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 1535 and TA 1537 and E.coli WP2 uvr A with and without metabolic activation

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

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:
22 Jan - 08 Feb 2016
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:
adopted 21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
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: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

The pre-experiment is reported as Experiment 1.

Experiment 2: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to bacteria.
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1); pre-incubation (Experiment 2)

DURATION
- Pre-incubation period: 1 h
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Evaluation criteria:
A test substance is considered as a mutagenic if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
Mean values and standard deviations were calculated.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(exp. 1: -S9: starting at 1000 µg/plate in TA98, TA100, TA1537 and at 2500 µg/plate in TA1535; +S9: starting at 2500 µg/plate in all strains; exp. 2: starting at 333 µg/plate in all strains with and without S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(exp. 1: starting at 2500 µg/plate with and without S9 mix; exp. 2: starting at 333 µg/plate with and without S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the overlay agar in the test tubes at the highest concentration of 5000 µg/plate in Experiment 1 and in the concentrations ranging from 2500 to 5000 µg/plate in Experiment 2. Precipitation of the test substance in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate with and without metabolic activation in both experiments.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I (all strains were tested in the pre-experiment).

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

Table 2. Test results of Experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration [μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA1535

TA100

WP2 uvrA

TA1537

TA98

-

0 (DMSO)

11 ± 5

193 ± 14

38 ± 5

12 ± 3

22 ± 4

-

0

11 ± 2

206 ± 8

46 ± 3

9 ± 2

31 ± 6

-

3

10 ± 3

192 ± 15

50 ± 10

10 ± 3

29 ± 8

-

10

12 ± 5

208 ± 9

34 ± 7

10 ± 2

26 ± 10

-

33

12 ± 2

196 ± 25

47 ± 7

9 ± 4

25 ± 10

-

100

15 ± 2

192 ± 20

41 ± 3

13 ± 3

22 ± 6

-

333

13 ± 2

196 ± 22

35 ± 5

9 ± 2

24 ± 5

-

1000

11 ± 2

13 ± 1

28 ± 1

5 ± 3

9 ± 2

-

2500

1 ± 1P

1 ± 0P

5 ± 2P

3 ± 0P

4 ± 1P

-

5000

0 ± 0P

1 ± 1P

0 ± 0P

1 ± 1P

0 ± 0P

Positive controls, –S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentration

[μg/plate]

10

10

2.0 µL

50

10

Mean No. of colonies/plate

(average of 3 ± SD)

1044 ± 127

1756 ± 164

912 ± 137

74 ± 22

357 ± 40

+

0 (DMSO)

11 ± 2

203 ± 2

47 ± 5

11 ± 2

28 ± 2

+

0

13 ± 4

211 ± 10

48 ± 5

16 ± 1

33 ± 11

+

3

9 ± 5

201 ± 12

49 ± 11

14 ± 3

35 ± 7

+

10

14 ± 6

196 ± 36

44 ± 7

11 ± 3

31 ± 12

+

33

11 ± 4

194 ± 23

54 ± 5

14 ± 1

40 ± 8

+

100

12 ± 4

205 ± 9

49 ± 9

10 ± 2

38 ± 11

+

333

12 ± 5

181 ± 16

46 ± 5

10 ± 6

32 ± 3

+

1000

10 ± 3

105 ± 26

47 ± 12

12 ± 4

47 ± 12

+

2500

1 ± 1P

2 ± 0P

8 ± 1P,M

2 ± 1P

9 ± 4P

+

5000

0 ± 0P

1 ± 2P

1 ± 1P

0 ± 0P

1 ± 1P

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

465 ± 42

4391 ± 109

463 ± 29

158 ± 28

5064 ± 226

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

P: precipitate

 

Table 3. Test results of Experiment 2 (preincubation).

