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
In vitro mammalian cell micronucleus test (OECD 487): negative in cultured human lymphocytes with and without metabolic activation
HPRT (OECD 476): negative in V79 cells with and without metabolic activation

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:
28 Jun - 09 Aug 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
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Umwelt und Verkehr Baden-Württemberg, Stuttgart, Germany
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 I:
15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Experiment II:
15, 50, 150, 500 and 1500 µg/plate without metabolic activation
15, 50, 150, 500, 1500 and 5000 µg/plate with 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:
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 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

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

Statistics:
The X²-test (Mohn and Ellenberger, 1977) was used.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate with and without S9 mix
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:
cytotoxicity
Remarks:
at 5000 µg/plate with and without S9 mix
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:
cytotoxicity
Remarks:
at 5000 µg/plate with and without S9 mix
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:
cytotoxicity
Remarks:
at 5000 µg/plate with and without S9 mix
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:
cytotoxicity
Remarks:
at 5000 µg/plate with and without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDY: Based on an initial toxicity test, the test substance was tested in concentrations of 15 to 5000 µg/plate.

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

NC

35 ± 8

99 ± 10

260 ± 17

33 ± 6

9 ± 2

Solvent control (DMSO)

36 ± 9

113 ± 6

259 ± 15

35 ± 4

12 ± 4

15

-

112 ± 25

-

-

-

50

33 ± 3

100 ± 13

239 ± 13

41 ± 4

9 ± 3

150

41 ± 6

111 ± 10

243 ± 12

45 ± 3

11 ± 6

500

31 ± 4

114 ± 10

244 ± 9

41 ± 5

9 ± 2

1500

30 ± 6

140 ± 12

263 ± 11

40 ± 6

13 ± 2

5000

14 ± 5 (T)

91 ± 19 (T)

109 ± 12 (T)

29 ± 5 (T)

2 ± 1 (T)

NaN3 (0.7)

-

465 ± 41

-

657 ± 71

-

2-NF (2.5)

269 ± 29

-

-

-

-

9-AA (50)

-

-

-

-

364 ± 29

Mitomycin C (0.15)

-

-

581 ± 12

-

-

S9-Mix

With

 

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

 

 

 

 

 

 

NC

40 ± 4

114 ± 4

280 ± 11

20 ± 3

14 ± 3

Solvent control (DMSO)

42 ± 7

114 ± 5

254 ± 26

23 ± 3

13 ± 3

15

-

120 ± 10

-

-

-

50

38 ± 5

103 ± 11

293 ± 16

16 ± 3

19 ± 3

150

36 ± 6

115 ± 8

298 ± 6

22 ± 5

16 ± 3

500

40 ± 4

107 ± 5

305 ± 13

19 ± 3

13 ± 3

1500

47 ± 10

117 ± 10

285 ± 5

20 ± 3

10 ± 2

5000

41 ± 7

70 ± 8 (T)

245 ± 27 (T)

18 ± 2 (T)

4 ± 3 (T)

2-AA (0.7)

1189 ± 70

1092 ± 92

-

-

-

2-AA (1.5)

-

-

725 ± 22

625 ± 38

508 ± 20

NC = Negative Control

T = bacteriotoxic

2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; 2-AA: 2-aminoanthracene

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

NC

47 ± 6

118 ± 10

269 ± 11

30 ± 8

16 ± 4

Solvent control (DMSO)

44 ± 12

108 ± 9

262 ± 15

35 ± 4

15 ± 5

15

47 ± 3

133 ± 10

283 ± 12

35 ± 3

14 ± 1

50

33 ± 9

121 ± 13

274 ± 19

29 ± 7

15 ± 5

150

52 ± 5

140 ± 19

265 ± 24

34 ± 8

18 ± 3

500

43 ± 5

132 ± 14

275 ± 8

39 ± 3

12 ± 4

1500

42 ± 11

149 ± 5

262 ± 13

43 ± 3

16 ± 2

NaN3 (0.7)

