Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The test substance was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.  

HPRT test:

The test substance is considered to be non-mutagenic in the HPRT assay.

Mirconucleus test:

The test substance is considered to be non-mutagenic in the in vitro micronucleus test, when tested up to phase separating concentrations.

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
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: 21' July 1997, OECD/OCDE Publication Service, Paris
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix induced with Aroclor 1254
Test concentrations with justification for top dose:
5- 5000 µg per plate presence of S9-mix
1.5- 5000 µg per plate absence of S9-mix
Vehicle / solvent:
- Vehicle used: Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours at 37°C

NUMBER OF REPLICATIONS: The experiment was repeated in full after an interval of at least 3 days.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
A reduction in the number of revertant colonies and/or a diminution of the background lawn was taken as an indication of bacteriotoxicity.
According Ames et al. (1975) as well as reported by De Serres and Shelby (1979).
Statistics:
XE2-test (Mohn and Ellenberger, 1977)
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9-mix at 50 µg/plate, with S9-mix at 500 µg/plate
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:
cytotoxicity
Remarks:
without S9 mix at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1: Induction of revertants (mean of 3 replicates) in S. typhimurium strains in the absence of a metabolizing system (Experiment 1)

 

S9 mix

TA98

TA100

TA102

TA1535

TA1537

Control

0 µg/plate

-

13

113

273

12

14

Solvent control

0 µg/plate

-

17

101

293

11

12

Test substance in µg/plate

 

5

-

15

-

277

14

10

15

-

19

108

296

13

11

50

-

19

93

268

9

9

150

-

11

96

131 (T)

6

8

500

-

16

85

99 (T)

8

6

1500

-

13

86

117 (T)

9

8

5000 (P)

-

12

89

-

-

-

NaN3 (0.7 µg/plate)

-

-

314

-

292

-

2-NF (2.5 µg/plate)

-

395

-

-

-

-

9-AA (50 µg/plate)

-

-

-

-

-

381

Mitomycin C (0.15 µg/plate)

-

-

-

1003

-

-

P: precipitation of test compound

T: bacteriotoxic

Solvent control: Ethanol, 50 µL/plate

2-NF: 2-nitrofluorene

9-AA: 9-aminoacridine

Table 2: Induction of revertants (mean of 3 replicates) in S. typhimurium strains in the presence of a metabolizing system (Experiment 1)

 

S9 mix

TA98

TA100

TA102

TA1535

TA1537

Control

0 µg/plate

+

22

96

294

7

15

Solvent control

0 µg/plate

+

22

101

290

15

15

Test substance in µg/plate

 

15

+

19

101

278

17

13

50

+

21

97

188

17

12

150

+

23

101

222

15

15

500

+

17

80

209

17

14

1500

+

22

89

226

17

10

5000 (P)

+

16

93

NA

15

13

2-AA (0.8 µg/plate)

+

501

839

683

123

 

2-AA (1.7µg/plate)

 

-

-

 

 

314

Solvent control: Ethanol, 50 µL/plate

2-AA: 2-aminoanthracene

Table 3: Induction of revertants (mean of 3 replicates) in S. typhimurium strains in the absence of a metabolizing system (Experiment 2)

 

S9 mix

TA98

TA100

TA1535

TA1537

Control

0 µg/plate

-

23

84

24

8

Solvent control

0 µg/plate

-

23

76

23

10

Test substance in µg/plate

 

5

-

21

-

-

-

15

-

14

86

20

10

50

-

23

80

27

8

150

-

19

79

21

6

500

-

12

76

20

10

1500

-

18

79

21

5

5000 (P)

-

-

86

22

6 (T)

NaN3 (0.7 µg/plate)

-

-

283

817

-

2-NF (2.5 µg/plate)

-

258

 

-

-

9-AA (50 µg/plate)

-

 

 

-

844

T: bacteriotoxic

Table 4: Induction of revertants (mean of 3 replicates) in S. typhimurium strains in the presence of a metabolizing system (Experiment 2)

 

S9 mix

TA98

TA100

TA102

TA1535

TA1537

Control

0 µg/plate

+

28

90

284

12

13

Solvent control

0 µg/plate

+

27

85

285

18

14

Test substance in µg/plate

 

5

+

-

-

324

-

-

15

+

25

-

337

-

14

50

+

29

85

325

17

13

150

+

15

82

226

17

13

500

+

26

84

247

18

7

1500

+

27

79

-

16

9

5000 (P)

