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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Mutagenicity (OECD 471, GLP), Ames: negative with and without metabolic activation

Cytogenicity/chromosome aberration in mammalian cells (OECD 473, GLP): negative with and without metabolic activation

 

Gene mutation in mammalian cells (OECD 476, GLP): negative 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:
30 May - 09 August 2007
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:
not specified
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (for S. typhimurium) and trp operon (for E. coli)
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:
Type and composition of metabolic activation system: cofactor supplemented post-mitochondrial fraction (S9 mix)
- source of S9 : liver of male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg bw, five days prior to sacrifice
- method of preparation of S9 mix: the S9 was lot prepared (Prep date: 14 February 2007) and purchased from Moltox. Each bulk preparation of S9 was assayed for its ability to metabolize at least two promutagens to forms mutagenic to Salmonella typhimurium TA100
- concentration or volume of S9 mix and S9 in the final culture medium: The S9 mix contained 10% S9. In the final medium the S9 concentration was 2% in the top agar.
- quality controls of S9: sterility, metabolic capability
Test concentrations with justification for top dose:
First experiment (part of the dose range finding experiment): 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (tested up to limit concentration)
Second experiment: 50, 150, 500, 1500 and 5000 μg/plate (tested up to limit concentration)
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: A solubility test using water and dimethyl sulfoxide (DMSO) was conducted to determine the vehicle. DMSO was selected as the solvent based on the solubility of the test substance and compatibility with the target cells. The test substance formed workable suspensions in DMSO from approximately 450 to 500 mg/mL and a soluble and clear solution at approximately 400 mg/mL.
- Other: Dosing solutions were adjusted to compensate for the purity of the test substance (92.2%, based on the initial characterization analysis on 22 December 2006) using a correction factor of 1.085. The Sponsor later provided a Certificate of Analysis with a purity of 90.5%, following reanalysis of the test substance on 27 August 2008. This resulted in the dosing solutions being prepared at concentrations that were lower than intended. However, the actual concentrations of the most concentrated dosing preparations remained within acceptable variation (±15%) from the nominal concentrations, even with the updated purity correction and, therefore, the regulatory-required top dose was achieved in each assay.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : duplicate (first experiment); triplicate (second experiment)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 to 72 h at 37 ± 2°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate and/or a thinning or disappearance of the bacterial background lawn

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- colony counting using an automated colony counter
Evaluation criteria:
Evaluation criteria
The test is considered positive if the test substance
- caused a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of 2 increasing concentrations of test substance.
- Tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was ≥ 3 x comapred to the vehicle control.
- Tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response ≥ 2 x comapred to the vehicle control.

Equivocal response: biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive (e.g. dose-responsive increase that does not achieve the respective threshold or a non-dose responsive increase that is ≥ the respective threshold)

A test will be evaluated as negative, if it is neither positive nor equivocal.

Validity criteria:
- Salmonella strains must demonstrate the presence of the deep rough mutation and the deletion in the uvrB gene, TA98 and TA100 strains must demonstrate the presence of the pKM101 plasmid R-factor, WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
- All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3 x 109 cells/mL.
- Mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the control.
- A minimum of 3 non-toxic dose levels is required.

A dose level is considered toxic if one or both of the following criteria are met:
- > 50 % reduction in the mean number of revertants/plate as compared to the mean vehicle control, accompanied by an abrupt dose-dependent drop in the revertant count.
- at least a moderate reduction in the background lawn.
Statistics:
Mean and standard deviation of the number of revertants per plate were calculated for each replicate/triplicate.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: workable stock concentration up to 50 mg/mL for aqueous solvents
- Precipitation and time of the determination: no precipitates observed up to limit concentration

RANGE-FINDING/SCREENING STUDIES
yes, reported as part of experiment 1
No precipitates or toxicity observed was observed up to the maximum dose of 5000 µg/plate

SOLUBILITY TEST
yes, The test substance formed workable suspensions in dimethyl sulfoxide (DMSO) from approximately 450 to 500 mg/mL and a soluble and clear solution at approximately 400 mg/mL.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : valid

Ames test:
- Signs of toxicity : no
- Individual plate counts: yes
- Mean number of revertant colonies per plate and standard deviation: yes
For details, please refer to table 1 and 2 in the "Any other infromation on results incl. tables" section.

HISTORICAL CONTROL DATA
- Positive historical control data: Please refer to table 3 in the "Any other infromation on results incl. tables" section.
- Negative (vehicle) historical control data: Please refer to table 3 in the "Any other infromation on results incl. tables" section.