-

515 ± 9

-

624 ± 39

-

2-NF (2.5)

222 ± 21

-

-

-

-

9-AA (50)

-

-

-

-

212 ± 19

Mitomycin C (0.15)

-

-

820 ± 53

-

-

S9-Mix

With

 

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

 

 

 

 

 

 

NC

48 ± 4

107 ± 7

307 ± 29

22 ± 4

15 ± 4

Solvent control (DMSO)

38 ± 7

91 ± 13

298 ± 9

22 ± 5

19 ± 5

15

-

124 ± 9

322 ± 21

18 ± 4

16 ± 5

50

37 ± 10

124 ± 9

330 ± 7

18 ± 3

17 ± 3

150

40 ± 6

120 ± 9

328 ± 13

21 ± 8

11 ± 3

500

39 ± 3

110 ± 11

296 ± 20

16 ± 3

17 ± 4

1500

45 ± 2

116 ± 8

347 ± 6

17 ± 2

17 ± 5

5000

27 ± 4 (T)

-

-

-

-

2-AA (0.7)

613 ± 33

903 ± 136

-

-

-

2-AA (1.5)

-

-

1106 ± 92

619 ± 51

259 ± 25

NC = Negative Control

T = bacteriotoxic

2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; 2-AA: 2-aminoanthracene

The test substance induced a slight increase in the mutation frequency in the tester strain TA 1535 and TA 100 in the absence of a metabolic activation system in both experiments. In the presence of S9 mix a slight increase in the frequency of revertants of strain TA102 was observed. However, the estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, using a X2-test did not reveal a significant effect at any of the test points.

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
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Dec 2014 - 12 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: 16.3, 32.7, 65.3, 130.6, 261.3, 522.5, 1045 and 2090 µg/mL without metabolic activation
4 h treatment: 16.3, 32.7, 65.3, 130.6, 261.3, 522.5, 1045 and 2090 µg/mL with metabolic activation

Experiment 1:
4 h treatment: 8.1, 16.3, 32.5*, 65*, 130*, 195*, 260* and 390 µg/mL without metabolic activation
4 h treatment: 32.5*, 65*, 130*, 260*, 390 and 520 µg/mL with metabolic activation

Experiment 2:
24 h treatment: 16.3*, 32.5*, 65*, 130*, 260*, 390 and 520 µg/mL without metabolic activation
4 h treatment: 8.1, 16.3, 32.5*, 65*, 130*, 195*, 260* and 325 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
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 260 µg/mL and higher following the 4 h treatment without S9 mix; at 390 µg/mL and higher following the 20 h treatment without S9 mix; at 325 µg/mL and higher following the 4 h treatment with 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: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 h) prior to removal to the test item. Phase separation occurred at 522.5 µg/mL and above after 4 and 24 h treatment with and without 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 16.3 µg/mL and 2090 µg/mL (10 mM) were used. The highest concentration of the pre-experiment was chosen with regard to the purity (98.6%) and the molecular weight (206.2 g/mol) of the test item. Relevant toxic mM) effects occurred at 522.5 µg/mL and above in the presence and absence of metabolic activation after 4 and 24 h treatment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in both cultures of Experiment 1 at the highest analyzable concentration of 260 µg/mL without metabolic activation. Due to a very steep gradient of toxicity, no relevant cytotoxicity was noted up to the maximum analyzable concentrations in all of the other experimental parts. Exceedingly severe cytotoxicity precluded analysis at the next higher concentration even though the concentrations were spaced by less than a factor of 2.0 recommended by the OECD guideline 476.