With or without S9-Mix

Test substance concentration [μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA1535

TA100

WP2 uvrA

TA1537

TA98

-

0 (DMSO)

12 ± 3

143 ± 22

39 ± 9

14 ± 1

29 ± 6

-

0

10 ± 2

205 ± 4

34 ± 9

11 ± 1

29 ± 1

-

3

14 ± 5

131 ± 22

33 ± 3

14 ± 5

21 ± 2

-

10

12 ± 3

166 ± 24

39 ± 13

13 ± 4

24 ± 5

-

33

12 ± 6

139 ± 13

35 ± 4

10 ± 4

21 ± 6

-

100

9 ± 2

116 ± 12

32 ± 2

10 ± 4

30 ± 3

-

333

5 ± 2R

4 ± 1R

8 ± 1R

2 ± 1R

2 ± 1R

-

1000

1 ± 1R

1 ± 1R

4 ± 1R

1 ± 1R

0 ± 0R

-

2500

0 ± 1P,R

0 ± 0P,R

0 ± 0P,R

0 ± 0P,R

0 ± 0P,R

-

5000

0 ± 0P,R

0 ± 0P,R

0 ± 0P,R

0 ± 0P,R

0 ± 0P,R

Positive controls, –S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentration

[μg/plate]

10

10

2.0 µL

50

10

Mean No. of colonies/plate

(average of 3 ± SD)

1135 ± 86

1765 ± 42

653 ± 30

86 ± 14

369 ± 19

+

0 (DMSO)

13 ± 2

165 ± 42

46 ± 6

15 ± 6

33 ± 4

+

0

16 ± 3

170 ± 23

49 ± 14

20 ± 4

39 ± 1

+

3

11 ± 3

126 ± 13

32 ± 10

16 ± 1

40 ± 5

+

10

13 ± 5

141 ± 22

25 ± 8

18 ± 3

36 ± 10

+

33

13 ± 3

141 ± 27

32 ± 8

15 ± 1

40 ± 6

+

100

12 ± 2

127 ± 12

61 ± 12

13 ± 3

39 ± 6

+

333

2 ± 1R

1 ± 1R

4 ± 1R

4 ± 2R

2 ± 1R

+

1000

1 ± 0R

0 ± 1R

2 ± 1R

1 ± 1R

1 ± 1R

+

2500

0 ± 0P,R

0 ± 1P,R

1 ± 1P,R

0 ± 1P,R

1 ± 1P,R

+

5000

0 ± 0P,R

0 ± 0P,R

0 ± 0P,R

0 ± 0P,R

0 ± 0P,R

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

342 ± 63

2934 ± 90

464 ± 2

140 ± 6

3950 ± 265

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

P: precipitate

R: reduced background growth

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Jan - 10 Mar 2016
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:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: 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“
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
other: mammalian 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 treatment: 9.4, 18.8, 37.6, 75.1, 150.3, 300.5, 601 and 1202 with and without metabolic activation

Main experiment:
4 h treatment: 18.8, 37.5, 75, 100, 150, 200 and 250 µg/mL without metabolic activation
4 h treatment: 18.8, 37.5, 75, 150, 200, 250 and 300 µg/mL with metabolic activation
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
- Exposure duration: 4 h exposure with and 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: duplicate cultures per concentration level

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the cloning efficiency below 50%
Evaluation criteria:
A test substance is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test substance 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 positive response is described as follows:
A test substance 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) 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:
at 150 µg/mL and above without metabolic activation and at 250 µg/mL and above with metabolic activation
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 substance.
- Precipitation: Phase separation was observed in the pre-experiment at 601 µg/mL and above with and without metabolic activation. In the main experiment no phase separation occurred up to the maximum concentration tested in the presence and absence of 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 and absence (4 h treatment) of metabolic activation. Test substance concentrations between 9.4 µg/mL and 1202 µg/mL (equal to a molar concentration of approx. 10 mM) were used. The highest concentration of the pre-experiment was chosen with regard to the molecular weight (120.16 g/mol) of the test substance. A relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below, was observed at 75.1 µg/mL and above in the absence of metabolic activation and at 300.5 µg/mL and above in the presence of metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A relevant cytotoxic effect indicated by an adjusted cloning efficiency below 50% in both cultures occurred at 150 µg/mL and above without metabolic activation and at 250 µg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10 - 20% relative adjusted cloning efficiency was covered without metabolic activation. In the experimental part with metabolic activation a very steep gradient of toxicity resulted in adjusted cloning efficiency values of 32.5% and 40.0% at 250 µg/mL and exceedingly severe cytotoxicity at the next higher concentration of 300 µg/mL.

