Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assey test using Salmonella typhimurium and Escherichia coli, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used with and without metabolic activation.

Results of a gene mutation assay in mammalian cells according to OECD 476 did also not indicate mutation potential of the test substance.

The test item tested up to cytotoxic concentrations, without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. The test item tested up to cytotoxic concentrations, with mammalian metabolic activation system, induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung cells. Thus, the test item is considered clastogenic in this system.


Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-03-18 to 2014-04-03
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:
adopted 21st July, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated May 30, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. Main DNA target GC.
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-mix (rat liver induced by Phenobarbital (PB) and β-naphthoflavone (BNF)
Test concentrations with justification for top dose:
5000; 1581, 500, 158, 50 and 15.8 μg/plate
Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test (Informatory Toxicity Test).
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: Test item is good soluble in DMSO
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
E.coli and all of Salmonella strains with S9-mix
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E.coli WP2 uvrA without S9-mix
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Salmonella TA1537 without S9-mix
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Salmonella TA100 and TA1535 without S9-mix
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine (NPD)
Remarks:
Salmonella TA98 without S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation) and pre-incubation
The Experiment I (Initial Mutation Test) of the main study the plate incorporation method was used. In the Experiment II (Confirmatory Mutation Test) the pre-incubation method was applied and the concentrations examined were the same as investigated in the Experiment I.

DURATION
- Preincubation period: 20 min at 37 °C (bacterial culture and the S9 Mix or phosphate buffer)
- Exposure duration: 48 hours in the dark at 37 °C

NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth; Toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment.
Evaluation criteria:
Evaluation of Experimental Data

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


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 Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia WP2 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
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the 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
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the Confirmatory Mutation the absence of revertant growth and reduced background lawn development was obtained in S. typhimurium TA1535 and TA1537 at 5000 μg/plate (-S9 Mix) and in S. typhimurium TA1535 at 1581 μg/plate (-S9 Mix).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES:
Pre-Experiment for Toxicity

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, 1581, 500, 158, 50, 15.8 and 5 μg/plate.
The obtained revertant colony numbers were slightly lower than the revertant colony numbers of the vehicle control in S. typhimurium TA100, in the whole examined concentration range of 5000-5 μg/plate, with addition of metabolic activation (+S9 Mix).
Slightly higher revertant colony counts were obtained in S. typhimurium TA98 at the concentrations of 50 μg/plate, with addition of metabolic activation (+S9 Mix) and in TA100 in the concentration range of 5000-5 μg/plate, without metabolic activation (-S9 Mix).

COMPARISON WITH HISTORICAL CONTROL DATA: The obtained changes, slightly lower or higher revertant counts remained in the corresponding historical control data and biological variability range of the applied test system.

Table 1 summary of results of 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

13.7

0.84

23.7

1.09

87.3

1.10

135.3

1.24

5.7

0.74

10.7

1.03

8.7

1.00

7.0

0.91

25.3

1.01

25.7

0.94

DMSO Control

16.3

1.00

21.7

1.00

79.7

1.00

109.0

1.00

7.7

1.00

10.3

1.00

8.7

1.00

7.7

1.00

25.0

1.00

27.3

1.00

Ultrapure Water Control

90.3

1.00

7.0

1.00

30.0

1.00

5000

11.3

0.69

21.0

0.97

114.0

1.43

132.7

1.22

10.0

1.30

9.3

0.90

12.3

1.42

10.3

1.35

20.7

0.83

26.3

0.96

1581

13.7

0.84

19.3

0.89

102.7

1.29

124.0

1.14

7.3

0.96

12.3

1.19

7.3

0.85

9.7

1.26

25.3

1.01

28.0

1.02

500

15.3

0.94

20.0

0.92

88.0

1.10

122.0

1.12

8.7

1.13

11.3

1.10

8.0

0.92

9.0

1.17

21.7

0.87

29.0

1.06

158

13.0

0.80

22.3

1.03

85.0

1.07

117.0

1.07

6.0

0.78

9.0

0.87

7.0

0.81

7.0

0.91

25.7

1.03

33.0

1.21

50

11.3

0.69

21.0

0.97

90.0

1.13

120.7

1.11

7.3

0.96

9.0

0.87

9.0

1.04

6.3

0.83

23.0

0.92

29.7

1.09

15.8

13.3

0.82

22.0

1.02

87.7

1.10

114.0

1.05

6.3

0.83

10.3

1.00

8.7

1.00

8.7

1.13

27.7

1.11

26.0

0.95

NPD (4mg)