+

28

83

-

14

14

2-AA (0.8 µg/plate)

+

583

511

-

136

-

2-AA (0.9 µg/plate)

 

 

 

747

 

-

2-AA (1.7µg/plate)

 

 

 

-

 

233

Table 5: Induction of revertants (mean of 3 replicates) in S. typhimurium strain TA 102 in the absence of a metabolizing system (Experiment 2)

 

S9 mix

TA102

Control

0 µg/plate

-

309

Solvent control

0 µg/plate

-

301

Test substance in µg/plate

-

-

1.5

-

286

5

-

298

15

-

281

50

-

140 (T)

150

-

142 (T)

500

-

135 (T)

Mitomycin C (0.15 µg/plate)

-

959

T: bacteriotoxic

Conclusions:
The test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.
 
Executive summary:

In the GLP and guideline compliant study the test substance was tested in reverse mutation study with the stains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100, TA 102). The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system.

The test substance was dissolved in ethanol and tested in concentrations of 5 to 5000 µg/plate in the presence and of 1.5 to 5000 µg per plate absence of S9.

The test substance was bacteriotoxic in the absence of S9-mix towards the strains TA 102 at 50 µg/plate and towards the strain TA 1537 at 5000 µg/plate.

In the presence of S9-mix the test substance was bacteriotoxic towards the strains TA 102 at 500 µg/plate. Precipitation of the test compound on the plates was observed at 5000 µg/plate.

 

The positive and negative controls were all valid and in the expected ranges.

In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system.

In conclusion, these results indicate that the test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-18 to 2016-08-08
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)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines: “Kanpoan No. 287 -- Environment Protection Agency“ “Eisei No. 127 -- Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry“.
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mammalian Cell Gene Mutation Assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years.
- Cell cycle doubling time x: 12 - 16 h in stock cultures
- Modal number of chromosomes: 22
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
- Culture media: MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1%).
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
pre-experiment: 10.7 μg/mL - 1364.0 μg/mL (equal to approximately 10 mM)
main test: without S9 mix: 1.3 - 30 μg/mL
with S9 mix: 85.3 - 1364 μg/mL
Vehicle / solvent:
- Vehicle used: acetone
- Justification for choice of solvent/vehicle:The solvent was chosen due to its solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone, purity 99.99%
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: 0.7 to 1.2×10E7

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Fixation time: The colonies used to determine the cloning efficiency were fixed and stained 6 to 8 days after treatment.

SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)

NUMBER OF REPLICATIONS: 2

STAINING TECHNIQUE USED:
The colonies were stained with 10% methylene blue in 0.01% KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

NUMBER OF CELLS EVALUATED: 50

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

A pre-test was conducted to establish the doses for the main test and evalute cytotxicity, precipitation and pH changes.
Evaluation criteria:
Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 106 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) the cloning efficiency II (absolute value) of the solvent controls must exceed 50%.
The data of this study comply with the above mentioned criteria (see tables of results, mutation rate and 95% confidence interval) and Historical Data.

Evaluation of Results
A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).
Statistics:
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9-mix: > 15.0 μg/mL; with S9-mix: 682.0 μg/mL toxic effects reduced the relative adjusted cloning efficiency to 20.7% or 30.0%, severe cytotoxicity occurred at even higher concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed down to the lowest concentration of 10.7 μg/mL without metabolic activation and at 682.0 μg/mL and above with metabolic activation.

HISTORICAL CONTROL DATA (2012-2015)
Number of mutant colonies per 10E6 cells; without metabolic activation (4 hours treatment time)
- Positive control EMS 150 and 225 μg/mL:
Range: 53.9 - 889.0
Mean value: 153.0
Standard deviation: 88.5
Number of studies: 147

- Negative solvent control (medium, acetone, water, DMSO, ethanol, THF)
Range: 1.6 - 42.8
Mean value: 15.0
Standard deviation: 7.4
95% confidence interval: 0.2 - 29.7
Number of studies: 147

with metabolic activation (4 hours treatment time)
- Positive control DMBA 1.1 and 2.2 μg/mL
Range: 59.6 - 2042.6
Mean value: 424.6
Standard deviation: 291.4
Number of studies: 142

- Negative solvent control (medium, acetone, water, DMSO, ethanol, THF)
Range: 2.4 - 44.2
Mean value: 14.6
Standard deviation: 7.0
95% confidence interval: 0.6 - 28.7
Number of studies: 142

