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 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with and without metabolic activation

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

Chromosome Aberration test (OECD 473): 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:
21 June - 04 July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Energie, 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:
Experiment I and II: 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 low toxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA), methylmethanesulfonate (MMS)
Remarks:
+S9: 2-AA (2.5 µg/plate, TA1535, TA1537, TA98, TA100; 10 µg/plate, WP2 uvrA); -S9: NaN3 (10 µg/plate, TA1535, TA100); 4-NOPD (10 µg/plate, TA98; 50 µg/plate, TA1537); MMS (2 µL/plate, WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 1 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiment

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 mutagen 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 deviation were calculated.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
exp. 1: -S9: starting at 1000 µg/plate, +S9: starting at 2500 µg/plate; exp. 2: -S9: starting at 333 µg/plate, +S9: starting at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
exp. 1: -S9 and +S9: starting at 2500 µg/plate; exp. 2: -S9: starting at 333 µg/plate, +S9: starting at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
exp. 1: -S9: starting at 1000 µg/plate, +S9: starting at 2500 µg/plate; exp. 2: -S9 and +S9: starting at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
exp. 1: -S9: starting at 1000 µg/plate, +S9: starting at 2500 µg/plate; exp. 2: -S9: starting at 100 µg/plate, +S9: starting at 1000 µg/plate
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. 2: +S9: at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as experiment I.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Reduced background growth was observed at the higher concentrations with and without metabolic activation in both experiments. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations with and without metabolic activation in both experiments.

Table 1. Test results of main test 1 (plate incorporation).

With or without S9 mix

Test substance concentration

g/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvrA

TA98

TA1537

-

0

134 ± 6

13 ± 1

46 ± 9

31 ± 4B

14 ± 5

-

0 (DMSO)

117 ± 22

16 ± 3

50 ± 5

22 ± 3B

14 ± 4

-

3

126 ± 10

17 ± 5

44 ± 6

25 ± 3B

13 ± 3

-

10

123 ± 25

17 ± 3

47 ± 2

22 ± 3B

13 ± 4

-

33

120 ± 20

19 ± 5

50 ± 2

21 ± 2B

10 ± 4

-

100

130 ± 6

18 ± 2

48 ± 3

22 ± 4B

11 ± 4

-

333

107 ± 18

10 ± 4

45 ± 10

20 ± 4B

11 ± 4

-

1000

26 ± 7R

9 ± 3R

49 ± 5

16 ± 3B R

8 ± 1

-

2500

9 ± 2R

5 ± 1R

54 ± 8

10 ± 2B R

11 ± 2R

-

5000

5 ± 1R

5 ± 2R

50 ± 17

7 ± 2B R

3 ± 1R

Positive controls, –S9 mix

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentrations

[μg/plate]

10

10

2.0 μl

10

50

Mean No. of colonies/plate

(average of 3 ± SD)

2232 ± 43

2026 ± 7

956 ± 36

341 ± 14

81 ± 2

+

0

196 ± 17

15 ± 7

58 ± 2

39 ± 4B

19 ± 6

+

0 (DMSO)

201 ± 4

18 ± 6

59 ± 8

44 ± 7B

17 ± 2

+

3

202 ± 24

20 ± 1

50 ± 3

41 ± 6B

19 ± 8

+

10

199 ± 25

18 ± 6

50 ± 7

43 ± 5B

20 ± 8

+

33

202 ± 24

24 ± 2

64 ± 9

41 ± 5B

16 ± 4

+

100

228 ± 13

21 ± 1

57 ± 7

42 ± 5B

21 ± 8

+

333

223 ± 30

17 ± 5

53 ± 9

32 ± 10B

17 ± 6

+

1000

189 ± 10

15 ± 4

57 ± 13

23 ± 5B

20 ± 3

+

2500

7 ± 2R

7 ± 1R

30 ± 6

4 ± 2B

2 ± 1R

+

5000

1 ± 1R

1 ± 1R

32 ± 7

1 ± 1B

1 ± 1R

Positive controls, +S9 mix

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

3418 ± 159

472 ± 45

431 ± 29

2905 ± 19

349 ± 101

NaN3: sodium azide

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

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

B: extensive bacterial growth

R: reduced background growth

Table 2. Test results of main test 2 (preincubation).

