Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

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

Micronucleus test (OECD 487): negative in 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:
24 Aug - 24 Sep 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
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:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone/ß-naphthoflavone (80/100 mg/kg bw/d, oral)
Test concentrations with justification for top dose:
Preliminary toxicity study:
0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation for TA100

First experiment:
15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for TA98
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for TA100, TA1535, TA102 and TA1537
5, 15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for TA1535
5, 15, 50, 150, 500 and 1500 µg/plate with metabolic activation for TA100, TA102, TA98 and TA1537

Second experiment:
15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for all strains
5, 15, 50, 150, 500 and 1500 µg/plate with metabolic activation for all strains
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
mitomycin C
other: 2-Aminoanthracene (2AA); 1,8- dihydroxyanthraquinon (DANTHRON)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) in experiment 1 and 2

DURATION
- Exposure duration: approx. 48 h

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the bacterial background lawn and/or a reduction in the frequency of revertant colonies
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.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
starting at 500 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: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. The test material was toxic at 5000 µg/plate to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be sterile.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the bacterial background lawn and/or a reduction in the frequency of revertant colonies in all of the tester strains both with and without metabolic activation. The first evidence of a weakened bacterial background lawn was observed at 500 µg/plate. Generally, the test material induced a greater toxic response after the addition of S9-mix. The sensitivity of the bacterial strains to the toxicity of the test material varied between strains and between exposures with or without S9-mix. The test material was, therefore, tested up to its toxic limit.

Table 1. Test results of experiment 1

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

TA100

TA1535

TA102

TA98

TA1537

0

128

20

247

24

7

0 (DMSO)

114 ± 13.7

30 ± 3.5

317 ± 36.4

28 ± 3.5

13 ± 2.3

15

NT

NT

NT

25 ± 4.0

NT

50

109 ± 4.4

19 ± 5.5

278 ± 3.6

22 ± 3.2

11 ± 2.9

150

112 ± 12.0

20 ± 2.6

281 ± 31.8

31 ± 6.5

12 ± 4.5

500

105 ± 2.1

15 ± 5.5

279 ± 22.2

28 ± 2.0

11 ± 1.0

1500

67 ± 6.1

5 ± 1.7

107 ± 12.1

10 ± 2.9

5 ± 4.7

5000

0

0

0T

0

0

Positive controls, –S9

Name

ENNG

ENNG

MMC

4-NQO

9-AA

Concentrations

(μg/plate)

3

5

0.5

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

539 ± 64.2

222 ± 39.2

751 ± 184.5

171 ± 19.0

784 ± 250.9

+

0

143

22

310

-

10

+

0 (DMSO)

145 ± 3.0

12 ± 3.5

310 ± 28.7

39 ± 1.7

16 ± 2.6

+

5

119 ± 9.8

12 ± 0.6

284 ± 63.4

40 ± 3.1

17 ± 7.8

+

15

118 ± 6.4

17 ± 4.5

318 ± 51.8

27 ± 8.1

15 ± 6.7

+

50

120 ± 14.7

10 ± 1.7

333 ± 21.7

29 ± 2.5

9 ± 2.1

+

150

98 ± 4.7

13 ± 2.6

296 ± 5.8

31 ± 4.0

10 ± 2.0

+

500

85 ± 6.1

6 ± 2.6

0V

12 ± 3.8

0V

+

1500

0V

0V

0V

0V

0T

+

5000

NT

0T

NT

NT

NT

Positive controls, +S9

Name

2-AA

2-AA

DAN

B[a]P

2-AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

1770 ± 182.4

192 ± 16.2

707 ± 65.6

255 ± 6.7

685 ± 40.8

DMSO: Dimethylsulphoxide

NT: Not tested at this dose level

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

ENNG: N-ethyl-N-nitro-N-nitrosoguanidine

4-NQO: 4-nitroquinoline-N-oxide

MMC: Mitomycin C

B[a]P: Benzo(a)pyrene

DAN: 1,8-dihydroxyanthraquinone

T: Toxic

V: Very weak lawn

Table 2. Test results of experiment 2.