173.3

10.61

SAZ (2mg)

948.0

10.49

729.3

104.19

9AA (50mg)

452.7

52.23

MMS (2mL)

826.0

27.53

2AA (2mg)

1725.3

79.63

1856.0

17.03

209.3

20.26

175.0

22.83

2AA (50mg)

284.3

10.40

 

Table 2 Summary of results of 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

16.7

1.09

23.3

1.06

108.7

1.32

139.3

1.23

9.3

0.93

17.7

1.20

6.3

0.79

7.3

0.79

31.3

1.12

42.0

1.09

DMSO Control

15.3

1.00

22.0

1.00

82.3

1.00

113.7

1.00

10.0

1.00

14.7

1.00

8.0

1.00

9.3

1.00

28.0

1.00

38.7

1.00

Ultrapure Water Control

78.7

1.00

9.3

1.00

40.0

1.00

5000

5.3

0.35

14.3

0.65

44.7

0.54

100.0

0.88

0.0

0.00

12.7

0.86

0.0

0.00

11.3

1.21

23.3

0.83

29.7

0.77

1581

13.0

0.85

20.7

0.94

82.7

1.00

112.7

0.99

5.7

0.57

14.3

0.98

7.3

0.92

9.0

0.96

32.7

1.17

39.7

1.03

500

13.7

0.89

20.0

0.91

95.3

1.16

114.3

1.01

10.3

1.03

13.0

0.89

6.3

0.79

7.0

0.75

35.0

1.25

43.7

1.13

158

14.3

0.93

23.0

1.05

89.7

1.09

115.7

1.02

10.0

1.00

11.0

0.75

6.7

0.83

8.3

0.89

31.3

1.12

41.0

1.06

50

13.7

0.89

20.3

0.92

84.7

1.03

127.3

1.12

10.3

1.03

13.3

0.91

7.7

0.96

7.0

0.75

28.0

1.00

43.3

1.12

15.8

14.3

0.93

24.3

1.11

87.3

1.06

122.3

1.08

11.7

1.17

14.0

0.95

8.7

1.08

8.0

0.86

24.0

0.86

40.0

1.03

NPD (4mg)

178.0

11.61

SAZ (2mg)

1230.0

15.64

497.3

53.29

9AA (50mg)

365.0

45.63

MMS (2mL)

1096.0

27.40

2AA (2mg)

709.3

32.24

1310.7

11.53

120.3

8.20

111.7

11.96

2AA (50mg)

261.7

6.77

 

Table Historical control data for Revertants/Plate (for the period of 2008-2013)

 

Bacterial strains

Historical control data of untreated control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

22.6

110.1

10.1

7.5

24.7

SD

3.3

31.0

1.4

2.7

4.2

Minimum

11

66

2

2

11

Maximum

40

162

23

19

41

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

29.6

117.7

12.0

8.7

34.2

SD

3.6

22.9

1.6

2.3

3.5

Minimum

13

76

4

2

20

Maximum

48

170

24

20

54

 

Bacterial strains

Historical control data of DMSO

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

22.1

106.0

9.9

7.4

23.8

SD

3.1

29.3

1.2

2.8

3.5

Minimum

11

66

3

2

10

Maximum

40

156

23

18

42

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

28.6

117.4

12.1

8.4

34.2

SD

3.4

22.1

1.6

2.2

3.8

Minimum

17

73

3

2

17

Maximum

49

168

25

18

55

 