With or without S9 mix

Test substance concentration

g/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvrA

TA98

TA1537

-

0

124 ± 12

13 ± 1

66 ± 4

31 ± 5

13 ± 6

-

0 (DMSO)

114 ± 3

14 ± 1

64 ± 7

27 ± 3

10 ± 3

-

3

98 ± 2

14 ± 6

63 ± 4

26 ± 7

12 ± 4

-

10

104 ± 11

15 ± 2

58 ± 1

28 ± 10

8 ± 2

-

33

98 ± 10

12 ± 4

59 ± 4

28 ± 7

13 ± 4

-

100

38 ± 4R

14 ± 3

57 ± 7

29 ± 7

8 ± 1

-

333

20 ± 4R

9 ± 2R

46 ± 6

26 ± 2

6 ± 0R

-

1000

10 ± 3R

8 ± 0R

47 ± 9

6 ± 2R

2 ± 2R

-

2500

7 ± 2R

0 ± 0R

40 ± 5

5 ± 1R

2 ± 1R

-

5000

3 ± 1R

0 ± 0R

49 ± 5

2 ± 1R

2 ± 1R

Positive controls, –S9 mix

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentrations

[μg/plate]

10

10

2.0 μl

10

50

Mean No. of colonies/plate

(average of 3 ± SD)

2128 ± 51

2106 ± 74

674 ± 19

325 ± 48

75 ± 2

+

0

167 ± 5

17 ± 7

62 ± 6

38 ± 9B

19 ± 3

+

0 (DMSO)

154 ± 14

21 ± 7

62 ± 6

28 ± 9B

20 ± 3

+

3

169 ± 13

19 ± 3

54 ± 9

28 ± 9B

23 ± 1

+

10

178 ± 16

23 ± 1

65 ± 9

27 ± 7B

16 ± 3

+

33

171 ± 9

19 ± 4

67 ± 10

29 ± 4B

21 ± 1

+

100

169 ± 3

19 ± 7

67 ± 4

26 ± 4B

17 ± 6

+

333

176 ± 7

15 ± 4

56 ± 4

27 ± 4B

18 ± 4

+

1000

13 ± 3R

12 ± 2R

35 ± 9

7 ± 2B R

10 ± 2R

+

2500

0 ± 0R

0 ± 0R

41 ± 6

0 ± 0B R

0 ± 0R

+

5000

0 ± 0R

0 ± 0R

7 ± 2R

0 ± 0B R

0 ± 0R

Positive controls, +S9 mix

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

3259 ± 99

231 ± 17

389 ± 25

405 ± 31

310 ± 23

NaN3: sodium azide

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

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

B: extensive bacterial growth

R: reduced background growth

Conclusions:
Under the conditions of the Ames 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 up to 5000 µg/plate.
Executive summary:

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2013). 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 plate incorporation or the preincubation method. Test substance concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate were selected for the first and second experiment (plate incorporation and preincubation method, respectively) with and without metabolic activation. No substantial increase in the mean number of revertants per plate was observed in any of the test strains compared to the control, neither in the presence nor absence of metabolic activation. Reduced background growth was observed at the higher concentrations with and without metabolic activation in both experiments. Furthermore, toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations with and without metabolic activation in both experiments. All positive and negative control values were found to be within the respective historical control ranges. 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 and absence of metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Mar - 19 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 26 September 2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED:
Blood samples were drawn from healthy non-smoking donors not receiving medication. Blood was collected from one single donor for each experiment, i.e. human lymphocytes in Experiment I from a male donor (31 years old) and in Experiment II from a female donor (35 years old). Human lymphocytes were stimulated for proliferation by the addition of the mitogen phytohemagglutinin (PHA) to the culture medium for a period of 48 hours. The cell harvest time point was approximately 1.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

