mix

 

number of aberrant cells/ 150cells

negative control

positive control
(Ethyl methanesulfonate)

incl. Gaps

excl. Gaps

incl. Gaps

excl. Gaps

Mean

5.45

2.75

45.95

36.85

SD

0.67

0.59

2.29

2.20

Lower confidence interval

3.93

1.41

40.77

31.87

Upper confidence interval

6.97

4.09

51.13

41.83

n

10

10

10

10

E. 3h/28h treatment/sampling time with S9-mix

 

number of aberrant cells/ 150 cells

negative control

positive control
(
Cyclophosphamide)

incl. Gaps

excl. Gaps

incl. Gaps

excl. Gaps

Mean

5.75

2.75

45.90

38.90

SD

0.50

0.59

2.07

3.29

Lower confidence interval

4.62

1.41

41.21

31.47

Upper confidence interval

6.88

4.09

50.59

46.33

n

10

10

10

10

n           = number of experiments

SD        = standard deviation

Conclusions:
The test item tested up to the maximum recommended concentration with and without mammalian metabolic activation system (three hours treatment) did not induce structural chromosome aberrations in Chinese Hamster lung cells. The test item tested up to cytotoxic concentrations, without mammalian metabolic activation system (twenty hours treatment) did not induced structural chromosome aberrations in Chinese Hamster lung cells. Thus, the test item is considered as not clastogenic in this system.
Executive summary:

A study according OECD TG 473 was conducted to detect chromosome aberrations in Chinese hamster lung cells (V79).

The test item was suspended in cell medium (DME, Dulbecco’s Modified Eagle’s) and the concentrations tested 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 the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 300 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below:

Experiment A with 3/20 h treatment/sampling time

without and with S9 mix: 500, 1000 and 2000 μg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 250, 500, 1000 and 2000 μg/mL

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 250, 500, 1000 and 2000 μg/mL

Experiment B with 3/28 h treatment/sampling time

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

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to the maximum recommended concentration. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent and historical controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to the maximum recommended concentration in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberrations were inside the distribution of the laboratory historical negative control data.

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.

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

The test item tested up to the maximum recommended concentration with and without mammalian metabolic activation system (three hours treatment) did not induce structural chromosome aberrations in Chinese Hamster lung cells. The test item tested up to cytotoxic concentrations, without mammalian metabolic activation system (twenty hours treatment) did not induced structural chromosome aberrations in Chinese Hamster lung cells. Thus, the test item is considered as not clastogenic in this system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 28, 2016 - July 6, 2017
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:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Deviations:
yes
Remarks:
Negative results were not confirmed as the confirmation of negative results is not required by the most current OECD Guideline; maximum recommended concentration was 2000 μg/mL instead of 5000 μg/mL.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Deviations:
yes
Remarks:
Negative results were not confirmed as the confirmation of negative results is not required by the most current OECD Guideline; maximum recommended concentration was 2000 μg/mL instead of 5000 μg/mL.
GLP compliance:
yes (incl. QA statement)
Remarks:
TOXI-COOP ZRT. 8230 Balatonfüred, Arácsi út 97. Hungary
Type of assay:
bacterial forward mutation assay
Target gene:
The method is based on the detection of mutations (either induced or spontaneously generated) in the hprt locus located on the X chromosome. HPRT (hypoxanthine-guanine phosphoribosyl transferase) is a cellular enzyme that allows cells to salvage hypoxanthine and guanine from surrounding medium for use in DNA synthesis.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
Sub-line (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: Ham's F12 medium 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 %), cell were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air. During treatment, serum content was reduced to 5 % (F12-5).
- 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 liver
Test concentrations with justification for top dose:
Treatment concentrations for the mutation assay were selected on the basis of the result of a pre-test on toxicity.
5-hour treatment period without S9-mix:
125, 250, 500, 1000 and 2000 μg/mL
5-hour treatment period with S9-mix:
125, 250, 500, 1000 and 2000 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: medium
- Justification for choice of solvent/vehicle: This vehicle is compatible with the survival of the CHO cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test, and its suitability is confirmed with the available laboratory’s historical database.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ham's F12 medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation, 1.0 μL/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation, 20 μg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 5x10E6 cells/dish

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 19 hours
- Selection time: 8 Days

SELECTION AGENT: hypoxanthine Ham's F12-SEL medium containing 3.4 μg/mL of thioguanine (6-TG)