Table 2: Main experiment - 4 h exposure - Without Metabolic Activation

Concentration
[µg/mL]

Rel. cloning efficiency

Rel. cell density

Rel. adjusted cloning efficiency

Mutant colonies per 106cells

Culture I

0 (DMSO)

100.0

100.0

100.0

17.6

18.8

95.1

105.9

100.7

13.5

37.5

95.1

93.7

89.1

16.0

75

85.1

102.8

87.6

19.5

100

59.8

109.9

65.7

10.8

150

14.0

106.3

14.9

5.9

200

1.0

61.3

0.6

Culture was not continued#

250

0.0

17.4

0.0

Culture was not continued#

EMS, 300

89.8

94.1

84.6

141.2

Culture II

0 (DMSO)

100.0

100.0

100.0

16.1

18.8

158.1

58.7

92.7

18.6

37.5

170.2

63.0

107.2

10.0

75

120.8

93.9

113.4

11.6

100

56.9

82.1

46.7

14.2

150

6.6

81.6

5.4

5.3

200

0.3

46.4

0.1

Culture was not continued#

250

0.0

22.8

0.0

Culture was not continued#

EMS, 300

132.2

69.6

91.9

278.5

DMSO: dimethyl sulfoxide

EMS: ethylmethane sulfonate

#: culture was not continued due to exceedingly severe cytotoxic effects

Table 3: Main experiment - 4 h exposure - With Metabolic Activation

Concentration
[µg/mL]

Rel. cloning efficiency

Rel. cell density

Rel. adjusted cloning efficiency

Mutant colonies per 106cells

Culture I

0 (DMSO)

100.0

100.0

100.0

15.8

18.8

74.7

84.5

63.1

Culture was not continued##

37.5

99.7

96.1

95.9

10.2

75

116.4

93.4

108.7

19.2

150

115.7

89.5

103.5

29.9

200

80.5

85.8

69.0

22.0

250

38.6

84.3

32.5

15.8

300

4.6

87.4

4.0

Culture was not continued#

DMBA, 2.3

73.5

93.2

68.5

183.2

Culture II

0 (DMSO)

100.0

100.0

100.0

11.0

18.8

92.5

105.6

97.6

Culture was not continued##

37.5

93.5

105.3

98.5

16.2

75

85.9

96.0

82.5

20.5

150

92.9

89.2

82.9

20.3

200

83.1

96.1

79.9

7.4

250

41.0

97.6

40.0

18.7

300

6.1

97.7

5.9

Culture was not continued#

DMBA, 2.3

98.7

109.0

107.6

150.6

DMSO: Dimethyl sulfoxide

DMBA: 7,12-dimethylbenzanthracene

#: culture was not continued due to exceedingly severe cytotoxic effects

##: culture was not continued since a minimum of only four concentrations required

No relevant and reproducible increase in mutant colony numbers/10E06 cells was observed in the main experiments up to the maximum concentration.