MEDIA USED:
Blood cultures were established by preparing an 11% mixture of whole blood in medium with 30 h after blood collection.
- Type and identity of media: DMEM/F12, mixture 1:1, supplemented with 200 mM GlutaMax, penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10% 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:
4 h treatment: 12.02, 21.03, 36.80, 64.41, 112.71, 197.25, 345.19, 604.08, 1057.14 and 1850 µg/mL with and without S9 mix

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

Experiment I:
36.80, 64.41 and 112.71 µg/mL were selected for scoring of chromosome aberrations without S9 mix
64.41, 112.71 and 197.25 µg/mL were selected for scoring of chromosome aberrations with S9 mix

Experiment II:
22 h treatment: 3.2, 5.7, 9.9, 17.4, 30.5, 53.3, 93.3, 163.3, 285.7 and 500 µg/mL without S9 mix
4 h treatment: 17.4, 30.5, 53.3, 93.3, 163.3, 285.7 and 500 µg/mL with S9 mix
53.3, 93.3 and 163.3 µg/mL were selected for scoring of chromosome aberrations without S9 mix
93.3, 163.3 and 285.7 µg/mL were selected for scoring of chromosome aberrations with S9 mix
Vehicle / solvent:
- Vehicle/solvent used: ethanol (final concentration in the culture medium was 0.5%)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative low 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
ethylmethanesulphonate
Remarks:
EMS: 825 µg/mL (4 h, -S9), 550 µg/mL (22 h, -S9); CPA: 7.5 and 15 µg/mL (4 h, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 22 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 22 h; 22 h treatment: 22 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid, 0.2 µg/mL
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 parallel cultures each in 2 independent experiments

NUMBER OF CELLS EVALUATED: At least 150 well-spread metaphases were evaluated per culture, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
− 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
The test substance is then considered unable to induce chromosomal aberrations in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
− 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
When all of the criteria are met, the test substance is then considered able to induce chromosomal aberrations in this test system.
Statistics:
Statistical significance will be confirmed by using the Fisher’s exact test (p < 0.05) using the validated R Script FisherMidP_V1.rnw for those values that indicate an increase in the number of cells with chromosomal aberrations compared to the concurrent solvent control. Other statistical methods may be used if appropriate.
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9: at 1057.14 µg/mL and above following 4 h treatment and at 285.7 µg/mL and above following 22 h treatment
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 was observed.
- Other confounding effects: Phase separation was observed in Experiment I at 112.71 µg/mL and above in the absence of S9 mix and at 197.25 µg/mL and above in the presence of S9 mix. In Experiment II phase separation was observed at 285.7 µg/mL and above in the absence and presence of S9 mix 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. With regard to the molecular weight of the test item, 1850 µg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures. Test item concentrations ranging from 12.02 to 1850 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 112.71 µg/mL and above in the absence of S9 mix and at 197.25 µg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Using reduced mitotic indices as an indicator for toxicity in Experiment I, clear toxic effects were observed after 4 h treatment with 1057.14 µg/mL and above, and in Experiment II, cytotoxicity was observed at 285.7 µg/mL and above, in the absence of S9 mix. In the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration.

Table 1. Results of the chromosomal aberration test.