NUMBER OF REPLICATIONS: 2

STAINING TECHNIQUE USED: After the selection period, the colonies were fixed, stained with Giemsa and counted for mutant selection and cloning efficiency determination.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Test item is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria 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 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:
no 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
- Solubility: The test item was suspended in Ham's F12 medium. A homogen suspension was obtained up to a concentration of 100 mg/mL.
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES:
A Pre-test on Toxicity was performed to establish an appropriate concentration range for the main mutation assay, both in the absence and in the presence of metabolic activation (rodent S9-mix). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control. The Relative survival in percent of cells treated with the test item in presence and absence of metabolic activation was not significantly different from the solvent control.

HISTORICAL CONTROL DATA: Please refer to "any other information including tables"

Table 1: Summarized results of the concentration selection cytotoxicity assay

Test group

Dose
µg/ml

S9-mix

Treatment/
time/ hour

Number of colonies/200 cells/dish

Mean

Relativea
survival
in percent

dish 1

dish 2

dish 3

Solvent Control (Ham's F12 medium)

5

204

205

202

203,7

100

Retardan 200 P

125

5

203

204

205

204,0

100

250

5

205

201

201

202,3

99

500

5

199

203

204

202,0

99

1000

5

204

204

200

202,7

100

1500

 

5

201

203

199

201,0

99

2000

5

202

200

204

202,0

99

Solvent Control (Ham's F12 medium)

+

5

202

206

200

202,7

100

Retardan 200 P

125

+

5

204

201

201

202,0

100

250

+

5

205

202

199

202,0

100

500

+

5

201

200

197

199,3

98

1000

+

5

197

204

201

200,7

99

1500

+

5

202

203

200

201,7

100

2000

+

5

200

198

203

200,3

99

a Relative to sovent control

Table 2 Main mutation assay summary of results without S9-Mix

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control a

204.7

±

1.53

100

100

1

0

1

1

3

6

101

5.94

Pos. control
(
EMS 1.0 µL/mL) a

51.7

±

1.53

25

60

198

194

198

203

206

999

61

1637.70**

TEST ITEM

 

125 µg/mL a

202,3

±

1,53

99

99

0

1

0

1

3

5

100

5.00

250 µg/mL a

203,3

±

2,89

99

99

3

1

3

0

0

7

100

7.00

500 µg/mL a

203,3

±

1,53

99

98

2

2

0

0

1

5

99

5.05

1000 µg/mL a

204,3

±

2,89

100

98

0

1

2

0

2

6

99

6.06

2000 µg/mL a

203,3

±

1,53

99

98

0

2

0

3

2

7

99

7.07

a = 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

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control b

203.0

±

0.00

100

100

1

0

3

0

2

6

100

6.00

Pos. control
(
EMS 1.0 µL/mL) b

52.3

±

1.53

26

60

191

195

200

203

197

986

60

1643.33**

TEST ITEM

 

125 µg/mL b

200,3

±

0,58

99

100

0

1

2

1

1

5

100

5.00

250 µg/mL b

201,3

±

1,53

99

99

1

0

1

3

3

8

99

8.08

500 µg/mL b

202,7

±

0,58

100

99

4

0

0

0

2

6

99

6.06

1000 µg/mL b

200,7

±

1,15

99

99

0

1

2

1

3

7

99

7.07

2000 µg/mL b

202,3

±

1,53

100

99

2

2

2

1

0

4

99

4.04

b = parallel of first culture;abs.C.E. = Absolute Cloning Efficiency;EMS=Ethyl methanesulfonate;** = p < 0.01 to the concurrent negative control and to the historical control

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control c

202.3

±

2.52

100

100

3

1

0

0

2

6

101

5.94

Pos. control
(
EMS 1.0µL/mL) c

53.0

±

2.65

26

61

204

210

199

203

196

1012

62

1632.25**

TEST ITEM

 

125 µg/mL c

202,0

±

2,65

100

99

1

0

4

0

1

6

101

5.94

250 µg/mL c

203,0

±

1,73

100

100

1

0

2

2

2

7

101

6.93

500 µg/mL c

201,0

±

3,46

99

98

0

0

3

3

2

8

99

8.08

1000 µg/mL c

202,3

±

2,08

100

99

1

1

1

1

1

5

100

5.00

2000 µg/mL c

200,0

±

2,65

99

98

3

1

2

0

1

7

99

7.07

c = 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

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control d

201.3

±

1.15

100

100

2

1

2

1

1

7

101

6.93

Pos. control
(
EMS 1.0 µL/mL) d

52.7

±

.53

26

61

204

199

196

201

207

1007

61

1650.81**

TEST ITEM

 