Bacterial strains

Historical control data of Water

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

23.8

110.3

9.9

6.5

25.2

SD

6.8

30.9

1.4

3.4

3.8

Minimum

9

70

2

1

12

Maximum

43

159

24

16

45

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

29.8

118.8

11.8

7.8

35.1

SD

7.4

23.0

1.5

3.4

4.9

Minimum

10

81

3

2

19

Maximum

48

174

24

20

55

 

Bacterial strains

Historical control data of positive controls

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

199.6

904.8

792.5

457.5

700.3

SD

35.1

107.9

106.0

111.8

58.9

Minimum

165

477

332

110

341

Maximum

248

1953

1278

1439

1236

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

199.6

1340.5

173.3

152.1

280.8

SD

35.1

347.7

37.6

23.3

80.0

Minimum

248

491

87

68

144

Maximum

165

2869

603

465

520

 

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 test item is considered non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

The Bacterial Reverse Mutation Assay (using Salmonella typhimurium and Escherichia coli) with the test item was conducted according to the OECD guideline 471 and GLP. The test item was suspended respectively dissolved in dimethyl sulfoxide (DMSO). Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential. 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. The tested test item concentrations were: 5000, 1581, 500, 158, 50 and 15.8 μg/plate.

In the performed experiments positive and negative (vehicle) controls were run concurrently. The revertant colony numbers of vehicle control plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants in the vehicle controls and were within the corresponding historical control data ranges. The reference mutagens showed a distinct increase of induced revertant colonies. In the performed experimental phases at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain were applied. The validity criteria of the study 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 mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. Slight, unequivocal inhibitory effect of the test item was observed in the Confirmatory Mutation Test (Pre-Incubation Test) in the examined Salmonella typhimurium strains at the concentration of 5000 μg/plate in absence of exogenous metabolic activation (-S9 Mix).

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

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-14 to 2017-05-02
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:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
yes
Remarks:
please refer to "Principles of method if other than guideline"
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
Deviations:
yes
Remarks:
please refer to "Principles of method if other than guideline"
Principles of method if other than guideline:
There are deviations from the guidelines regarding the confirmation of negative results and the maximum recommended concentration.
Negative results were not confirmed as the confirmation of negative results is not required by the most current Guideline (OECD 476, 29 July 2016).
The maximum recommended concentration was 2000 μg/mL, according to current Guideline (OECD 476, 29 July 2016) instead of 5000 μg/mL.
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian gene mutation assay
Target gene:
HPRT locus
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 %).
During the 5 hour treatment with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (F12-5). The selection medium for TG resistant mutants contained 3.4 μg/mL of 6-thioguanine (6-TG)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
without S9: 125, 250, 500, 1000, 2000, µg/mL (Solvent (DMSO) 10 µL/mL; positive control 1.0 µL/mL)
with S9: 125, 250, 500, 1000, 2000, µg/mL (Solvent (DMSO) 10 µL/mL; positive control 20.0 µL/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
- Cell density at seeding: 5x10^6 cells per dish

DURATION
- Preincubation period: 24 h
- Exposure duration: 5 h
- Expression time: 19 h
- Selection time: 8 d

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


NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
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:
cytotoxicity
Remarks:
without S9 (1000 and 2000 µg/mL); with S9 (250, 500, 1000 and 2000 µg/mL)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES: yes

HISTORICAL CONTROL DATA
please refer to "any other information including tables"

Table 1 Main mutation assay summary of results with S9

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
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control a

202.0

±

2.0

100

100

0

2

2

2

0

6

100

6.00

Pos. control
(EMS 1.0 µL/mL) a

55.3

±

3.21

27

63

202

198

188

211

202

1001

63

1588.89**

TEST ITEM

 

125g/mL a

198.7

±

1.53

98

99

1

1

2

0

0

4

99

4.04

250g/mL a

199.3

±

1.53

99

99

1

0

1

2

1

5

99

5.05

500g/mL a

199.3

±

3.51

99

98

2

1

0

1

1

5

98

5.10

1000g/mL a

181.0

±

2.00

90

99

1

1

1

2

1

6

99

6.06

2000g/mL a

149.3

±

2.08

74

98

2

2

1

1

0

6

98

6.12

 

 

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
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control b