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

TA100

TA1535

TA102

TA98

TA1537

0

145

20

306

32

12

0 (DMSO)

147 ± 5.7

20 ± 1.5

339 ± 3.2

33 ± 4.0

12 ± 5.9

15

127 ± 17.7

18 ± 0.6

295 ± 23.0

38 ± 6.5

12 ± 4.9

50

146 ± 7.6

18 ± 2.6

300 ± 16.1

28 ± 5.3

5 ± 1.7

150

124 ± 4.5

15 ± 0.6

297 ± 29.4

22 ± 5.0

9 ± 4.6

500

110 ± 26.5

10 ± 0.6

310 ± 14.9

22 ± 5.5

6 ± 3.1

1500

63 ± 5.6

4 ± 2.1

158 ± 18.7

9 ± 3.5

6 ± 3.6

5000

0

2 ± 1.5

0

1 ± 1.2

4 ± 3.2S

Positive controls, –S9

Name

ENNG

ENNG

MMC

4-NQO

9-AA

Concentrations

(μg/plate)

3

5

0.5

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

471 ± 47.3

144 ± 20.9

1054 ± 39.6

139 ± 52.3

1032 ± 104.8

+

0

90

-

-

-

-

+

0 (DMSO)

107 ± 9.5

17 ± 5.0

302 ± 17.1

32 ± 3.5

10 ± 2.3

+

5

103 ± 3.8

15 ± 0.6

302 ± 34.8

28 ± 2.6

10 ± 4.4

+

15

100 ± 4.6

11 ± 2.9

310 ± 9.2

28 ± 4.6

8 ± 0.6

+

50

88 ± 20.3

16 ± 5.7

289 ± 12.9

38 ± 7.1

8 ± 0.6

+

150

85 ± 7.6

15 ± 4.2

265 ± 11.1

29 ± 1.5

3 ± 1.0

+

500

59 ± 2.5

11 ± 0.6

0S

10 ± 7.4S

0V

+

1500

21 ± 4.0V

2 ± 2.1S

0V

9 ± 3.1S

0T

Positive controls, +S9

Name

2-AA

2-AA

DAN

B[a]P

2-AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

1277 ± 77.7

124 ± 7.4

727 ± 165.4

188 ± 13.2

169 ± 12.0

DMSO: Dimethylsulphoxide

NT: Not tested at this dose level

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

ENNG: N-ethyl-N-nitro-N-nitrosoguanidine

4-NQO: 4-nitroquinoline-N-oxide

MMC: Mitomycin C

B[a]P: Benzo(a)pyrene

DAN: 1,8-dihydroxyanthraquinone

S: Sparse lawn

T: Toxic

V: Very weak lawn

Table 3. Historical control data

positive control values up to and including 1997
Strain TA100 TA98 TA1535 TA1538 TA1537 WP2uvrA TA102
S9-Mix +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9
Mean 117 120 28 33 24 18 21 25 10 11 20 21 264 283
SD 25 22 7 7 5 4 7 6 2 2 5 s 30 33
Min 61 63 13 14 10 11 6 11 4 5 12 11 216 205
Max 196 177 56 52 39 39 41 50 17. 20 .39 40 339 343
Values 1603 863 1047 837 948 718 309 190 938 703 634 436 197 100
vehicle control values up to and including 1997
Strain  TA100 TA98 TA1535 TA1538 TA1537 WP2uvrA TA102
S9-Mix +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9
Mean 613 949 203 420 466 291 484 454 812 266 834 734 785 705
SD 176 190 57 113 208 64 146 131 201 90 197 211 172 127
Min 277 412 127 198 163 139 190 212 161 123 342 225 540 511
M ax 1126 1315 698 757 1005 516 799 915 1149 718 1209 1089 1188 1090
Values 195 195 194 195 180 181 93 92 182 183 162 157 76 72
positive control values up to and including 1998
Strain TA100 TA98 TA1535 TA1538 TA1537 WP2uvrA TA102
S9-Mix +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9
Mean 124 125 31 36 26 18 26 28 11 14 23 25 262 285
SD 16 16 6 6 5 4 6 5 3 3 4 4 31 32
Min 73 81 15 22 12 10 15 18 5 6 13 13 191 231
Max 173 170 45 54 38 37 36 36 20 23 34 41 304 363
Values 342 339 337 340 341 331 16 16 323 325 287 285 20 22
vehicle control values up to and including 1998
Strain TA100 TA98 TA1535 TA1538 TA1537 WP2uvrA TA102
S9-Mix +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9
