Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 with and without metabolic activation

HPRT (OECD 476): negative in V79 cells with and without metabolic activation

In vitro mammalian cell micronucleus test (OECD 487): negative in cultured human lymphocytes with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Aug - 17 Nov 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Directive 92/69/EEC
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
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:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1 and 2: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation


Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene (2AA); 1,8-dihydroxyanthraquinone (Danthron)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: approx. 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett´s method of linear regression) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Mean values and standard deviation were calculated.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In order to select appropriate dose levels for the main study, a preliminary test was carried out in S. typhimurium strain TA 100 to determine the toxicity of the test substance. The dose range for the test substance used was 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate wiht and without metabolic activation. The test substance was non-toxic to strain TA 100 at the tested concentrations.

Table 1: Test Results of Experiment 1

Experiment 1

S9-Mix

Without

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

Solvent control (DMSO)

30 ± 4

124 ± 18

299 ± 20

28 ± 7

18 ± 4

50

30 ± 5

123 ± 7

286 ± 4

20 ± 6

11 ± 0

150

26 ± 8

122 ± 10

301 ± 14

23 ± 6

15 ± 3

500

31 ± 7

145 ± 13

297 ± 27

27 ± 9

19 ± 3

1500

25 ± 4

110 ± 14

287 ± 26

30 ± 6

16 ± 1

5000

20 ± 6

128 ± 5

307 ± 35

33 ± 2

9 ± 1

ENNG (3)

-

539 ± 64

-

-

-

ENNG (5)

-

-

-

222 ± 39

-

9-AA (80)

-

-

-

-

784 ± 251

4NQO (0.2)

160 ± 2

-

-

-

-

MMC (0.5)

-

-

751 ± 185

-

-

S9-Mix

With

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

Solvent control (DMSO)

44 ± 6

143 ± 21

330 ± 24

24 ± 4

22 ± 2

50

50 ± 17

129 ± 16

313 ± 26

13 ± 5

22 ± 1

150

48 ± 11

138 ± 8

321 ± 9

18 ± 7

23 ± 3

500

56 ± 14

144 ± 5

331 ± 22

20 ± 1

24 ± 1

1500

37 ± 5

131 ± 17

316 ± 19

16 ± 5

22 ± 3

5000

38 ± 5

127 ± 3

289 ± 12

17 ± 5

20 ± 1

2-AA (1)

-

2057 ± 428

-

-

-

2-AA (2)

-

-

-

274 ± 43

814 ± 11

DAN (10)

-

-

813 ± 53

-

-

BP (5)

814 ± 11

-

-

-

-

EENG: N-ethyl-N-nitro-N-nitrosoguanidin

4NQO: 4-Nitroquinoline-1-oxide

9-AA: 9-Aminoacridine

MMC: Mitomycin C

2-AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

DAN: 1,8-Dihydroxyanthraquinone

Table 2: Test Results of Experiment 2

Experiment 2

S9-Mix

Without

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

Solvent control (DMSO)

27 ± 4

119 ± 11

352 ± 4

25 ± 2

16 ± 4

50

29 ± 3

123 ± 4

338 ± 5

20 ± 3

16 ± 5

150

29 ± 7

137 ± 15

331 ± 15

25 ± 7

17 ± 3

500

23 ± 6

135 ± 6

350 ± 12

23 ± 1

14 ± 2

1500

27 ± 7

121 ± 7

323 ± 56

24 ± 2

11 ± 3

5000

22 ± 8

124 ± 16

324 ± 17

32 ± 3

15 ± 5

ENNG (3)

-

481 ± 25

-

-

-

ENNG (5)

-

-

-

167 ± 15

-

9-AA (80)

-

-

-

-

926 ± 324

4NQO (0.2)

168 ± 14

-

-

-

-

MMC (0.5)

-

-

955 ± 75

-

-

S9-Mix

With

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

Solvent control (DMSO)

39 ± 2

127 ± 15

311 ± 20

15 ± 7

20 ± 1

50

34 ± 3

112 ± 11

308 ± 17

14 ± 1

19 ± 5

150

40 ± 8

157 ± 18

318 ± 25

18 ± 1

22 ± 3

500

31 ± 4

132 ± 29

317 ± 3

16 ± 3

17 ± 4

1500

29 ± 7

119 ± 11

317 ± 3

17 ± 4

14 ± 2

5000

38 ± 7

138 ± 19

269 ± 18

14 ± 1

12 ± 1

2-AA (1)