201.0

±

2.0

100

100

4

0

0

1

1

6

100

6.00

Pos. control
(EMS 1.0 µL/mL) b

55.7

±

1.15

28

63

194

199

196

189

209

987

63

1566.67**

TEST ITEM

 

125g/mL b

199.3

±

1.15

99

99

2

1

1

1

0

5

99

5.05

250g/mL b

199.7

±

2.08

99

99

1

0

4

2

0

7

99

7.07

500g/mL b

199.0

±

2.00

99

99

2

2

2

0

0

6

99

6.06

1000g/mL b

182.3

±

2.08

91

98

0

1

0

1

2

4

99

4.04

2000g/mL b

149.7

±

1.53

74

98

1

1

1

2

0

5

99

5.05

 

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
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control c

201.0

±

2.00

100

100

0

1

2

2

2

7

100

7.00

Pos. control
(EMS 1.0 µL/mL) c

50.3

±

1.15

25

65

189

193

197

201

189

969

65

1490.77**

TEST ITEM

 

125g/mL c

197.3

±

2.31

98

100

1

1

3

0

0

5

100

5.00

250g/mL c

200.3

±

1.53

100

98

1

1

2

1

0

5

98

5.10

500g/mL c

196.0

±

1.00

98

99

3

2

0

1

0

6

99

6.06

1000g/mL c

186.3

±

2.08

93

98

1

1

1

2

2

7

98

7.14

2000g/mL c

151.3

±

4.04

75

98

1

0

0

3

2

6

98

6.12

 

 

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
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control d

200.7

±

1.15

100

100

1

3

0

1

1

6

100

6.00

Pos. control
(EMS 1.0 µL/mL) d

53.3

±

1.53

27

64

196

192

194

187

184

953

64

1489.06**

TEST ITEM

 

125g/mL d

198.0

±

1.73

99

100

3

0

2

0

1

6

100

6.00

250g/mL d

199.7

±

1.53

100

99

0

2

1

1

0

4

98

4.08

500g/mL d

198.0

±

1.00

99

99

0

1

4

0

1

6

99

6.06

1000g/mL d

186.7

±

2.31

93

99

1

2

0

1

1

5

98

5.10

2000g/mL d

151.7

±

2.89

76

99

1

0

1

2

2

6

98

6.12

 

a = parallel of first culture 

b = parallel of first culture.

c = parallel of second culture.

d = parallel of second culture.

 

abs.C.E. = Absolute Cloning Efficiency

EMS= Ethyl methanesulfonate

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

 

 

 

Table 2 Main mutation assay summary of results without S9

 

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
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control a

199.7

±

0.58

100

100

1

1

2

1

1

6

100

6.00

Pos. control
(DMBA 20 µg/mL) a

123.3

±

2.08

62

69

104

109

114

112

105

544

69

788.41**

TEST ITEM

 

125g/mL a

191.3

±

1.53

96

98

1

1

2

2

1

7

97

7.22

250g/mL a

178.7

±

1.53

89

99

1

1

2

1

0

5

99

5.05

500g/mL a

169.0

±

1.73

85

98

1

2

3

0

2

8

97

8.25

1000g/mL a

140.7

±

1.15

70

98

0

1

2

1

2

6

98

6.12

2000g/mL a

119.0

±

1.00

60

97

1

1

3

1

2

8

97

8.25

 

 

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
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control b

200.0

±

1.73

100

100

2

0

2

1

2

7

100

7.00

Pos. control
(DMBA 20 µg/mL) b

123.7

±

1.53

62

70

122

102

110

100

107

541

70

772.86**

TEST ITEM

 

125g/mL b

193.0

±

1.00

97

98

1

0

3

0

2

6

98

6.12

250g/mL b

180.3

±

1.53

90

98

1

1

2

1

1

6

98

6.12

500g/mL b

170.3

±

2.08

85

98

2

1

1

1

1

6

98

6.12

1000g/mL b

142.3

±

0.58

71

99

1

0

2

3

2

8

99

8.08

2000g/mL b

121.0

±

1.00

61

98

1

2

2

1

1

7

98

7.14

 

 