Test item

Concentration

[µg/mL]

Mitotic Index

(% of control)

Aberrant cells (%)

incl. gaps

excl. gaps

Exposure period 4 h, fixation time 22 h, without S9 mix

Ethanol

0.5% (v/v)

100

1.3

1.0

EMS

825

76.2

18.3

18.0S

Test substance

36.80

105.9

2.0

2.0

64.41

109.9

2.0

2.0

112.71PS

112.1

1.7

1.7

Exposure period 4 h, fixation time 22 h, with S9 mix

Ethanol

0.5% (v/v)

100

2.3

2.0

CPA

15

47.7

24.0

23.7S

Test substance

64.41

120.1

3.0

2.7

112.71##

116.6

2.5

2.5

197.25PS

89.9

2.3

1.7

Exposure period 22 h, fixation time 22 h, without S9 mix

Ethanol

0.5% (v/v)

100

2.7

2.3

EMS

550#

42.5

40.0

39.0S

Test substance

53.3

95.5

3.0

2.7

93.3

103.9

3.7

3.0

163.3

82.3

5.3

4.7

Exposure period 4 h, fixation time 22 h, with S9 mix

Ethanol

0.5% (v/v)

100

3.0

2.3

CPA

7.5

77.9

15.7

15.3S

Test substance

93.3

101.0

1.0

1.0

163.3

111.2

3.0

3.0

285.7PS

114.6

1.3

1.3

EMS: ethylmethane sulfonate

CPA: cyclophosphamide

S: aberration frequency statistically significant higher than corresponding control values

PS: phase separation occurred at the end of treatment

#: evaluation of 50 metaphases per culture

##: evaluation of 300 metaphases per culture

In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix neither statistically significant nor biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test substance.

In Experiment II in the absence of S9 mix one increase in chromosomal aberrations (4.7 % aberrant cells, excluding gaps) was observed after treatment with 163.3 µg/mL. The value exceeded the laboratory historical solvent control data range (0.0 – 3.0 % aberrant cells, excluding gaps) and dose dependency was observed, but the value is not statistically significant.

 

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

 

In both experiments, either EMS or CPA were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:
Under the experimental conditions of the in vitro chromosome aberration test, the test substance did not induce structural chromosomal aberrations in human lymphocytes with and without metabolic activation.
Executive summary:

The clastogenic activity of the test substance was investigated in an in vitro mammalian chromosome aberration test in cultured human lymphocytes performed according to OECD Guideline 473 and GLP (2015). The test substance was dissolved in ethanol and two independent experiments were performed. In Experiment I, the exposure period was 4 h with and without S9 metabolic activation. In Experiment II, the exposure periods were 4 h with and 22 h without metabolic activation. The chromosomes were prepared 22 h after start of treatment with the test substance. In each experimental group two parallel cultures were analysed. At least 150 metaphases per culture were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of metabolic activation, where only 50 metaphases were evaluated due to strong clastogenic effects. 1000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration in this study, 1850.0 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test substance. Phase separation was observed in Experiment I at 112.71 µg/mL and above in the absence and at 197.25 µg/mL and above in the presence of metabolic activation. In Experiment II, phase separation was observed at 285.7 µg/mL and above in the absence and presence of metabolic activation at the end of treatment. In the presence of metabolic activation, no cytotoxicity was observed up to the highest evaluated concentration. In Experiment I and II in the absence of metabolic activation concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In Experiment I in the absence and presence of metabolic activation and in Experiment II with metabolic activation neither statistically significant nor biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test substance. In Experiment II without metabolic activation one increase in chromosomal aberrations (4.7% aberrant cells, excluding gaps) was observed after treatment with 163.3 µg/mL. The value exceeded the laboratory historical solvent control data range (0.0 - 3.0% aberrant cells, excluding gaps) and dose dependency was observed, but the value was not statistically significant. No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance as compared to the control cultures. In both experiments, either ethylmethane sulfonate or cyclophosphamide were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, the test substance did not induce structural chromosomal aberrations in human lymphocytes in vitro under the experimental conditions reported. Therefore, the test substance is not considered to be clastogenic in this chromosome aberration test, when tested up to phase separating or the highest evaluable concentrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Mar - 11 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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: 14.5, 28.9, 57.8, 115.6, 231.3, 462.5, 925 and 1850 µg/mL without metabolic activation
4 h treatment: 14.5, 28.9, 57.8, 115.6, 231.3, 462.5, 925 and 1850 µg/mL with metabolic activation