OTHER:
Dose formulation analyses were performed. The actual concentrations of the analyzed dosing formulations were between 73.8 and 101 % of target. The most concentrated dosing preparation, 100 mg/mL, in each assay was 91.8 and 99.4% of target, meeting the acceptance criteria of 85 to 115% of target concentration. These results were determined based on the original test substance purity value (92.2%) provided by the Sponsor in the characterization analysis conducted on 22 December 2006. The Sponsor later provided a Certificate of Analysis with a purity of 90.5%, following reanalysis of the test substance on 27 August 2008. This resulted in the dosing solutions being prepared at concentrations that were lower than intended. The Study Director has concluded that the discrepancy between the two purity values did not have any adverse impact on the integrity of the data or the validity of the study conclusion since the discrepancy in the purity values was minimal (1.7%) and the most concentrated dosing preparations remained within acceptable variation (± 15%) from the nominal concentrations, even with the updated purity correction. Therefore, the required top dose was achieved in each assay and the results support the validity of the study conclusion.


Table 1: Summary of Experiment 1

Average revertants/plate ± standard deviation

without S 9

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

WP2 uvrA

Vehicle control

28

±

4

179

±

25

19

±

6

8

±

1

34

±

7

1.5

25

±

11

195

±

6

22

±

1

9

±

1

30

±

6

5.0

22

±

0

181

±

27

21

±

6

8

±

1

24

±

4

15

24

±

1

210

±

13

20

±

3

11

±

1

27

±

6

50

19

±

2

154

±

25

22

±

1

5

±

1

32

±

5

150

11

±

1

184

±

8

21

±

4

6

±

1

31

±

7

500

23

±

7

228

±

23

20

±

3

14

±

1

34

±

2

1500

24

±

4

204

±

16

18

±

2

9

±

4

35

±

4

5000

23

±

2

186

±

9

19

±

2

7

±

3

32

±

0

Positive control

283

±

42

717

±

129

729

±

148

794

±

69

439

±

83

with S 9

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

WP2 uvrA

Vehicle control

26

±

3

201

±

47

16

±

3

10

±

2

36

±

8

1.5

19

±

4

203

±

13

17

±

2

11

±

1

39

±

4

5.0

24

±

6

226

±

9

19

±

2

11

±

3

36

±

3

15

25

±

6

194

±

15

18

±

1

10

±

4

44

±

1

50

20

±

0

217

±

24

18

±

4

12

±

4

44

±

7

150

19

±

4

211

±

33

25

±

2

8

±

6

34

±

3

500

26

±

11

199

±

30

21

±

1

8

±

2

34

±

1

1500

25

±

4

187

±

1

15

±

3

9

±

4

41

±

6

5000

35

±

5

201

±

1

19

±

3

9

±

2

41

±

4

Positive control

666

±

134

1190

±

329

154

±

13

150

±

14

353

±

97

Positive Control

2-nitrofluorene 1.0 μg per plate for TA 98(without S 9)

sodium azide 1.0 μg per plate for TA 100, TA 1535(without S 9)

9-aminoacridine 75 μg per plate for TA 1537(without S 9)

methyl methanesulfonate 1000 μg per plate for WP2 uvra (without S 9)

2-aminoanthracene: 10 μg per plate for WP2 uvra, 1.0 μg per plate for TA98, 100, 1535 and 1537 (with S 9)

 

Table 2: Summary of Experiment 2

Average revertants/plate ± standard deviation

without S 9

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

WP2 uvrA

Vehicle control

14

±

4

124

±

11

10

±

5

8

±

4

26

±

3

50

16

±

7

143

±

5

13

±

5

5

±

3

25

±

8

150

17

±

2

194

±

28

13

±

3

5

±

1

16

±

4

500

12

±

2

136

±

27

12

±

5

6

±

2

26

±

2

1500

15

±

3

153

±

2

10

±

3

6

±

2

17

±

2

5000

17

±

3

130

±

8

13

±

1

7

±

1

28

±

9

Positive control

239

±

44

573

±

21

512

±

35

800

±

97

346

±

7

with S 9

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

WP2 uvrA

Vehicle control

25

±

4

179

±

12

13

±

6

9

±

2

24

±

3

50

21

±

8

167

±

12

8

±

2

9

±

3

29

±

6

150

24

±

3

162

±

15

10

±

4

6

±

2

35

±

4

500

22

±

5

148

±

23

12

±

4

7

±

2

36

±

7

1500

21

±

2

152

±

13

11

±

3

9

±

1

34

±

2

5000

21

±

3

172

±

10

12

±

5

10

±

3

31

±

4

Positive control

307

±

53

725

±

238

114

±

16

64

±

5

247

±

10

Positive Control

2-nitrofluorene 1.0 μg per plate for TA 98(without S 9)

sodium azide 1.0 μg per plate for TA 100, TA 1535(without S 9)