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
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control c

201.7

±

1.53

100

100

3

0

1

1

2

7

100

7.00

Pos. control
(DMBA 20 µg/mL) c

122.7

±

0.58

61

71

100

105

102

117

109

533

70

761.43**

TEST ITEM

 

125g/mL c

194.0

±

3.61

96

98

1

2

1

1

0

5

98

5.10

250g/mL c

178.7

±

1.53

89

99

1

2

1

1

1

6

99

6.06

500g/mL c

169.7

±

0.58

84

98

1

2

2

1

2

8

98

8.16

1000g/mL c

142.7

±

1.15

71

98

1

1

2

2

0

6

98

6.12

2000g/mL c

123.0

±

1.00

61

98

3

1

1

2

1

8

97

8.25

 

 

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
NUMBERS.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Solvent control d

201.3

±

2.08

100

100

2

0

1

4

0

7

100

7.00

Pos. control
(DMBA 20 µg/mL) d

121.7

±

1.53

60

71

104

116

112

108

112

552

70

788.57**

TEST ITEM

 

125g/mL d

194.7

±

4.16

97

99

1

1

1

0

3

6

98

6.12

250g/mL d

180.3

±

1.53

90

98

1

1

1

1

2

6

98

6.12

500g/mL d

171.0

±

1.73

85

98

1

3

1

0

0

5

98

5.10

1000g/mL d

144.0

±

1.00

72

99

2

1

3

0

1

7

98

7.14

2000g/mL d

124.0

±

1.00

62

98

2

0

0

2

1

5

98

5.10

 

 

Table 3 historical control data of solvent control (2015 – 2016)

 

 

Without S9 mix

With S9 mix

5-hour treatment

5-hour treatment

Mean

6.23

6.55

SD

0.57

0.89

Range

4.90 - 8.82

4.12 – 11.76

Lower 95% confidence interval

4.94

4.53

Upper 95% confidence interval

7.53

8.57

n

10

10

 

 

 

Table 4 historical control data of positive control

Without S9 mix

EMS

With S9 mix

DMBA

5-hour treatment

5-hour treatment

Mean

1526.96

755.62

SD

27.21

15.16

Range

1357.81 – 1636.92

690.00 - 810.29

Lower 95% confidence interval

1465.41

721.32

Upper 95% confidence interval

1588.50

789.92

n

10

10

EMS    =         Ethyl methanesulphonate

DMBA =         7,12-Dimethylbenzanthracene

SD      =         Standard deviation

n         =         number of experiments

Range: minimum value-maximum value

 

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

The test item, dissolved in Dimethyl sulfoxide (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:

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

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-09 to 2017-02-02
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:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
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)
- Lot. No.: 10H016
- Suitability of cells: The V79 cell line is well established in toxicology studies. 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: The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 °C in a humidified atmosphere containing 5 % CO2. The V79 cells for this study were grown in 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 %).
- 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 (PB) and β-naphthoflavone (BNF) induced rat liver
Test concentrations with justification for top dose:
Experiment A
with S9: 125, 250, 1000 µg/mL (solvent control 10 µL/mL; positive control 5 µg/mL)
without S9: 125, 250, 500 µg/mL (solvent control 5 µL/mL; positive control 1 µg/mL)

Experiment B
with S9: 125, 250, 500, 1000 µg/mL (solvent control 10 µL/mL; positive control 5 µg/mL)
without S9: 31.3, 62.5, 125.0 µg/mL (solvent control 1.25 µL/mL; positive control 0.4 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:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 5 x 10^5 cells per replicate

DURATION
- Exposure duration: Experiment A: 3h; experiment B: 3h (with S9); 20 h without S9
- Expression time (cells in growth medium): 20h

SPINDLE INHIBITOR: colchicine (0.2 μg/mL)

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 300

DETERMINATION OF CYTOTOXICITY
- Method: Relative increase in cell count (RICC)

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 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 inside the distribution of the laboratory historical negative control data.
Statistics:
For statistical analysis CHI^2 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:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RICC < 50 at 500 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RICC < 50 at 1000 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RICC < 50 at 125 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES: yes