The 95% confidence interval was slightly exceeded at 150 µg/mL in the first culture with metabolic activation. This increase was judged as irrelevant as it was not reproduced in the parallel culture (the mean value of both parallel cultures, 29.9 and 20.3 equal to a mean of 25.1 colonies per 10E06 cells easily remained within the 95% confidence interval) and there was no dose dependent increase as indicated by a lacking statistical significance of the trend test. Furthermore, the mutation frequency of the experimental part with metabolic activation remained within the range of historical 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 with 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 substance 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:
20 Jan - 30 Mar 2016
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 Sep 2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
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 phytohemagglutinin (PHA) to the culture medium for a period of 48 h.
- 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:
Without S9 mix:
Experiment IA (4 hours exposure): 7.81, 13.66, 23.91, 41.85*, 73.23*, 128.2*, 224.3, 392.5, 686.9 and 1202 µg/mL
Experiment II (20 hours exposure): 7.81, 13.66, 23.91*, 41.85*, 73.23*, 128.2, 224.3, 392.5, 686.9 and 1202 µg/mL

With S9 mix:
Experiment IB (4 hours exposure): 7.81, 13.66, 23.91, 41.85*, 73.23*, 128.2*, 224.3, 392.5, 686.9 and 1202 µg/mL

* evaluated for cytogenetic damage
Vehicle / solvent:
- Vehicle/solvent used: DMSO (final concentration in cell culture medium was 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
- Preparation time (start of exposure up to fixation or harvest preparation 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: Two parallel cultures in 3 independent experiments (IA, IB and II)

NUMBER OF CELLS EVALUATED: 1000 binucleated cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)

Evaluation criteria:
A test substance is considered to be negative if:
− none of the test substance concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− there is no concentration-related increase
− the results in all evaluated test substance concentrations should be within the range of the laboratory historical solvent control data

A test substance is considered to be positive if:
− at least one of the test substance concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− the increase is concentration-related in at least one experimental condition
− the results are outside the range of the laboratory historical solvent control data
Statistics:
Statistical significance was confirmed by means of the Chi square test.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
no cytotoxicity was observed up to the highest evaluated concentration
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 and osmolarity: No relevant influence on osmolarity or pH value was observed.
- Precipitation: In the experiments IA, IB and II in the absence and presence of S9 mix, phase separation of the test substance in the culture medium was observed at 686.9 µg/mL and above at the end of treatment.

RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described for the mutagenicity assay. The pre-experiment was performed with 10 concentrations of the test substance and the solvent and positive controls. All cell cultures were set up in duplicate. Exposure time was 4 hours (with and without S9 mix) and the cells were prepared 40 hours after start of the exposure. Since the cultures fulfilled the requirements for cytogenetic evaluation in the absence of S9 mix, the preliminary test was designated Experiment IA. Due to strong cytotoxic effects, the experimental part with S9 mix was repeated with the same top dose (Exp. IB).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration.

Table 2: Results of Experiment IA, IB and II.

Test item

Concentration[µg/mL]

Proliferation index CBPI

Cystostasis (%)1)

Micronucleated cells (%)2)

Experiment IA

(Exposure period 4 h, preparation interval 40 h, without S9 mix)

DMSO

0.5% (v/v)

1.99

-

0.15

MMC

1.0

1.79

20.9

5.15S

Test substance

41.85

1.90

9.7

0.10

73.23

1.84

15.2

0.40

128.2

1.73

27.1

0.40

Experiment IB

(Exposure period 4 h, preparation interval 40 h, with S9 mix)

DMSO

0.5% (v/v)

2.05

-

0.45

CPA

15.0

1.80

24.5

4.75S

Test substance

41.85

2.10

n.c.

0.70

73.23

2.07

n.c.

0.55

128.2

2.03

2.1

0.80

Experiment II

(Exposure period 20 h, preparation interval 40 h, without S9 mix)

DMSO

0.5% (v/v)

1.89

-

0.25

Demecolcin

100 ng/mL

1.57

36.1

2.75S

Test substance

23.91

2.01

n.c.

0.55

41.85

1.97

n.c.

0.20

73.23

1.87

2.5

0.70S

1) for the positive control groups and the test substance treatment groups the values are related to the solvent controls

2) The number of micronucleated cells was determined in a sample of 2000 binucleated cells.