Experiment I:
4 h treatment: 14.4, 28.8, 57.5, 115, 230, 345 and 460 µg/mL without metabolic activation
4 h treatment: 28.8, 57.7, 115, 230, 460, 690 and 920 µg/mL with metabolic activation

Experiment II:
24 h treatment: 7.2, 14.4, 28.8, 57.5, 115, 172.5 and 230 µg/mL without metabolic activation
4 h treatment: 115, 320, 460, 690, 805, 920 and 1150 µg/mL with metabolic activation
Vehicle / solvent:
- Vehicle/solvent used: ethanol (0.5% (v/v))
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative low toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
EMS: 0.15 mg/mL (4 and 24 h, -S9); DMBA: 1.1 µg/mL (4 h, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

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

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

NUMBER OF REPLICATIONS: duplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency I or cell density below 50%
Evaluation criteria:
A test substance 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 substance 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 substance 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 substance 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.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9: at 115, 230 and 460 µg/mL following 4 h treatment; +S9: at 920 µg/mL following 4 h treatment
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 and 24 h) prior to removal to the test substance.
Pre-Experiment:
Phase separation occurred at the two highest concentrations of 925 and 1850 µg/mL following 4 h treatment with and without metabolic activation. After 24 h treatment without metabolic activation phase separation was noted at 462.5 µg/mL and above.
Main Experiment:
Phase separation was noted at the two highest analysable concentrations of Experiment II with metabolic activation (805.0 and 1150 µg/mL).

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 14.5 µg/mL and 1850 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 (99.68%) and the molecular weight (184.3 g/mol) of the test item. Strong cytotoxic effects were observed at 462.5 µg/mL and above without metabolic activation and at 925 µg/mL and above with metabolic activation following 4 h treatment indicated by a completely inhibited cell growth. Following 24 h treatment without metabolic activation no cytotoxic effect occurred up to 115.6 µg/mL. At all of the higher concentrations the cell growth was completely stopped.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in the first experiment at 230 μg/mL (culture I) and at 345.0 μg/mL (both cultures) without metabolic activation. In the second experiment exceedingly severe cytotoxic effects were noted at 115.0 μg/mL and above without metabolic activation. In the second experiment with metabolic activation relevant or severe cytotoxicity was noted at 460 μg/mL and above.
The recommended cytotoxic range of approximately 10 - 20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. The unusual, not strictly dose dependent response of cytotoxicity was based on phase separation of the test substance. At the closely spaced concentrations of Experiment II with metabolic activation, relevant cytotoxic effects indicated by a drop of the relative cloning efficiency I were observed at 460 and 690 µg/mL. At the next higher concentration of 805 µg/mL macroscopic visible phase separation occurred and the relative cloning efficiency I showed no cytotoxicity at all. The reason is that small droplets of an organic phase are present in the medium before a macroscopic phase separation is notable. These small droplets of organic phase easily get into contact with the cells leading to damage of cell membranes. At macroscopically visible phase separation the organic layer forms on top of the medium and chances are that the cells attached to the bottom of the bottles are not getting into contact with the organic layer on top. The relative cell density shows a similar course of toxicity but shifted to higher concentrations. Based on the high cell density, phase separation is shifted towards higher concentrations. A similar course of cytotoxicity was noted in Experiment I without metabolic activation even though the range of macroscopically visible phase separation was not reached.

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 106cells

Induction factor

Culture I

0 (DMSO)

100.0

100.0

100.0

8.8

1.0

14.4

95.6

94.2

98.9

5.4

0.6

28.8

91.9

107.2

100.4

7.6

0.9

57.5

68.0

101.4

108.0

6.3

0.7

115

0.0

7.2

Culture was not continued#

230

3.1

18.7

115.4

8.6

1.0

345

7.7

11.4

97.1

9.1

1.0

460

0.0

2.6

Culture was not continued#

EMS, 150

76.3

91.1

82.8

289.3

32.8

Culture II

0 (DMSO)