HISTORICAL CONTROL DATA: please refer to "any other information on results incl. tabkes"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC, when cytokinesis block is not used
Remarks on result:
other: experiment A

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
(DMSO)

-

3 h

20 h

6

3

Test item

125 µg/mL

-

3 h

20 h

7

3

250 µg/mL

-

3 h

20 h

8

5

500 µg/mL

-

3 h

20 h

9

5

Pos. Control (EMS)

-

3 h

20 h

35**

26**

Solvent control
(DMSO)

+

3 h

20 h

6

3

Test item

125 µg/mL

+

3 h

20 h

9

3

250 µg/mL

+

3 h

20 h

8

3

500 µg/mL

+

3 h

20 h

11

7

1000 µg/mL

+

3 h

20 h

18*

12*

Pos. Control (Cycl.)

+

3 h

20 h

49**

40**

 

 

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
(DMSO)

-

20 h

20 h

6

3

Test item

31.3 µg/mL

-

20 h

20 h

8

4

62.5 µg/mL

-

20 h

20 h

8

3

125 µg/mL

-

20 h

20 h

8

5

Pos. Control (EMS)

-

20 h

20 h

47**

38**

Solvent control
(DMSO)

-

20 h

28 h

7

3

Test item

31.3 µg/mL

-

20 h

28 h

8

4

62.5 µg/mL

-

20 h

28 h

6

3

125 µg/mL

-

20 h

28 h

12

8

Pos. Control (EMS)

-

20 h

28 h

42**

35**

Positive control (-S9): Ethyl methanesulfonate (1.0L/mL)

Positive control (+S9): Cyclophosphamide (5.0g/mL)

*: p<0.05

**: p<0.01

 

 

Table 3 mean percentage of cells with structural chromosome aberrations experiment B

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Mean aberrant cells/150 cells

incl. gaps

excl. gaps

Solvent control
(DMSO)

+

3 h

28 h

6

3

Test item

125 µg/mL

+

3 h

28 h

10

5

250 µg/mL

+

3 h

28 h

9

4

500 µg/mL

+

3 h

28 h

16*

7

1000 µg/mL

+

3 h

28 h

16*

12*

Pos. Control (Cycl.)

+

3 h

28 h

47**

40**

 

 

 

Historical control data

 

Table 4 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

2.56

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

 

Table 5 3h/20h 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

5.57

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

 

 

Table 6 20h/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

2.85

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

 

 

Table 7 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

5.41

2.65

45.95

37.59

SD

0.65

0.60

1.70

1.92

Lower confidence interval

3.93

1.30

42.11

33.23

Upper confidence interval

6.89

4.00

49.79

41.94

n

10

10

10

10

 

Table 8 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

5.45

2.75

45.90

38.90

SD

0.67

0.59

2.07

3.29

Lower confidence interval

3.93

1.41

41.21

31.47

Upper confidence interval

6.97

4.09

50.59

46.33

n

10

10

10

10

 

Conclusions:
The test item tested up to cytotoxic concentrations, without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells.
The test item tested up to cytotoxic concentrations, with mammalian metabolic activation system, induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung cells.
Thus, the test item is considered clastogenic in this system.
Executive summary:

The test item was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In 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 S9 mix: 125, 250 and 500 μg/mL

with S9 mix: 125, 250, 500 and 1000 μg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 31.3, 62.5 and 125 μg/mL

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 31.3, 62.5 and 125 μg/mL

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 125, 250, 500 and 1000 μg/mL

 

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, in the absence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.

In Experiment A, biologically significant increase in the number of cells showing structural chromosome aberrations at the cytotoxic concentration of 1000 μg/mL, in the presence of metabolic activation. There were statistical differences between treatment and concurrent solvent and historical control groups, too. At concentrations of 500 and 1000 μg/mL dose-response relationships were noted in the number of cells showing structural chromosome aberrations and endoreduplication.

 

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. A 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment caused biologically and statistically significant increase in the number of cells showing structural chromosome aberrations and endoreduplication at concentrations of 1000 μg/mL. In the number of cells showing structural chromosome aberrations and endoreduplication a dose-response relationships were observed at concentrations of 500 and 1000 μg/mL.