CPA: cyclophosphamide

DMSO:dimethylsulfoxide

MMC: mitomycin C

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

No relevant increase in the number of micronucleated cells was observed after treatment with the test substance with and without metabolic activation. However, in Experiment II a statistical significant increase in micronucleate cells (0.70 %) was observed at the highest evaluated concentration (73.23 µg/mL). Since the value is within the 95% control limit (0.05 – 1.05 % micronucleate cells), the finding can be regarded as biologically irrelevant.

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 (2016). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and the Escherichia coli strain WP2 uvrA were exposed to the test substance using either the pre-incubation or the plate incorporation method. Each concentration, including the controls, was tested in triplicate. In both experiments, test substance concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate were selected for the incubation with and without metabolic activation. Precipitation was observed in the overlay agar in the test tubes at the highest concentration of 5000 µg/plate in Experiment 1 and in the concentrations ranging from 2500 to 5000 µg/plate in Experiment 2. Precipitation of the test substance in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate with and without metabolic activation in both experiments. In Experiment 1, cytotoxicity was observed starting at 1000 µg/plate in TA 98, TA 100 and TA 1537 and at 2500 µg/plate in TA1535 and WP2 uvrA without metabolic activation. With metabolic activation, cytotoxicity was noted in all strains starting at 2500 µg/plate. In Experiment 2, reduced background growth was observed from 333 to 5000 µg/plate with and without metabolic activation in all strains. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation. In the concentration range investigated, no substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance, neither in the presence nor absence of metabolic activation. Furthermore, no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance was noted. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium strains and in the E. coli strain in the presence or 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 (2016). 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 and absence of metabolic activation with an incubation time of 4 h. Test substance concentrations between 9.4 and 1202 µg/mL (equal to 10 mM) were used. Phase separation was observed at 601 µg/mL and above in the presence and absence of metabolic activation. Relevant toxic effects occurred at 75.1 µg/mL and above in the absence of metabolic activation and at 300.5 µg/mL and above in the presence 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 with or without metabolic activation up to concentrations of 300 µg/mL and 250 µg/mL, respectively. Relevant cytotoxic effects indicated by a relative cloning efficiency or cell density below 50% occurred in both cultures at 150 µg/mL and above without metabolic activation and at 250 µg/mL and above with metabolic activation. Very steep gradient of toxicity in the experimental part with metabolic activation resulted in adjusted cloning efficiency I values of 32.5% and 40.0% at 250 μg/mL. Exceedingly severe cytotoxicity precluded analysis at the next higher concentration of 300 µg/mL 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/10E06 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 95% confidence interval was slightly exceeded at 150 μg/mL in the first culture with metabolic activation. This increase was judged as irrelevant as it was not reproduced in the parallel culture and there was no dose dependent increase as indicated by a lacking statistical significance of the trend test. 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 with 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 micronuclei was investigated in an in vitro mammalian cell micronucleus test in cultured peripheral human lymphocytes performed according to OECD Guideline 487 and GLP (2016). The test substance was dissolved in DMSO and three independent experiments were performed in total. A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Test substance concentrations ranging from 7.81 to 1202 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. Due to strong cytotoxic effects, the experimental part with metabolic activation was repeated with the same top dose (Exp. IB). In Experiment II, cultures were exposed to the test substance for 20 h without metabolic activation up to a concentration of 1202 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. In experiments IA, IB and II with and without metabolic activation, phase separation of the test substance in the culture medium was observed at 686.9 μg/mL and above at the end of treatment. No relevant influence on osmolarity or pH was observed. However, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. No cytotoxicity was observed with and without metabolic activation up to 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. However, in Experiment II a statistical significant increase in micronucleate cells (0.70 %) was observed at the highest evaluated concentration (73.23 μg/mL). Since the value is within the 95% control limit (0.05 – 1.05 % micronucleate cells), the finding can be regarded as biologically irrelevant. 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.