100.0

100.0

100.0

16.4

1.0

14.4

101.2

111.8

103.5

7.0

 

28.8

99.9

103.9

58.2

38.5

 

57.5

65.7

120.9

21.8

42.4

2.6

115

0.0

3.2

Culture was not continued#

230

2.1

4.5

Culture was not continued#

345

1.3

51.7

86.3

35.9

2.2

460

0.0

5.8

Culture was not continued#

EMS, 150

86.9

96.6

109.6

221.8

13.5

DMSO: dimethyl sulfoxide

EMS: ethylmethane sulfonate

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

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

 

Table 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 106cells

Induction factor

Culture I

0 (DMSO)

100.0

100.0

100.0

13.3

1.0

28.8

96.1

Culture was not continued##

57.5

94.6

94.3

Culture was not continued##

115

96.2

131.2

92.1

23.0

1.7

23

93.2

117.8

86.7

12.5

0.9

460

92.0

77.0

87.2

9.7

0.7

690

94.1

133.6

95.9

15.7

1.2

920

92.2

75.2

92.6

18.0

1.4

DMBA, 1.1

100.0

156.4

91.8

298.6

22.5

Culture II

0 (DMSO)

100.0

100.0

100.0

6.9

1.0

28.8

93.9

Culture was not continued##

57.5

97.7

159.5

109.6

10.1

1.5

115

94.5

155.0

92.5

13.7

2.0

23

94.1

122.8

101.7

13.2

1.9

460

96.5

125.4

90.7

4.9

0.7

690

50.0

119.2

117.6

11.4

1.6

920

46.5

5.2

Culture was not continued#

DMBA, 1.1

97.4

201.0

108.7

262.7

37.9

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 analysable concentrations is required.

 

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 106cells

Induction factor

Culture I

0 (DMSO)

100.0

100.0

100.0

24.0

1.0

7.2

84.2

101.1

88.4

17.3

0.7

14.4

88.6

92.2

90.9

33.3

1.4

28.8

83.8

102.6

90.7

25.8

1.1

57.5

84.2

113.6

89.0

16.4

0.7

115.0

0.0

3.9

Culture was not continued#

172.5

0.0

1.6

Culture was not continued#

230

0.0

1.3

Culture was not continued#

EMS, 150

79.1

101.1

92.0

424.5

17.7

Culture II

0 (DMSO)

100.0

100.0

100.0

17.5

1.0

7.2

96.4

102.0

88.7

14.6

0.8

14.4

95.5

106.0

87.9

24.6

1.4

28.8

98.3

112.2

84.4

19.4

1.1

57.5

94.7

119.8

83.3

14.4

0.8

115.0

0.0

4.6

Culture was not continued#

172.5

0.0

0.0

Culture was not continued#

230

0.0

0.0

Culture was not continued#

EMS, 150

93.6

108.4

125.6

302.0

17.3

DMSO: dimethyl sulfoxide

EMS: ethylmethane sulfonate

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

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

 

Table 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 106cells

Induction factor

Culture I

0 (DMSO)

100.0

100.0

100.0

17.3

1.0

115

105.6

93.6

Culture was not continued#

320

53.7

96.1

98.2

29.6

1.7

460

19.8

91.2

98.9

36.8

2.1

690

13.5

87.3

98.4

28.2

1.6

805 (PS)

97.8

19.5

97.7

19.8

1.1

920 (PS)

0.0

3.4

Culture was not continued#

1150 (PS)

0.0

91.6

99.9

21.2

1.2

DMBA, 1.1

100.6

86.0

98.9

130.0

7.5

Culture II

0 (DMSO)

100.0

100.0

100.0

30.4

1.0

115

101.8

85.9

Culture was not continued#

320

50.3

88.5

98.9

19.7

0.6

460

22.0

101.9

99.3

15.0

0.5

690

12.1

87.3

100.8

21.9

0.7

805 (PS)

90.9

16.6

103.3

18.5

0.6

920 (PS)