 

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 was shown as the concurrent positive controls Ethyl methanesulfonate (0.4 or 1.0 μL/mL) and Cyclophosphamide (5.0 μg/mL) caused the expected increases in cells with structural chromosome aberrations and were compatible with the historical control range.

 

The test item tested up to cytotoxic concentrations, without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells.

The test item tested up to cytotoxic concentrations, with mammalian metabolic activation system, induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung cells.

Thus, the test item is considered clastogenic in this system. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A testing proposal for a Mammalian Erythrocyte Micronucleus Test in vivo (according to OECD 474) is provided and currently assessed by ECHA.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study planned
Study period:
The study will be performed in 2020 or in the following years depending on ECHA decision.
Justification for type of information:
Testing proposal Mammalian Erythrocyte Micronucleus Test

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out:
3-((5-(3-Acetoxy-2,2-dimethylpropylideneamino)-1,3,3-trimethylcyclohexyl)methylimino)-2,2-dimethylpropyl acetate

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: There are no GLP studies available for this substance covering the endpoint of gene mutation in vivo
- Available non-GLP studies: There are no non-GLP studies available for this substance covering the endpoint of gene mutation in vivo
- Historical human data: There are no historical human data available on gene mutation for the substance.
- (Q)SAR: There is no valid (Q)SAR model available to address gene mutation and or chromosome aberration in vivo and which outcome would be reliable enough to rule out the concern raised from a positive in vitro Chromosome Aberration test (ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R 7a: Endpoint specific guidance, 2017)
- In vitro methods: Three in vitro tests in regards to genotoxicity were conducted with the registered substance:
- Bacterial reverse mutation assay (Ames): negative
- Gene mutation study in mammalian cells (HPRT): negative
- Chromosome Aberration study in mammalian cells: positive

The test item indicated a clastogenic potential under the conditions of a chromosome aberration assay in vitro. Based on ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R 7a (2017) the follow-up procedure on a positive chromosome aberration assay in vitro is to conduct a chromosome aberration study in vivo such as a mammalian erythrocyte micronucleus test in vivo (OECD 474).

- Weight of evidence: There is no data available which is sufficient for a weight of evidence approach.
- Grouping and read-across: There are no substances which apply for read across addressing gene mutation in vivo in this case.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:

The substance is fully registered according to REACH Annex VIII. Therefore, three in vitro tests on mutation in bacteria, mammalian cells as well as chromosome aberration are legally required. Moreover, according to REACH Annex VIII Section 8.4 “Further mutagenicity studies shall be considered in case of a positive result”. Thus, the registrant considers it his duty to conduct a respective mammalian erythrocyte micronucleus test in vivo.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
Details on study design / methodology proposed:

Based on the available in vitro data, mammalian erythrocyte micronucleus test in vivo according to OECD Test Guideline 474 is proposed to assess the clastogenic properties of the test substance in vivo. This study is considered to be the appropriate test system to investigate clastogenic potential of bioavailable substances, i. e. substances or their metabolites (hydrolysis products) that become readily systemically available upon ingestion. Furthermore, historical control data are available for various tissues. The test substance is proposed to be administered orally by gavage to mice. Doses will be based on data available for acute as well as repeated oral toxicity studies.
Based on the results of the acute and repeated oral toxicity studies and taken the OECD requirements into consideration, a maximum dose of 2000 mg/kg bw/day, and two additional doses (separated by a factor of 2 to 4) are proposed at a single administration.
It is further proposed to assess the erythrocytes of the bone marrow, rather than peripheral blood, since there is more experience and historical control data available for this approach which makes it more reliable.
Six animals per dose or control group are proposed to reach a minimum of five analysable animals for one sex according to the Guideline. Since the available data did not demonstrate relevant differences between males and females, the use of males only is proposed.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Type of assay:
mammalian erythrocyte micronucleus test
Species:
rat
Route of administration:
oral: gavage
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Genetic toxicity in vitro

Bacterial Reverse Mutation Assay

The Bacterial Reverse Mutation Assay (using Salmonella typhimurium and Escherichia coli) with the test item was conducted according to the OECD guideline 471and GLP. The test item was suspended respectively dissolved in dimethyl sulfoxide (DMSO). Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential. 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. The tested test item concentrations were: 5000, 1581, 500, 158, 50 and 15.8 μg/plate.