Mean 556 1209 143 472 375 260 403 556 1052 298 851 837 863 747
SD 114 318 24 119 173 64 93 197 259 106 202 223 127 116
Min 349 681 101 198 112 105 305 344 173 123 415 260 646 591
Max 1147 2740 225 733 1143 601 604 1073 1912 652 1486 1380 1096 1096
Values 141 141 140 143 142 141 12 12 142 141 138 138 24 25

SD = standard deviation

Min = minimal value

Max = maximal value

Conclusions:
Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA1535, TA1537, TA98, TA100 and TA102) with and without metabolic activation tested.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Aug - 21 Oct 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, Klimaschutz, 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: 15.5, 31, 62, 123.9, 247.9, 495.8, 991.5 and 1983 µg/mL without metabolic activation
4 h treatment: 15.5, 31, 62, 123.9, 247.9, 495.8, 991.5 and 1983 µg/mL with metabolic activation

Experiment 1:
4 h treatment: 15.5, 31, 62, 124, 248, 372 and 496 µg/mL without metabolic activation
4 h treatment: 7.8, 15.5, 31, 62, 124, 186 and 248 µg/mL with metabolic activation

Experiment 2:
24 h treatment: 31, 62, 124, 248, 496 and 744 µg/mL without metabolic activation
4 h treatment: 15.5, 31, 62, 124, 155 and 186 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
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 186 µg/mL in Exp. I and 124 µg/mL and above in Exp. II following 4 h treatment with S9; at 372 µg/mL in Exp. I and at 496 µg/mL and above in Exp. II following 4 and 24 h treatment, respectively, without S9
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.
Pre-Experiment:
Phase separation occurred at 991.5 µg/mL and above after 4 h treatment with and without metabolic activation. Following 24 h treatment phase separation was observed at 495.8 µg/mL and above.

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 15.5 µg/mL and 1983 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 purity (99.97%) and the molecular weight (198.29 g/mol) of the test item. Strong cytotoxic effects occurred after 4 h treatment at 247.9 µg/mL and above with and without metabolic activation. Following 24 h treatment, severe cytotoxicity was noted at 991.5 µg/mL and above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The cultures at the highest concentration with metabolic activation were not continued based on exceedingly severe cytotoxic effects in Experiment I. In Experiment II the cultures at the highest concentration without metabolic activation were not continued for the same reason.

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

Concentration
[µg/mL]

Rel. cloning efficiency I

Rel. cell density

Rel. cloning efficiency II

Mutant colonies per 10E6 cells

Induction factor

Culture I

0 (DMSO)

100.0

100.0

100.0

9.6

1.0

15.5

97.5

117.8

Culture was not continued#

31

98.3

120.4

93.9

8.9

0.9

62

93.0

112.2

95.2

17.1

1.8

124

83.4

119.2

86.7

16.1

1.7

248

56.9

98.3

91.2

18.5

1.9

372

2.5

10.6

61.7

11.9

1.2

496

0.0

Culture was not continued##

EMS, 150

92.6

89.6

91.5

153.2

16.0

Culture II

0 (DMSO)