125 µg/mL d

200,0

±

1,00

99

100

0

0

5

1

0

6

101

5.94

250 µg/mL d

201,3

±

1,15

100

100

3

2

1

1

1

8

101

7.92

500 µg/mL d

200,7

±

2,89

100

99

2

0

1

1

1

5

100

5.00

1000 µg/mL d

200,3

±

0,58

100

99

3

0

0

3

2

8

100

8.00

2000 µg/mL d

199,7

±

0,58

99

99

1

1

0

2

2

6

99

6.06

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 3 Main mutation assay summary of results with S9-Mix

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control a

201.3

±

2.52

100

100

0

3

2

3

0

8

100

8.00

Pos. control
(
DMBA 20 µg/mL) a

113.0

±

2.65

56

75

103

111

114

101

117

546

75

728.00**

TEST ITEM

 

125 µg/mL a

202,3

±

1,53

100

100

0

1

4

1

0

6

99

6.06

250 µg/mL a

199,3

±

1,15

99

100

1

2

0

0

4

7

100

7.00

500 µg/mL a

202,0

±

1,00

100

98

0

3

0

1

3

7

98

7.14

1000 µg/mL a

201,7

±

3,51

100

98

2

3

0

3

0

8

98

8.16

2000 µg/mL a

199,7

±

0,58

99

100

1

2

2

0

2

7

100

7.00

a = parallel of first culture; abs.C.E. = Absolute Cloning Efficiency; DMBA=7,12-Dimethyl benzanthracene; ** = p < 0.01 to the concurrent negative control and to the historical control

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control b

202.0

±

1.00

100

100

0

1

1

1

2

5

100

5.00

Pos. control
(
DMBA 20 µg/mL) b

112.0

±

2.00

55

74

113

117

99

108

116

553

75

737.33**

TEST ITEM

 

125 µg/mL b

200,3

±

0,58

99

99

1

0

3

0

2

6

99

6.06

250 µg/mL b

200,0

±

0,00

99

99

1

1

1

3

1

7

99

7.07

500 µg/mL b

201,3

±

0,58

100

99

0

0

2

4

1

7

99

7.07

1000 µg/mL b

200,0

±

2,00

99

99

2

1

0

2

0

5

99

5.05

2000 µg/mL b

200,7

±

0,58

99

99

2

2

1

1

2

8

99

8.08

b = parallel of first culture;abs.C.E. = Absolute Cloning Efficiency;DMBA=7,12-Dimethyl benzanthracene;** = p < 0.01 to the concurrent negative control and to the historical control

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control c

202.3

±

2.89

100

100

0

0

3

3

1

7

101

6.93

Pos. control
(
DMBA20 µg/mL) c

114.3

±

1.53

57

70

120

114

110

118

112

574

71

808.45**

TEST ITEM

 

125 µg/mL c

204,3

±

1,15

101

100

0

1

1

2

2

6

101

5.94

250 µg/mL c

203,3

±

2,08

100

98

4

1

1

0

1

7

99

7.07

500 µg/mL c

200,3

±

2,52

99

99

4

0

0

1

2

7

100

7.00

1000 µg/mL c

199,7

±

3,06

99

100

2

0

2

1

0

5

101

4.95

2000 µg/mL c

200,3

±

2,08

99

99

0

3

1

1

2

7

100

7.00

c = parallel of second culture;abs.C.E. = Absolute Cloning Efficiency;DMBA=7,12-Dimethyl benzanthracene

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control d

202.0

±

1.00

100

100

0

1

1

4

2

8

101

7.92

Pos. control
(
DMBA 20 µg/mL) d

112.0

±

2.00

55

70

119

110

107

115

112

563

70

804.28**

TEST ITEM

 