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

6.5

1.0

8.1

81.9

Culture was not continued#

16.3

82.6

Culture was not continued#

32.5

79.5

90.8

113.6

14.8

2.3

65

78.9

70.4

113.8

10.0

1.5

130

71.7

84.9

103.8

28.8

4.4

195

68.4

91.8

85.4

16.4

2.5

260

43.8

61.0

103.4

16.0

2.5

390

Culture was not continued##

EMS, 150

64.4

91.7

96.4

173.6

26.7

Culture II

0 (DMSO)

100.0

100.0

100.0

27.6

1.0

8.1

92.6

Culture was not continued#

16.3

80.9

Culture was not continued#

32.5

82.5

77.7

105.1

20.8

0.8

65

80.2

72.1

109.2

32.9

1.2

130

77.5

82.5

117.6

21.1

0.8

195

73.5

86.3

108.2

19.8

0.7

260

44.2

36.6

120.6

17.5

0.6

390

Culture was not continued##

EMS, 150

64.1

87.0

113.2

128.6

4.7

DMSO: Dimethylsulfoxide

EMS: Ethylmethanesulphonate

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

##: 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

26.4

1.0

32.5

101.5

106.3

80.0

15.9

0.6

65

100.7

140.9

89.9

18.9

0.7

130

94.5

123.6

92.3

23.0

0.9

260

85.2

112.8

86.7

10.5

0.4

390

14.0

2.8

Culture was not continued##

520

0.0

Culture was not continued##

DMBA, 1.1

94.5

106.3

83.4

388.8

14.7

Culture II

0 (DMSO)

100.0

100.0

100.0

16.5

1.0

32.5

101.8

108.6

77.7

15.7

1.0

65

97.2

74.5

110.9

36.8

2.2

130

93.0

86.6

90.3

31.3

1.9

260

87.0

74.6

110.9

25.6

1.6

390

20.4

Culture was not continued##

520

0.0

Culture was not continued##

DMBA, 1.1

99.4

62.9

100.6

162.8

9.9

DMSO: Dimethylsulfoxide

DMBA: 7,12-dimethylbenzanthracene

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

 

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

26.4

1.0

16.3

101.3

85.5

99.2

29.2

1.1

32.5

100.5

96.4

92.5

35.9

1.4

65

101.6

85.7

105.3

10.7

0.4

130

103.6

87.9

108.3

11.4

0.4

260

92.3

90.2

109.6

15.1

0.6

390

0.0

2.2

Culture was not continued##

520

0.0

Culture was not continued##

EMS, 150

96.9

72.6

92.8

534.9

20.3

Culture II

0 (DMSO)

100.0

100.0

100.0

19.5

1.0

16.3

97.2

102.7

76.4

15.8

0.8

32.5

96.8

97.9

86.6

14.6

0.7

65

97.6

101.8

82.5

19.1

1.0

130

95.7

99.5

81.7

29.3

1.5

260

90.7

96.3

90.4

30.8

1.6

390

0.0

4.7

Culture was not continued##

520

0.0

Culture was not continued##

EMS, 150

96.2

95.1

73.6

398.2

20.4

DMSO: Dimethylsulfoxide

EMS: Ethylmethanesulphonate

##: 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

24.7

1.0

8.1

97.4

Culture was not continued#

16.3

103.0

Culture was not continued#

32.5

104.1

102.1

80.4

40.1

1.6

65

96.7

96.0

82.9

16.4

0.7

130

93.9

81.5

82.6

12.1

0.5

195

95.0

70.3

89.6

15.8

0.6

260

76.8

63.5

84.7

18.1

0.7

325

43.8

5.4

Culture was not continued##

DMBA, 1.1

100.5

100.4

92.3

171.7

7.0

Culture II

0 (DMSO)

100.0

100.0

100.0

22.4

1.0

8.1

91.5

Culture was not continued#

16.3

95.9

Culture was not continued#

32.5

96.5

96.1

101.5

30.5

1.4

65

116.2

95.5

93.6

24.9

1.1

130

91.8

73.9

89.5

29.2

1.3

195

78.1

66.3

98.5

12.5

0.6

260

80.9

67.5

100.2

4.7

0.2

325

52.8

Culture was not continued##

DMBA, 1.1

92.1

101.8

94.1

139.3

6.2

DMSO: Dimethylsulfoxide

DMBA: 7,12-dimethylbenzanthracene

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

##: 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.