-

1484 ± 94

-

-

-

2-AA (2)

-

-

-

279 ± 41

551 ± 35

DAN (10)

-

-

875 ± 204

-

-

BP (5)

257 ± 6

-

-

-

-

EENG: N-ethyl-N-nitro-N-nitrosoguanidin

4NQO: 4-Nitroquinoline-1-oxide

9-AA: 9-Aminoacridine

MMC: Mitomycin C

2-AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

DAN: 1,8-Dihydroxyanthraquinone

Conclusions:
Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA 100, TA 1535, TA 102, TA 98 and TA 1537) tested with and without metabolic activation up to 5000 µg/plate.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Dec 2014 - 03 Mar 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM containing Hank's salts, 10% FBS (except during 4 h treatment), neomycin (5 µg/mL) and amphotericin B (1%)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Pre-Experiment:
4 h and 24 h treatment: 13.8, 27.5, 55, 110, 220, 440, 880 and 1760 µg/mL without metabolic activation
4 h treatment: 13.8, 27.5, 55, 110, 220, 440, 880 and 1760 µg/mL with metabolic activation

Experiment 1:
4 h treatment: 55*, 110*, 220*, 440*, 880* and 1760* µg/mL with and without metabolic activation

Experiment 2:
24 h treatment: 110*, 220*, 440*, 880*, 1320 and 1760 µg/mL without metabolic activation
4 h treatment: 110*, 220*, 440*, 880*, 1320* and 1760 µg/mL with metabolic activation

*concentrations chosen for mutation rate analysis
Vehicle / solvent:
- Vehicle/solvent used: DMSO (0.5% (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
ethylmethanesulphonate (EMS): 0.15 mg/mL (4 and 24 h, -S9); 7,12-dimethylbenzanthracene (DMBA): 1.1 µg/mL (4 h, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
1st experiment: 4 h exposure with and without S9 mix.
2nd experiment: 4 h exposure with S9 mix and 24 h without S9 mix
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 11 µg/mL 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: duplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency I or cell density below 50%
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations of the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) 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.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1320 µg/mL and higher following the 24 h treatment without S9 mix; at 1760 µg/mL following the 4 h treatment with and without S9 mix
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 and osmolarity: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: Precipitation was noted in Experiment 1 at 1760 µg/mL with metabolic activation and in Experiment 2 at 1320 µg/mL with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
A pre-experiment was performed in order to determine the concentration range for the mutagenicity experiments. The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 13.8 µg/mL and 1760 µg/mL were used. The highest concentration of the pre-experiment was chosen with regard to the purity (100%) and the molecular weight (176.2 g/mol) of the test item. No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 hours of treatment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects solely occurred in the Experiment 2 with metabolic activation at 1320 µg/mL. Based on the steep gradient of toxicity no relevant cytotoxicity was noted at the maximum analyzable concentration of the other experimental parts. Exceedingly severe cytotoxicity occurred at the next higher concentration even though the individual concentrations were spaced by a factor of 2 or below.

Table 1: Experiment 1 - 4 h exposure - Without Metabolic Activation

Concentration
[µg/mL]

Rel. cloning efficiency I

Rel. cell density

Rel. cloning efficiency II

Mutant colonies per 106cells

Induction factor

Culture I

0 (DMSO)

100.0

100.0

100.0

11.8

1.0

55

97.6

101.3

83.0

23.3

2.0

110

94.0

131.0

87.1

14.9

1.3

220

94.8

115.9

78.8

19.2

1.6

440

95.4

100.3

93.2

9.0

0.8

880

62.2

112.3

108.7

7.4

0.6

1760 (P)

22.9

Culture was not continued#

EMS, 150

95.9

78.3

91.4

77.1

6.5

Culture II

0 (DMSO)

100.0

100.0

100.0

17.2

1.0

55

106.1

90.5

100.3

8.2

0.5

110

108.7

81.0

106.7

30.3

1.8

220

103.5

79.9

107.1

23.2

1.3

440

122.0

106.8

105.2

33.9

2.0

880

103.1

108.1

102.7

34.1

2.0

1760 (P)

43.9

Culture was not continued##

EMS, 150

117.2

93.3

92.5

148.7

8.6

DMSO: Dimethylsulfoxide

EMS: Ethylmethanesulphonate

(P): Precipitation

#: Culture was not continued due to exceedingly severe cytotoxic effects.