125 µg/mL d

201,0

±

1,73

100

99

3

3

0

0

1

7

100

7.00

250 µg/mL d

200,7

±

0,58

99

99

0

0

0

4

2

6

99

6.06

500 µg/mL d

200,7

±

2,08

99

99

1

0

1

3

3

8

100

8.00

1000 µg/mL d

200,7

±

2,08

99

99

0

1

5

2

0

8

100

8.00

2000 µg/mL d

200,7

±

1,15

99

99

0

1

0

4

0

5

100

5.00

d = parallel of second culture;abs.C.E. = Absolute Cloning Efficiency;DMBA=7,12-Dimethyl benzanthracene

Table 4 Historical control data (for solvent control mutant frequency 2016 -2017)

 

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

Table 5 Historical control data (for positive control mutant frequency)

 

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

690.00-810.29

Lower confidence interval

1457.87

718.55

Upper confidence interval

1574.32

783.30

n

11

11

EMS    =   Ethyl methanesulphonate; DMBA=   7,12-Dimethylbenzanthracene; SD =   Standard deviation; n =   number of experiments

Conclusions:
The test item tested both without and with metabolic activation, did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells. Thus, it was not mutagenic under the conditions of this study.
Executive summary:

A study was conducted according OECD TG 476 to determine the mutagenic potential of the test item in mammalian Chinese hamster Ovary cells (CHO-K1). 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.

5-hour treatment period without S9-mix:

125, 250, 500, 1000 and 2000 μg/mL

5-hour treatment period with S9-mix:

125, 250, 500, 1000 and 2000 μg/mL

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

Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was 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 suspension 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 substance tested 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 maximum recommended concentration. Thus, it was not mutagenic under the conditions of this study.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to section 13 for "Read-Across justification".
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to section 13 for "Read-Across justification".
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames Test

A bacterial reverse mutation assay according OECD TG 471, EU method B.13/14 and EPA OTS 789.5100 was performed to investigate the mutagenic potential in two independent experiments, in a plate incorporation test (Initial Mutation Test) and in a pre-incubation test (Confirmatory Mutation Test).

In the Initial and Confirmatory Mutation Tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were investigated. The test item was dissolved in ultrapure water. In the Initial and Confirmatory Mutation Tests the following concentrations were examined: ±S9 Mix: 5000, 1600, 500, 160, 50 and 16 μg/plate.

Five bacterial strains were used to investigate the mutagenic potential in two independent experiments. 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.

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.

The positive controls showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the substance is considered non-mutagenic in this bacterial reverse mutation assay.

Two further in vitro genotoxicity studies with the structural analogue substance Reaction mass of calcium 2,6-bis(3-carboxylatopropanamido)hexanoate and isomers of calcium amino-(3-carboxylatopropanamido)hexanoate are available:

Chromosome Aberration Assay

A study according OECD TG 473 was conducted to detect chromosome aberrations in Chinese hamster lung cells (V79). The test item was suspended in cell medium (DME, Dulbecco’s Modified Eagle’s) and the concentrations tested were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital andβ-naphthoflavone induced rat liver). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 300 well-spread metaphase cells were analysed at concentrations and incubation/ expression intervals given below:

Experiment A with 3/20 h treatment/sampling time

without and with S9 mix: 500, 1000 and 2000 μg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 250, 500, 1000 and 2000 μg/mL

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 250, 500, 1000 and 2000 μg/mL

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 500, 1000 and 2000 μg/mL

 In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to the maximum recommended concentration. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent and historical controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to the maximum recommended concentration in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberrations were inside the distribution of the laboratory historical negative control data.

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.

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

The test item tested up to the maximum recommended concentration with and without mammalian metabolic activation system (three hours treatment) did not induce structural chromosome aberrations in Chinese Hamster lung cells. The test item tested up to cytotoxic concentrations, without mammalian metabolic activation system (twenty hours treatment) did not induced structural chromosome aberrations in Chinese Hamster lung cells. Thus, the test item is considered as not clastogenic in this system.

HPRT Assay

A study was conducted according OECD TG 476 to determine the mutagenic potential of the test item in mammalian Chinese hamster Ovary cells (CHO-K1). 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.

5-hour treatment period without S9-mix:

125, 250, 500, 1000 and 2000 μg/mL

5-hour treatment period with S9-mix:

125, 250, 500, 1000 and 2000 μg/mL

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

Phenotypic expression was evaluated up to 8 days following exposure. In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was 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 suspension 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 (1.0μL/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 substance tested 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 maximum recommended concentration. Thus, it 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 for the target substance and a structural analogue, the test item is not classified as mutagenic or clastogenic according to Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.