100.0

100.0

100.0

5.0

1.0

15.5

101.9

82.1

Culture was not continued#

31

99.6

93.8

86.2

12.5

2.5

62

90.9

132.2

88.6

14.3

2.9

124

85.8

75.5

89.3

18.9

3.8

248

54.0

69.5

90.3

20.8

4.2

372

4.4

17.9

67.3

6.1

1.2

496

0.0

Culture was not continued##

EMS, 150

91.9

58.0

90.9

182.7

36.9

DMSO: Dimethyl sulfoxide

EMS: Ethylmethane sulfonate

#: 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 I - 4 h exposure - With Metabolic Activation

Concentration
[µg/mL]

Rel. cloning efficiency I

Rel. cell density

Rel. cloning efficiency II

Mutant colonies per 10E6 cells

Induction factor

Culture I

0 (DMSO)

100.0

100.0

100.0

17.8

1.0

7.8

105.0

95.5

Culture was not continued#

15.5

107.9

106.5

85.4

13.5

0.8

31

99.3

101.3

94.6

10.4

0.6

62

79.9

106.4

99.3

19.7

1.1

124

51.6

119.6

106.8

16.3

0.9

186

2.1

56.0

106.4

29.3

1.6

248

0.0

12.3

Culture was not continued##

DMBA, 2.2

86.1

87.5

86.7

210.9

11.8

Culture II

0 (DMSO)

100.0

100.0

100.0

17.8

1.0

7.8

106.8

98.5

Culture was not continued#

15.5

102.3

105.0

102.9

18.5

1.2

31

102.5

95.2

98.9

20.3

1.3

62

82.0

90.4

97.6

15.4

1.0

124

54.1

83.2

101.1

15.9

1.0

186

4.1

46.7

93.3

22.5

1.5

248

0.0

7.6

Culture was not continued##

DMBA, 2.2

80.7

105.6

94.2

144.1

9.5

DMSO: Dimethyl sulfoxide

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.

 

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

Concentration
[µg/mL]

Rel. cloning efficiency I

Rel. cell density

Rel. cloning efficiency II

Mutant colonies per 10E6 cells

Induction factor

Culture I

0 (DMSO)

100.0

100.0

100.0

16.5

1.0

31

98.8

89.4

94.3

8.5

0.6

62

91.8

68.9

87.6

12.9

0.9

124

94.3

105.8

83.4

22.5

1.5

248

91.5

54.0

83.9

13.4

0.9

496

48.7

80.1

80.8

12.1

0.8

744

0.0

0.0

Culture was not continued##

EMS, 150

93.6

84.9

72.8

392.7

26.8

Culture II

0 (DMSO)

100.0

100.0

100.0

14.9

1.0

31

98.5

123.0

105.6

15.3

0.8

62

94.9

81.9

103.1

13.7

0.7

124

95.9

116.0

100.7

28.1

1.5

248

90.3

118.8

96.3

13.4

0.7

496

58.1

87.1

99.4

15.3

0.8

744

0.0

3.3

Culture was not continued##

EMS, 150

93.2

108.8

97.6

301.4

16.0

DMSO: Dimethyl sulfoxide

EMS: Ethylmethane sulfonate

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

 

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

Concentration
[µg/mL]

Rel. cloning efficiency I

Rel. cell density

Rel. cloning efficiency II

Mutant colonies per 10E6 cells

Induction factor

Culture I

0 (DMSO)

100.0

100.0

100.0

12.6

1.0

15.5

97.5

Culture was not continued#

31

97.1

108.8

106.3

11.9

0.9

62

91.9

88.1

100.5

12.0

0.9

124

33.4

90.7

99.3

10.5

0.8

155

20.7

94.8

96.8

9.2

0.7

186

0.0

32.1

97.5

12.1

0.9

DMBA, 2.2

93.0

76.6

92.9

186.4

14.5

Culture II

0 (DMSO)