The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of Experiment 1 at 130 µg/mL without metabolic activation. This effect however, was based upon the rather low solvent control of 6.5 mutant colonies/10E6 cells and thus, judged as biologically irrelevant.

A significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in culture I of the second experiment without metabolic activation. Since the mutation frequency neither exceeded the historical range of solvent controls nor the threshold as indicated above, the statistical result was judged as biologically irrelevant.

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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Jan - 14 Apr 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 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 phytohemeagglutinine (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 1A:
4 h treatment: 13.2, 23.1, 40.4, 70.7*, 123.7, 216.4*, 378.8*, 662.9*, 1160 and 2030 µg/mL with and without metabolic activation
Since the cultures fulfilled the requirements for cytogenetic evaluation in the presence of S9 mix, this preliminary test was designated Experiment 1A. The experimental part without S9 mix was repeated with the same top dose to obtain evaluable concentrations in a cytotoxic range.

Experiment 1B:
4 h treatment: 31.3, 62.5, 125*, 250*, 500*, 600, 700, 800, 900, 1000 and 2030 µg/mL without metabolic activation

Experiment 2:
20 h treatment: 35.2, 52.8, 79.2, 118.8, 178.2*, 267.3*, 401*, 601.5, 902.2, 1353.3 and 2030 µg/mL without metabolic activation
4 h treatment: 52.8, 79.2, 118.8, 178.2, 267.3*, 401*, 601.5*, 902.2, 1353.3 and 2030 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
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: No precipitation of the test substance in the culture medium was observed. Phase separation was observed at the end of treatment in Experiment IA at 662.9 µg/mL and above in the absence of S9 mix and at 123.7 µg/mL and above in the presence of S9 mix, in Experiment IB at 600.0 µg/mL and above in the absence of S9 mix and in Experiment II at 902.2 µg/mL and above in the absence and at 601.5 µg/mL and above in the presence of S9 mix.

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 13.2 to 2030 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-experiment for toxicity, no precipitation, but phase separation of the test substance was observed at the end of treatment at 662.9 µg/mL and above in the absence of S9 mix and at 123.7 µg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation in the presence of S9 mix, this preliminary test was designated Experiment 1A.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment IA and IB in the absence and presence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment II in the absence of S9 mix clear cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix cytotoxicity of 42.3% cytostasis was observed at the highest evaluated concentration. Higher concentrations were not evaluable for cytogenetic damage.

Table 1: Results of Experiment 1A and 1B.

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 (Exp. 1A)

DMSO

0.5% (v/v)

1.63

0.70

MMC

3.0

1.15

7.85S

Test substance

70.7

1.65

0.55

216.4

1.54

0.30

378.8

1.59

0.40

662.9PS

1.59

0.20

Exposure period 4 h, fixation time 40 h, without S9-mix (Exp. 1B)

DMSO

0.5% (v/v)

1.86

0.95

MMC

2.0

1.35

6.85S

Test substance

125

1.80

0.75

250

1.83

0.80

500

1.56

0.80

Exposure period 4 h, fixation time 40 h, with S9 mix (Exp. 1A)

DMSO

0.5% (v/v)

1.77

0.75

CPA

15.0

1.71

3.00S

Test substance

216.4PS

1.78

0.55

378.8PS

1.80

0.35

662.9PS

1.61

0.40

CPA: Cyclophosphamide

DMSO:Dimethylsulfoxide

MMC: Mitomycin C

PS: Phase separation occurred at the end of the treatment

S: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)

1.91

0.25

Demecolcin

125 ng

1.65

3.05S

Test substance

178.2

1.82

0.20

267.3

1.74

0.15

401

1.35

0.15

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

DMSO

0.5% (v/v)

1.94

1.55

CPA

15.0

1.80

3.40S

Test substance

267.3PS

1.81

0.35

401PS

1.80

0.65

601.5PS

1.54

0.45

CPA: Cyclophosphamide

DMSO:Dimethylsulfoxide

PS: Phase separation occurred at the end of the treatment

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

Neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test substance.