 

Table 2: Experiment 1 - 4 h exposure - With Metabolic Activation

Concentration
[µg/mL]

Rel. cloning efficiency I

Rel. cell density

Rel. cloning efficiency II

Mutant colonies per 106cells

Induction factor

Culture I

0 (DMSO)

100.0

100.0

100.0

11.5

1.0

55

101.0

110.2

93.2

17.0

1.5

110

97.2

107.0

90.8

4.3

0.4

220

95.1

107.8

90.9

12.0

1.0

440

94.4

109.0

86.6

15.6

1.4

880

92.6

94.9

76.5

12.0

1.0

1760 (P)

0.0

2.3

Culture was not continued#

DMBA, 1.1

96.1

81.5

79.7

125.1

10.9

Culture II

0 (DMSO)

100.0

100.0

100.0

12.7

1.0

55

73.2

135.6

Culture was not continued##

110

88.7

127.1

93.7

15.7

1.2

220

108.6

136.7

87.0

5.7

0.4

440

88.7

144.5

68.3

28.4

2.2

880

72.4

129.2

81.2

22.6

1.8

1760 (P)

0.0

15.8

78.6

3.2

0.3

DMBA, 1.1

75.9

115.8

82.3

127.3

10.1

DMSO: Dimethylsulfoxide

DMBA: 7,12-dimethylbenzanthracene

(P): Precipitation

#: Culture was not continued due to exceedingly severe cytotoxic effects.

##: Culture was not continued since a minimum of only four analysable concentrations is required.

 

Table 3: Experiment 2 - 24 h exposure - Without Metabolic Activation

Concentration
[µg/mL]

Rel. cloning efficiency I

Rel. cell density

Rel. cloning efficiency II

Mutant colonies per 106cells

Induction factor

Culture I

0 (DMSO)

100.0

100.0

100.0

23.4

1.0

110

98.9

117.0

104.5

4.5

0.2

220

97.1

107.4

97.5

12.2

0.5

440

96.1

105.6

109.8

8.1

0.3

880

79.2

81.8

116.8

17.0

0.7

1320

0.1

6.6

Culture was not continued#

1760 (P)

0.0

1.3

Culture was not continued#

EMS, 150

88.2

107.0

97.4

271.8

11.6

Culture II

0 (DMSO)

100.0

100.0

100.0

19.4

1.0

110

100.8

125.6

96.0

7.8

0.4

220

100.4

120.7

100.8

15.4

0.8

440

100.0

115.1

95.0

23.9

1.2

880

88.0

81.8

109.5

20.5

1.1

1320

0.3

8.2

Culture was not continued#

1760 (P)

0.0

2.1

Culture was not continued#

EMS, 150

94.2

115.0

92.5

285.2

14.7

DMSO: Dimethylsulfoxide

EMS: Ethylmethanesulphonate

(P): Precipitation

#: Culture was not continued due to exceedingly severe cytotoxic effects.

 

Table 4: Experiment 2 - 4 h exposure - With Metabolic Activation

Concentration
[µg/mL]

Rel. cloning efficiency I

Rel. cell density

Rel. cloning efficiency II

Mutant colonies per 106cells

Induction factor

Culture I

0 (DMSO)

100.0

100.0

100.0

22.9

1.0

110

99.2

102.0

100.0

19.9

0.9

220

101.1

94.7

98.6

17.0

0.7

440

98.6

93.1

99.1

27.3

1.2

880

88.0

122.0

101.0

41.5

1.8

1320 (P)

20.4

12.3

102.2

21.8

1.0

1760 (P)

0.6

0.7

Culture was not continued#

DMBA, 1.1

98.3

105.4

100.1

452.8

19.7

Culture II

0 (DMSO)

100.0

100.0

100.0

13.6

1.0

110

101.1

62.4

117.7

12.6

0.9

220

100.3

50.4

104.1

15.3

1.1

440

100.8

37.8

104.0

20.1

1.5

880

87.9

38.3

103.3

7.8

0.6

1320

19.0

8.9

94.4

14.0

1.0

1760 (P)

1.3

1.0

Culture was not continued#

DMBA, 1.1

101.1

58.0

108.0

222.1

16.3

DMSO: Dimethylsulfoxide

DMBA: 7,12-dimethylbenzanthracene

(P): Precipitation

#: Culture was not continued due to exceedingly severe cytotoxic effects.

No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency did not exceed the historical range of solvent controls.

No significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups.