In the performed experiments positive and negative (vehicle) controls were run concurrently. The revertant colony numbers of vehicle control plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants in the vehicle controls and were within the corresponding historical control data ranges. The reference mutagens showed a distinct increase of induced revertant colonies. In the performed experimental phases at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain were applied. The validity criteria of the study 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 mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. Slight, unequivocal inhibitory effect of the test item was observed in the Confirmatory Mutation Test (Pre-Incubation Test) in the examined Salmonella typhimurium strains at the concentration of 5000 μg/plate in absence of exogenous metabolic activation (-S9 Mix). 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.

Gene mutation in mammalian cells

The test item, dissolved in Dimethyl sulfoxide (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:

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

In vitro mammalian chromosome aberration test

The test item was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DMSO and the following concentration 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 S9 mix: 125, 250 and 500 μg/mL

with S9 mix: 125, 250, 500 and 1000 μg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 31.3, 62.5 and 125 μg/mL

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 31.3, 62.5 and 125 μg/mL

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 125, 250, 500 and 1000 μg/mL

 

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, in the absence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.

In Experiment A, biologically significant increase in the number of cells showing structural chromosome aberrations at the cytotoxic concentratio of 1000 μg/mL, in the presence of metabolic activation. There were statistical differences between treatment and concurrent solvent and historical control groups, too. At concentrations of 500 and 1000 μg/mL dose-response relationships were noted in the number of cells showing structural chromosome aberrations and endoreduplication.

 

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. A 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment caused biologically and statistically significant increase in the number of cells showing structural chromosome aberrations and endoreduplication at concentrations of 1000 μg/mL. In the number of cells showing structural chromosome aberrations and endoreduplication a dose-response relationships were observed at concentrations of 500 and 1000 μg/mL.

 

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 was shown as the concurrent positive controls Ethyl methanesulfonate (0.4 or 1.0 μL/mL) and Cyclophosphamide (5.0 μg/mL) caused the expected increases in cells with structural chromosome aberrations and were compatible with the historical control range.

 

The test item tested up to cytotoxic concentrations, without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. The test item tested up to cytotoxic concentrations, with mammalian metabolic activation system, induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung cells. Thus, the test item is considered clastogenic in this system. 

Endpoint conclusion

The test item did not indicate mutagenic potential in both in vitro bacterial reverse mutation assay and mammalian cell gene mutation assay.

However, clastogenic potential was observed in an in vitro chromosome aberration assay in mammalian cells.

The test item is known to undergo hydrolysis quickly in contact with water (please refer to IUCLID section 5.1.2). Therefore, it is likely that the hydrolysis products, 3-(aminomethyl)-3,5,5-trimethylcyclohexan-1-amine (isophorone diamine) and 2,2-dimethyl-3-oxopropyl acetate are mainly present in the actual assay. Isophorone diamine was demonstrated to be not clastogenic in a corresponding chromosome aberration assay in vitro as well as in a micronucleus assay in vivo. For more details please refer to REACH registration dossier of isophorone diamine.

In regards to 2,2-dimethyl-3-oxopropyl acetate no data on clastogenic potential is available. However, results of an available in vivo comet assay according to OECD 489 revealed a non-mutagenic activity of this hydrolysis product.

Available data demonstrated that both hydrolysis products of the registered substance are of no concern in regards to genotoxicity. Therefore, and against the backgound of a positive chromosome aberration assay in vitro with the registered substance, a testing proposal for mammalian erythrocyte micronucleus test in vivo with the registered substance is provided in order to elucidade its clastogenic potential.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable but inconclusive for classification purposes under Regulation (EC) No 1272/2008. More information is needed and a testing proposal is provided.