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 15, 50, 150, 500, 1500 and 5000 µg/plate were selected for the incubation with and without metabolic activation in the first experiment. In the second experiment, 15, 50, 150, 500 and 1500 µg/plate were selected for the incubation without metabolic activation and 15, 50, 150, 500, 1500 and 5000 µg/plate for the incubation with metabolic activation. In the absence and presence of metabolic activation the test substance was bacteriotoxic towards all tester strains at 5000 µg/plate. In the concentration range investigated, the test substance induced a slight increase in the mutation frequency in strains TA 1535 and TA 100 in the absence of a metabolic activation in both experiments. In the presence of metabolic activation a slight increase in the frequency of revertants of strain TA 102 was observed. However, the estimation of the statistical significance did not reveal a significant effect at any of the test points. The results with the positive control substances confirmed the known reversion properties and specifity of the tester strains as well as the full activity of the metabolizing system. Under the conditions of this experiment, the test substance did not show mutagenicity 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 16.3 and 2090 µg/mL (equal to 10 mM) were used. Relevant toxic effects occurred at 522.5 µg/mL and above in the presence and absence of metabolic activation after 4 and 24 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 520 and 390 µg/mL, respectively. The second experiment was performed with a treatment time of 4 h with and 24 h without metabolic activation up to concentration of 325 and 520 µg/mL, respectively. Relevant cytotoxic effects indicatedby a relative cloning efficiency or cell density below 50% occurred in both cultures of the first experiment at the highest analysable concentration of 260 µg/mL without metabolic activation. Due to a very steep gradient of toxicity, no relevant cytotoxicity was noted up to the maximum analysable concentrations in all of the other experimental parts. Exceedingly severe cytotoxicity precluded analysis at the next higher concentration even though the concentrations were spaced by less than a factor of 2.0 recommended by the OECD guideline 476.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutant frequency generally did not exceed the historical range of solvent controls. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of the first experiment at 130 µg/mL without metabolic activation. This effect however, was based upon the rather low solvent control of 6.5 mutant colonies/106 and thus, judged as biologically irrelevant. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in culture I of the second experiment without metabolic activation. Since the mutation frequency neither exceeded the historical range of solvent controls nor the threshold as indicated above, the statistical result was judged as biologically irrelevant. 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 in total three 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 13.2 to 2030 µg/mL (with and without metabolic activation) were chosen for the evaluation of cytotoxicity. Since the cultures fulfilled the requirements for cytogenetic evaluation in the presence of metabolic activation, this preliminary test was designated Experiment IA. The experimental part without metabolic activation was repeated with closer concentrations and reported as Experiment IB. In Experiment II, cultures were exposed to the test substance for 4 h with and 20 h without metabolic activation up to concentration of 2030 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 binucleate cells per culture were evaluated for cytogenetic damage. No precipitation of the test substance in the culture medium was observed. Phase separation was observed at the end of treatment in Experiment IA at 662.9 µg/mL and above in the absence and at 123.7 µg/mL and above in the presence of metabolic activation. In Experiment IB, phase separation occurred at 600 µg/mL and above in the absence and in Experiment II at 902.2 µg/mL and above in the absence and at 601.5 µg/mL and above in the presence of metabolic activation. No relevant influence on osmolarity or pH was observed. In Experiment IA and IB in the absence and presence of metabolic activation, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment II in the presence and absence of metabolic activation clear cytotoxicity was observed at the highest evaluated concentration. No biologically relevant increase in the number of micronucleated cells was observed after treatment with the test substance with and without metabolic activation. Mitomycin C, demecolcin and cyclophosphamide 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-clastogenic and non-aneugenic in this in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentrations.


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.