EMS and DMBA were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:
Under the experimental conditions of the gene mutation assay the test item did not induce gene mutations at the HPRT locus in V79 cells with and without metabolic activation.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Dec 2014 - 20 Feb 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 26 September 2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Cell proliferation: Blood was collected from healthy non-smoking donors not receiving medication. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 h. The cell harvest time point was approx. 2 – 2.5 x AGT (average generation time).
- Type and identity of media: 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
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Pre-Experiment / Experiment 1:
4 h treatment: 11.4, 20, 35, 61.3, 107.2, 187.7, 328.4, 574.7*, 1005.7* and 1760* µg/mL with and without metabolic activation
Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment 1.

Experiment 2:
20 h treatment: 11.4, 20, 35, 61.3, 107.2, 187.7, 328.4, 574.7*, 1005.7* and 1760* µg/mL without metabolic activation
4 h treatment: 187.7, 328.4, 574.7*, 1005.7* and 1760.0* µg/mL with metabolic activation

*evaluated for cytogenetic damage
Vehicle / solvent:
- Vehicle/solvent used: DMSO (0.5% (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: demecolcin
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 40 h; 20 h treatment: 40 h

ACTIN POLYMERISATION INHIBITOR (cytogenetic assays): cytochalasin B, 4 µg/mL
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1000 binucleated cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)
Evaluation criteria:
The micronucleus assay will be considered acceptable if it meets the following criteria:
a) The rate of micronuclei in the solvent controls falls within the historical laboratory control data range.
b) The rate of micronuclei in the positive controls is statistically significant increased.
c) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells.

A test item can be classified as non-clastogenic and non-aneugenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
- no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent control.

A test item can be classified as clastogenic and aneugenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.
Statistics:
Chi square test (p < 0.05)
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1760 µg/mL following the 20 h treatment without S9 mix
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 and osmolarity: No relevant influence on osmolarity or pH value was observed.
- Precipitation: In all experimental parts no precipitation of the test item in the culture medium was observed.

RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Test item concentrations ranging from 11.4 to 1760 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-experiment for toxicity, no precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment 1.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment 1 in the absence and presence of S9 mix and in Experiment 2 in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment 2 in the absence of S9 mix clear cytotoxicity was observed at the highest evaluated concentration.

Table 1: Results of Experiment 1.

Test item

Concentration

in µg/mL

Proliferation index

CBPI

Number of cells with MN

in %

Exposure period 4 h, fixation time 40 h, without S9 mix

DMSO

0.5% (v/v)

1.79

0.75

MMC

2.0

1.19

12.25#

Test substance

574.7

1.87

0.60

1005.7

1.84

0.20

1760

1.72

0.70

Exposure period 4 h, fixation time 40 h, with S9 mix

DMSO

0.5% (v/v)

1.80

0.75

CPA

15.0

1.35

6.50#

Test substance

574.7

1.70

0.90

1005.7

1.71

0.45

1760

1.72

0.80

CPA: Cyclophosphamide

DMSO: Dimethylsulfoxide

MMC: Mitomycin C

#: The number of micronucleated cells is statistically significantly higher than corresponding control values.


Table 2: Results of Experiment 2.

Test item

Concentration

in µg/mL

Proliferation index

CBPI

Number of cells with MN

in %

Exposure period 20 h, fixation time 40 h, without S9 mix

DMSO

0.5% (v/v)

1.76

0.65

Demecolcin

50.0 ng

1.49

4.15#

Test substance

574.7

1.61

0.75

1005.7

1.54

0.20

1760

1.14

0.90

Exposure period 4 h, fixation time 40 h, with S9 mix

DMSO

0.5% (v/v)

1.95

0.55

CPA

17.5

1.70

6.30#

Test substance

574.7

1.97

0.35

1005.7

1.88

0.70

1760

1.84

1.15#

CPA: Cyclophosphamide

DMSO:Dimethylsulfoxide

#: The number of micronucleated cells is statistically significantly higher than corresponding control values.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. The micronucleus rates of the cells after treatment with the test item (0.20 – 1.15% micronucleated cells) exceeded the range of the solvent control values (0.55 – 0.75% micronucleated cells), but were within the range of the laboratory historical control data.

However, in Experiment 2 in the presence of S9 mix, one single statistically significant increase in micronucleated cells was observed after treatment with 1760 µg/mL (1.15%). Since the value is clearly within the range of the laboratory historical solvent control data (0.15 – 1.70% micronucleated cells), the finding has to be regarded as biologically irrelevant.