The 95% confidence interval is derived from the mean value plus/minus 2 times the standard deviation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Strong and not really reproducible cytotoxic effects indicated by an adjusted cloning efficiency I below 50% occurred in the insoluble range showing phase separation. This is often seen in a phase separating range as the organic phase may come into direct contact with the cells upon handling of the cell culture bottles damaging the cell membranes. No relevant cytotoxicity was noted without metabolic activation up to a concentration of 10.0 μg/mL.
Exceedingly severe cytotoxicity occurred at the next higher, phase separating concentration of 15.0 μg/mL. In the presence of metabolic activation the cultures were analyzable up to the second phase separating concentration of 682.0 μg/mL even though toxic effects reduced the relative adjusted cloning efficiency I to 20.7% or 30.0% at this concentraton. Exceedingly severe cytotoxicity occurred at even higher concentrations. The striking difference of cytotoxicity with and without metabolic activation indicates possible protein binding capacity of the test item as toxicity with metabolic activation (in the presence of protein rich S9 mix) was shifted to much higer concentrations.

Summary of Results

 

 

conc.

µg/mL

PS

S9 mix

Relative coloning efficiency I

%

Relative cell density

%

Rel. adjusted cloning efficiency I

%

Mutant colonies/ 106cells

95% confidence interval

Relative coloning efficiency I

%

Relative cell density

%

Rel. adjusted cloning efficiency I

%

Mutant colonies/ 106cells

95% confidence interval

Column

1

2

3

4

5

6

7

8

9

10

11

12

13

Experiment I /4 h treatment

culture I

culture II

Solvent control with acetone

 

 

-

100.0

100.0

100.0

21.3

0.2 – 29.7

100.0

100.0

100.0

14.4

0.2 – 29.7

Positive control (EMS)

300.0

 

-

98.0

79.8

78.2

230.8

0.2 – 29.7

97.5

94.1

91.7

192.6

0.2 – 29.7

Test item

1.3

 

-

104.1

106.3

110.6

8.3

0.2 – 29.7

98.3

98.9

97.2

11.6

0.2 – 29.7

Test item

2.5

 

-

99.3

94.0

93.3

9.5

0.2 – 29.7

100.2

113.1

113.3

28.9

0.2 – 29.7

Test item

5.0

 

-

101.2

101.8

103.0

12.4

0.2 – 29.7

102.5

91.1

93.4

10.0

0.2 – 29.7

Test item

10.0

 

-

91.2

85.7

78.1

13.9

0.2 – 29.7

90.2

101.9

91.9

22.7

0.2 – 29.7

Test item

15.0

PS

-

#

7.9

#

68.6

35.8

24.6

#

Test item

20.0

PS

-

#

6.5

#

#

8.1

#

#

Test item

25.0

PS

-

culture not continued #

culture not continued #

Test item

30.0

PS

-

culture not continued #

culture not continued #

Solvent control with acetone

 

 

+

100.0

100.0

100.0

7.6

0.6 – 28.7

100.0

100.0

100.0

21.8

0.6 – 28.7

Positive control (DMBA)

2.3

 

+

92.3

82.0

75.6

86.0

0.6 – 28.7

85.8

120.8

103.6

107.1

0.6 – 28.7

Test item

85.3

 

+

87.2

74.3

64.8

14.9

0.6 – 28.7

71.7

112.5

80.7

7.2

0.6 – 28.7

Test item

170.5

 

+

100.9

82.7

83.4

11.3

0.6 – 28.7

84.7

120.2

101.8

14.6

0.6 – 28.7

Test item

341.0

PS

+

105.9

65.1

68.9

6.6

0.6 – 28.7

76.3

113.5

86.6

19.3

0.6 – 28.7

Test item

682.0

PS

+

51.0

58.8

30.0

6.4

0.6 – 28.7

22.1

93.8

20.7

10.9

0.6 – 28.7

Test item

1023.0

PS

+

44.5

53.3

23.7

#

#

1.3

#

#

Test item

1364.0

PS

+

19.5

19.2

3.7

#

#

0.6

#

#

 

PS =  phase separation visible at the beginning and at the end of treatment

#        culture was not continued due to exceedingly severe cytotoxic effects

Conclusions:
The test substance is considered to be non-mutagenic in this HPRT assay with and without metabolic activation.
Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according OECD 476 and GLP. The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (1364 μg/mL) was equal to 10 mM. The analysed concentration range of the main experiment without S9-mix: 1.3 -10 μg/mL with S9-mix: 85.3 – 682 μg/mL was limited by cytotoxicity of the test item and phase separation.