100.0

100.0

100.0

11.7

1.0

15.5

101.3

Culture was not continued#

31

103.9

95.6

76.7

32.8

2.2

62

92.0

84.9

63.6

21.7

1.4

124

39.3

85.6

89.7

27.0

1.8

155

25.4

87.8

102.9

17.9

1.2

186

0.0

41.1

99.8

15.3

1.0

DMBA, 2.2

95.0

87.4

96.5

186.5

12.4

DMSO: Dimethyl sulfoxide

DMBA: 7,12-dimethylbenzanthracene

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

Table 5. Histrorical control data

Number of mutant colonies per 10E6 cells
without metabolic activation (4 h treatment time)
  Positive control EMS 150 µg/mL Solvent control (medium, acetone, water, DMSO, ethanol, THF)
Range 54.88 - 889.0 1.6 - 45.7
Mean value 149.2 14.7
Standard deviation 99 7.8
Number of studies 108 108
with metabolic activation (4 h treatment time)
  Positive control DMBA 1.1 and 2.2 µg/mL Solvent control (medium, acetone, water, DMSO, ethanol, THF)
Range 77.7 - 2042.6 2.4 - 44.2
Mean value 495 13.9
Standard deviation 302.1 6.9
Number of studies 108 108
without metabolic activation 2(4 h treatment time)
  Positive control EMS 150 µg/mL Solvent control (medium, acetone, water, DMSO, ethanol, THF)
Range 108.5 - 786.1 2.4 - 41.8
Mean value 344.1 13.7
Standard deviation 126.4 7.1
Number of studies 103 103

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

In the second culture of Experiment I the mutant colonies/10E6 cells exceeded the induction factor of three times the mutation frequency of the corresponding solvent control at 124.0 and 248.0 µg/mL in the absence of metabolic activation. The effects however, were judged as biologically irrelevant as they were clearly based upon a rather low solvent control of 5.0 mutant colonies/10E6 cells. Furthermore, the threshold was not reached at any other, even higher concentration or in the parallel culture under identical conditions.

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.

 

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:
22 July - 08 Sep 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, 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:
Pre-Experiment / Experiment 1:
4 h treatment: 12.9, 22.5, 39.5, 69.0, 120.8, 211.4*, 370*, 647.5*, 1133.1 and 1983 µg/mL with and without metabolic activation
Since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment 1.

Experiment 2:
20 h treatment: 12.9, 22.5, 39.5, 69.0, 120.8, 211.4*, 370*, 647.5*, 1133.1 and 1983 µg/mL without 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
- 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 2 independent experiments

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.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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.
- Other confounding effects: Phase separation of the test substance was observed at the end of treatment at 647.5 µg/mL and above with and without metabolic activation.

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. Since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment 1.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed up to 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 %1)

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

DMSO

0.5% (v/v)

1.89

0.50

MMC

1.0

1.35

9.40S

Test substance

211.4

1.85

0.80

370

1.82

0.40

647.5PS

1.84

0.30

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

DMSO

0.5% (v/v)

1.92

0.95

CPA

12.5

1.43

3.05S

Test substance

211.4

1.86

1.00

370

1.91

0.55

647.5PS

1.84

0.85

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

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 %1)

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

DMSO

0.5% (v/v)

1.58

0.80

Demecolcin

50 ng/mL

1.35

2.15S

Test substance

211.4

1.75

0.95

370

1.72

0.40

647.5PS

1.64

0.55

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

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.

Either demecolcin, MMC or CPA were used as positive controls and showed distinct increases in cells with micronuclei.