In both experiments, either demecolcin, MMC or CPA were used as positive controls and showed distinct increases in cells with micronuclei.

Conclusions:
Under the experimental conditions of the in vitro micronucleus test the test substance did not induce micronuclei in human lymphocytes with and without metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

 Gene mutation in bacteria

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (1999). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were exposed to the test substance using the plate incorporation method. Based on the results of a pre-experiment, test substance concentrations of 50, 150, 500, 1500 and 5000 µg/plate were selected for the incubation with and without metabolic activation in both experiments. The test substance showed no bacterial toxicity at any dose in both experiments with or without metabolic activation. No biologically relevant increase in revertant numbers was observed after treatment with the test substance in any bacterial strain and at any concentration tested in the presence and absence of metabolic activation. The revertant frequencies of the vehicle control were within the expected range and the positive control chemicals induced marked increases in revertant colonies, demonstrating the effective performance of the experiments. Under the conditions of this experiment, the test substance is considered non-mutagenic in the selected S. typhimurium strains in the presence and absence of metabolic activation.

 

Gene mutation in mammalian cells

The mutagenic activity of the test substance was evaluated in an in vitro mammalian cell gene mutation test according to OECD Guideline 476 and in compliance with GLP (2015). The test substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. A pre-experiment was performed in order to determine the concentration range for the mutagenicity experiments. The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test substance concentrations between 13.8 and 1760 µg/mL (equal to 10 mM) were used. No relevant toxic effects occurred up to the maximum concentration tested in the presence and absence of metabolic activation after 4 h treatment. Based on the results of the pre-experiment, cells were exposed to the test substance for 4 h in the first experiment with and without metabolic activation up to concentration of 1760 µg/mL. The second experiment was performed with a treatment time of 4 h with and 24 h without metabolic activation up to concentration of 1760 µg/mL, respectively. Precipitation was noted in the first experiment at 1760.0 μg/mL with metabolic activation and in the second experiment at 1320 μg/mL with metabolic activation.The maximum concentration of 1760 μg/mL was solely analysable in culture II of the first experiment with metabolic activation. In all other experimental parts, exceedingly severe cytotoxicity did not permit analysis of the mutation frequency. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures solely occurred in the second experiment with metabolic activation at 1320 μg/mL. Based on the steep gradient of toxicity no relevant cytotoxicity was noted at the maximum analysable concentration of the other experimental parts. Exceedingly severe cytotoxicity occurred at the next higher concentration even though the individual concentrations were spaced by a factor of 2 or below. No relevant and reproducible increase in mutant colony numbers/10E06 cells was observed in the main experiments up to the maximum concentration. The mutant frequency did not exceed the historical range of solvent controls. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups. 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, the test substance did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Cytogenicity in mammalian cells

The potential of the test substance to induce mirconuclei was investigated in an in vitro mammalian cell micronucleus test in cultured peripheral human lymphocytes performed according to OECD Guideline 487 and GLP (2015). The test substance was dissolved in DMSO and two independent experiments were performed. A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Test substance concentrations ranging from 11.4 to 1760 µg/mL (with and without metabolic activation) were chosen for the evaluation of cytotoxicity. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test with an exposure period of 4 h was designated as Experiment I. In Experiment II, cultures were exposed to the test substance for 4 h with and 20 h without metabolic activation up to a concentration of 1760 µg/mL. The cells were prepared 40 h after start of treatment. In each experimental group two parallel cultures were analysed and at least 1000 binucleated cells per culture were evaluated for cytogenetic damage. No precipitation of the test substance in the culture medium was observed. No relevant influence on osmolarity or pH was observed. In Experiment I with and without metabolic activation and in Experiment II with metabolic activation, no cytotoxic effects were observed up to highest dose tested. In Experiment II clear cytotoxicity was observed without metabolic activation at the highest evaluated concentration. No relevant increase in the number of micronucleated cells was observed after treatment with the test substance with or without metabolic activation in Experiment I. However, in Experiment II, one single statistically significant increase in micronucleated cells was observed after treatment with 1760.0 μg/mL (1.15 %) with metabolic activation. Since the value is clearly within the range of the laboratory historical solvent control data (0.15 - 1.70% micronucleated cells), the finding has to be regarded as biologically irrelevant. Appropriate mutagens were used as positive controls and induced statistically significant increases in cells with micronuclei. In conclusion, the test substance did not induce micronuclei in the in vitro micronucleus test in human lymphocytes under the experimental conditions reported. Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

 

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.