In the main experiment with and without S9 mix the range of the solvent controls was from 7.6 up to 21.8 mutants per 10E6 cells; the range of the groups treated with the test item was from 6.4 up to 28.9 mutants per 10E6 cells.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2016-07-06 to 2016-11-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
primary culture, other: human lymphocytes
Remarks:
Blood was collected from a male donor (22 years old) for Experiment I and from a female donor (27 years old) for Experiment II.
Details on mammalian cell type (if applicable):
- Type and identity of media: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
- Properly maintained: yes
- Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.
Additional strain / cell type characteristics:
other: established low incidence of micronuclei
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I: 4 hrs: with and without S9 mix: 5.1, 8.9, 15.5, 27.1, 47.5, 83.1, 145, 255, 445, 799, 1364 μg/mL
Experiment II: 20 hrs: without S9 mix: 17.4, 30.5, 53.3, 93.3, 163, 286, 500 μg/mL

With regard to the molecular weight of the test item, 1364 μg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 255 μg/mL and above in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Considering the phase separation data in Experiment I, 500 μg/mL (without S9 mix) were chosen as top concentration in Experiment II.
The cytogenetic evaluation of concentrations in Experiment II (without S9 mix) higher than 53.3 μg/mL was impossible due to strong test item-induced toxic effects (low cell numbers). Phase separation occurred at 163 μg/mL and above.
Vehicle / solvent:
- Vehicle used: acetone; The final concentration of acetone in the culture medium was 0.5 %.
- Justification for choice of solvent: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
culture medium with 0.5% acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation, pulse treatment, dissolved in deionized water to concentration of 1.0 μg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
culture medium with 0.5% acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Demecolcin
Remarks:
Without metabolic activation, continuous treatment, dissolved in deionized water to concentration of 100.0 ng/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
culture medium with 0.5% acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation, dissolved in saline (0.9 % NaCl) at concentration level 12.5 μg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 (pulse exposure) and 20 h (continuous exposure)
- Expression time: 16 h (pulse exposure only)
- Recovery period: 20 h
- Fixation time: 40 h

SPINDLE INHIBITOR: Cytochalasin B (4 μg/mL)
STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were harvested by centrifugation 40 hrs after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is characterized by the percentages of reduction in the CBPI (Cytokinesis-block proliferation index) in comparison with the controls (% cytostasis) by counting 500 cells per culture.

A pre-test was conducted to establish the doses for the main test.
Evaluation criteria:
The test item was considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibited a statistically significant increase compared with the concurrent solvent control
− There was no concentration-related increase
− The results in all evaluated test item concentrations was within the range of the laboratory historical solvent control data
The test item was then considered unable to induce chromosome breaks and/or gain or loss in this test system.
The test item was considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibited a statistically significant increase compared with the concurrent solvent control
− The increase was concentration-related in at least one experimental condition
− The results were outside the range of the laboratory historical solvent control data

When all of the criteria were met, the test item was then considered able to induce chromosome breaks and/or gain or loss in this test system.
Statistics:
Statistical significance was confirmed by using the Chi-squared test (α < 0.05) using a validated R Script for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Key result
Species / strain:
primary culture, other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
primary culture, other: human lymphocytes
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II, concentrations of 93.3 μg/mL and above, showing clear cytotoxic effects, were not evaluable for cytogenetic damage.
In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. However, in the presence of S9 mix one statistical significant increase (0.85%) in micronucleate cells was observed at 145 μg/mL. This can be declared as biologically irrelevant because this value is clearly within the historical laboratory control data (0.08 – 1.20%).
In both experiments, either Demecolcin (100.0 ng/mL), MMC (1.0 μg/mL) or CPA (12.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.
Remarks on result:
other: 4 hrs treatment

Summary of results

 

Exp.