0.0

1.5

Culture was not continued#

1150 (PS)

0.0

90.5

101.6

27.8

0.9

DMBA, 1.1

94.2

90.3

101.3

128.5

4.2

DMSO: dimethyl sulfoxide

DMBA: 7,12-dimethylbenzanthracene

(PS): phase separation

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

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

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

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

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

Conclusions:
Under the experimental conditions of the gene mutation assay the test substance did not induce gene mutations at the HPRT locus in V79 cells with and without metabolic activation.
Executive summary:

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 were exposed for 4 and 24 h to the test substance up to concentrations of 460 µg/mL in the absence and 1150 µg/mL in the presence of metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in Experiment I at 230 µg/mL (culture I) and at 345 µg/mL (both cultures) without metabolic activation. In Experiment II, exceedingly severe cytotoxic effects were noted at 115 µg/mL and above without metabolic activation and at 460 µg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10 - 20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. 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. 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 not considered to be mutagenic in this HPRT assay.

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 (2013). 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 plate incorporation or the preincubation method. Test substance concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate were selected for the first and second experiment (plate incorporation and preincubation method, respectively) with and without metabolic activation. No substantial increase in the mean number of revertants per plate was observed in any of the test strains compared to the control, neither in the presence nor absence of metabolic activation. Reduced background growth was observed at the higher concentrations with and without metabolic activation in both experiments. Furthermore, toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations with and without metabolic activation in both experiments. All positive and negative control values were found to be within the respective historical control ranges. 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 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 were exposed for 4 and 24 h to the test substance up to concentrations of 460 µg/mL in the absence and 1150 µg/mL in the presence of metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in Experiment I at 230 µg/mL (culture I) and at 345 µg/mL (both cultures) without metabolic activation. In Experiment II, exceedingly severe cytotoxic effects were noted at 115 µg/mL and above without metabolic activation and at 460 µg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10 - 20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. 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. 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 not considered to be mutagenic in this HPRT assay.

 

Cytogenicity in mammalian cells

The clastogenic activity of the test substance was investigated in an in vitro mammalian chromosome aberration test in cultured human lymphocytes performed according to OECD Guideline 473 and GLP (2015). The test substance was dissolved in ethanol and two independent experiments were performed. In Experiment I, the exposure period was 4 h with and without S9 metabolic activation. In Experiment II, the exposure periods were 4 h with and 22 h without metabolic activation. The chromosomes were prepared 22 h after start of treatment with the test substance. In each experimental group two parallel cultures were analysed. At least 150 metaphases per culture were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of metabolic activation, where only 50 metaphases were evaluated due to strong clastogenic effects. 1000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration in this study, 1850.0 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test substance. Phase separation was observed in Experiment I at 112.71 µg/mL and above in the absence and at 197.25 µg/mL and above in the presence of metabolic activation. In Experiment II, phase separation was observed at 285.7 µg/mL and above in the absence and presence of metabolic activation at the end of treatment. In the presence of metabolic activation, no cytotoxicity was observed up to the highest evaluated concentration. In Experiment I and II in the absence of metabolic activation concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In Experiment I in the absence and presence of metabolic activation and in Experiment II with metabolic activation neither statistically significant nor biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test substance. In Experiment II without metabolic activation one increase in chromosomal aberrations (4.7% aberrant cells, excluding gaps) was observed after treatment with 163.3 µg/mL. The value exceeded the laboratory historical solvent control data range (0.0 - 3.0% aberrant cells, excluding gaps) and dose dependency was observed, but the value was not statistically significant. No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance as compared to the control cultures. In both experiments, either ethylmethane sulfonate or cyclophosphamide were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, the test substance did not induce structural chromosomal aberrations in human lymphocytes in vitro under the experimental conditions reported. Therefore, the test substance is not considered to be clastogenic in this chromosome aberration test, when tested up to phase separating 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) No 1272/2008, and are therefore conclusive but not sufficient for classification.