Table 3. Historical control data (2009-2013)

Without metabolic activation    
    Micronucleated cells (%)
Concentration No. of experiments Range Mean ±Standard deviation
Solvent control (pulse treatment)
Aqueous solvent 1) 41 0.10 - 1.30 0.57 0.22
Organic solvent 2) 39 1.15 - 1.40 0.59 0.3
Total 80 0.10 - 1.40 0.58 0.26
Solvent control (continuous treatment)
Aqueous solvent 1) 49 0.30 - 1.45 0.3 0.19
Organic solvent 2) 47 0.05 - 1.35 0.44 0.21
Total 96 0.05 - 1.45 0.43 0.2
Total 3)# 10 0.10 - 0.80 0.43 0.21
Positive control (pulse treatment)
Mitomycin C 0.3 - 3.0 µg/mL 47 2.85 - 33.35 11.35 5.18
Positive control (continuous treatment)
Demecolcin 50 - 150 ng/mL 52 1.40 - 6.85 3.62 1.05
Demecolcin# 50 - 175 ng/mL 11 1.30 - 5.45 2.79 1.18
With metabolic activation    
Solvent control (pulse treatment)
Aqueous solvent 1) 79 0.15 - 1.70 0.65 0.27
Organic solvent 2) 70 0.15 - 1.65 0.66 0.29
Total 149 0.15 - 1.70 0.65 0.28
Positive control (pulse treatment)
CPA 7.5 - 20.0 µg/mL 81 2.15 - 11.05 5.22 1.89

1) Aqueous solvents: DMEM/Ham’s F12, deionised water (10 % v/v)

2) Organic solvents: DMSO (0.5 or 1.0 %), acetone, ethanol and THF (0.5 %)

3) Aqueous and organic solvents

# Micronucleated mononucleate cells

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 (Bowles, 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 suspended in DMSO using the plate incorporation method. The dose range for the first experiment was determined in a preliminary toxicity assay and ranged from 5 to 5000 µg/plate depending on bacterial strain type and presence or absence of metabolic activation. The dose range of the second experiment was based on the results observed in Experiment 1 and ranged from 5 to 5000 µg/plate depending on bacterial strain type and presence or absence of metabolic activation. The test substance caused a visible reduction in the bacterial background lawn and/or a reduction in the frequency of revertant colonies in all of the tester strains both with and without metabolic activation. The first evidence of a weakened bacterial background lawn was observed at 500 µg/plate. Generally, the test substance induced a greater toxic response after the addition of S9-mix. The sensitivity of the bacterial strains to the toxicity of the test material varied between strains and between exposures with or without S9-mix. The test substance was, therefore, tested up to its toxic limit. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The vehicle control plates gave counts of revertant colonies within the normal range. A marked increase in the frequency of revertant colonies, both with and without metabolic activation, was observed as a result of treatment with positive control chemicals.

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. The study was performed in two independent experiments, using identical experimental procedures. Based on the results of a pre-test, cells of the first experiment were exposed to the test substance for 4 h at concentrations ranging from 31 - 372 µg/mL without metabolic activation and from 15.5 - 186 µg/mL with metabolic activation. In the second experiment cells were exposed to the test substance for 4 h at concentrations ranging from 31 - 186 µg/mL and for 24 h at concentrations ranging from 31 - 496 µg/mL. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both cultures occurred in the first experiment at 372 μg/mL without metabolic activation and at 186 μg/mL with metabolic activation. In the second experiment cytotoxicity as described above was noted at 124 μg/mL and above with metabolic activation. In the second experiment without metabolic activation a steep gradient of cytotoxicity occurred with only moderate toxic effects at 496 μg/mL. No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The range of the historical solvent control data was not exceeded. 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 human lymphocytes performed according to OECD Guideline 487 and GLP (Sokolowski, 2016). The test substance was dissolved in DMSO and cytotoxicity of the test substance was investigated in a preliminary test. Cells were incubated for 4 hours with and without metabolic activation up to 1983 µg/mL (approx. 10 mM). Since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment 1. In Experiment 2, cells were incubated for 20 hours without S9 mix up to 1983 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. Phase separation of the test substance was observed at the end of treatment at 647.5 µg/mL and above with and without metabolic activation. No relevant influence of the test substance on osmolarity or pH value was observed. No cytotoxicity was observed up to the highest evaluated concentration. No biologically relevant increase in the number of micronucleated cells was observed after treatment with the test substance. 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 phase separating 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.