Preparation interval

Test item concentration

in µg/mL

Proliferation index

CBPI

Cytostasis

in %*

Micronucleated cells

in %**

Exposure period 4 hrs without S9 mix

I

40 hrs

Solvent control1

2.06

 

0.75

Positive control2

1.77

27.5

8.25S

83.1

1.89

16.0

0.25

145

1.99

7.1

0.35

255PS

2.04

2.2

0.55

Exposure period 20 hrs without S9 mix

II

40 hrs

Solvent control1

1.92

 

0.40

Positive control3

1.75

18.1

2.55S

17.4

1.87

5.1

0.45

30.5

1.90

1.6

0.40

53.3

1.64

30.4

0.30

Exposure period 4 hrs with S9 mix

I

40 hrs

Solvent control1

1.83

 

0.25

Positive control4

1.89

n.c.

2.65S

83.1

1.79

5.0

0.35

145

1.70

15.8

0.85S

255PS

1.85

n.c.

0.05

 

*       For the positive control groups and the test item treatment groups the values are related to the solvent controls

**      The number of micronucleated cells was determined in a sample of 2000 binucleated cells

PS     Phase separation occurred at the end of treatment

n.c.   Not calculated as the CBPI is equal or higher than the solvent control value

S       The number of micronucleated cells is statistically significantly higher than corresponding control values

1       Acetone 0.5 % (v/v)

2       MMC 1.0 μg/mL

3       Demecolcin 100.0 ng/mL

4       CPA 12.5 μg/mL

Conclusions:
Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating or the highest evaluable concentrations.
Executive summary:

An in vitro micronucleus test according to OECD Guideline 487 was conducted in primary human lymphocytes. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity was described as % cytostasis. The highest treatment concentration in this study, 1364 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item. Phase separation was observed in Experiment I at 255 μg/mL and above in the absence and presence of S9 mix and in Experiment II at 163 μg/mL in the absence of S9 mix and above at the end of treatment. In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. In Experiment II concentrations of 93.3 μg/mL and above, showed clear cytotoxic effects and were not evaluable for cytogenetic damage. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix one single statistically significant increase in micronucleated cells (0.85 %) was observed at 145 μg/mL. Since the value is in the range of the laboratory historical control data (0.08 – 1.20 % micronucleated cells), the finding can be regarded as biologically irrelevant. The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, it is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genotoxicity in vitro

Ames test:

The mutagenicity of the test substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102) according to OECD 471. The investigations were carried out using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system.

The test substance was dissolved in ethanol and tested in concentrations of 5 to 5000 µg/plate,in the presence and of 1.5 to 5000 µg per plate absence of S9. In the absence of S9-mix the test substance was bacteriotoxic towards the strains TA102 at 50 µg/plate and towards the strain TA1537 at 5000 µg/plate. In the presence of S9-mix the test substance was bacteriotoxic towards the strains TA102 at 500 μg/plate. Precipitation of the test compound on the plates was observed at 5000 μg/plate.

The positive controls confirmed the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system.

In conclusion, these results indicate that the test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

HPRT test:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according OECD 476 and GLP. The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (1364 μg/mL) was equal to 10 mM. The analysed concentration range of the main experiment without S9-mix: 1.3 -10 μg/mL with S9-mix: 85.3 – 682 μg/mL was limited by cytotoxicity of the test item and phase separation.

In the main experiment with and without S9 mix the range of the solvent controls was from 7.6 up to 21.8 mutants per 10E6 cells; the range of the groups treated with the test item was from 6.4 up to 28.9 mutants per 10E6 cells.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, the test substance is considered to be non-mutagenic in the HPRT assay.

Mirconucleus test:

An in vitro micronucleus test according to OECD Guideline 487 was conducted in primary human lymphocytes.

Two independent experiments were performed.

In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity was described as % cytostasis. The highest treatment concentration in this study, 1364 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item. Phase separation was observed in Experiment I at 255 μg/mL and above in the absence and presence of S9 mix and in Experiment II at 163 μg/mL in the absence of S9 mix and above at the end of treatment. In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration.

In Experiment II concentrations of 93.3 μg/mL and above, showed clear cytotoxic effects and were not evaluable for cytogenetic damage. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item.

However, in Experiment I in the presence of S9 mix one single statistically significant increase in micronucleated cells (0.85 %) was observed at 145 μg/mL. Since the value is in the range of the laboratory historical control data (0.08 – 1.20 % micronucleated cells), the finding can be regarded as biologically irrelevant. The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, it is considered to be non-mutagenic in the in vitro micronucleus test, when tested up to phase separating concentrations.

Justification for classification or non-classification

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

Based on the available experimental test data - Ames test, HPRT test, Micronucleus test, the test substance is not considered to be classified and labelled for genotoxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.