9-aminoacridine 75 μg per plate for TA 1537(without S 9)

methyl methanesulfonate 1000 μg per plate for WP2 uvra (without S 9)

2-aminoanthracene: 10 μg per plate for WP2 uvra, 1.0 μg per plate for TA98, 100, 1535 and 1537 (with S9)

 

Table 3: Historical positive and negative controls(2003 - 2005)

Revertants per plate

 

 

without S9

with S 9

Strain

Control

Mean

SD

Min

Max

Mean

SD

Min

Max

TA98

Negative

18

6

5

51

26

9

8

72

 

Positive

212

175

44

1981

781

434

42

2669

TA100

Negative

146

32

63

253

156

34

67

267

 

Positive

586

136

239

2373

959

427

168

2652

TA1535

Negative

18

7

4

49

15

5

2

45

 

Positive

370

137

31

1050

139

70

21

985

TA1537

Negative

7

3

1

23

7

3

1

25

 

Positive

690

373

14

2216

120

113

13

2021

WP2 uvrA

Negative

17

6

5

58

18

7

6

52

 

Positive

151

110

32

1741

520

271

35

1392

SD=standard deviation; Min=minimum value; Max=maximum value

 

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five tester strains (TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA) tested with and without metabolic activation up to 5000 µg/plate.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 June - 09 August 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
(according to study report: 1998)
Deviations:
yes
Remarks:
no data on proficiency provided
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes:
Remarks:
human
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: human peripheral lymphocytes

For lymphocytes:
- Sex, age and number of blood donors: healthy female, 25 years (main study), 30 years (preliminary study), 2 donors
- Whether whole blood or separated lymphocytes were used: whole blood, heparinized
- Whether blood from different donors were pooled or not: no
- Mitogen used for lymphocytes: 1 % Phytohemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: RPMI 1640 serum-free medium supplemented with 100 units penicillin/mL and 100 μg streptomycin/mL, and 2 mM L-glutamine, 5±1% CO2, humidified atmosphere, 37±1°C
Cytokinesis block (if used):
Colcemid (0.1 µg/mL) was added two hours before scheduled cell harvest.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : liver from male rats treated with Aroclor 1254 (Moltox, Lot number: No. 2074)
- concentration or volume of S9 mix and S9 in the final culture medium: 20 µL S 9 per mL medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each bulk preparation of S9 was assayed for sterility and its ability to metabolize 2-aminoanthracene and 7,12-dimethyl-benzanthracene.
Test concentrations with justification for top dose:
Preliminary toxicity test (4 h exposure: +/- S9, 20 h exposure: - S9): 0.2136, 0.6408, 2.136, 6.408, 21.36, 64.08, 213.6, 640.8 and 2136 µg/mL (tested up to top concentration of 2 mg/mL)
Main study (4 h exposure: +/- S9, 20 h exposure: - S9): 534, 1068 and 2136 μg/mL (tested up to top concentration of 2 mg/mL)
Vehicle / solvent:
- Vehicle used: DMSO

- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The vehicle of choice was the solvent, selected in order of preference that permitted preparation of the highest soluble stock concentration, up to 50 mg/mL in water and 500 mg/mL in DMSO. The test substance was soluble in DMSO at a maximum concentration of approximately 400 mg/mL, in the solubility test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF APPLICATION: in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 0.6 mL heparinized blood were incubated with 1% Phytohemagglutinin (PHA) for 44-48 h.
- Exposure duration/duration of treatment: 4 h and 20 h
- Harvest time after the end of treatment (sampling/recovery times): 20 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colcemid (0.1 µg/mL) was added to cultures 2 h before scheduled cell harvest.

- Methods of slide preparation and staining technique used including the stain used: 1 to 2 drops of the fixed cell suspension were dropped on clean microscope slides and were allowed to air dry at room temperature. Subsequently slides were stained with 5% Giemsa, air dried and permanently mounted.

- Number of cells spread and analysed per concentration: a minimum of 200 metaphase spreads (100 per duplicate treatment condition). The number of metaphase spreads that were examined and scored per duplicate flask was reduced when the percentage of aberrant cells reached a significant level (at least 10%) before 100 cells were scored.

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
Aberrant Cells: numerical findings includes polyploid and endoreduplicated cells; structural effects exclude cells with only gaps. Chromatid breaks include chromatid and isochromatid breaks and fragments; chromatid exchange figures (Ex) include quadriradials, triradials and complex rearrangements. Chromosome breaks include breaks and acentric fragments; Dic, dicentric chromosome. Severely damaged cells includes cells with one or more pulverized chromosomes and cells with 10 or more aberrations. Average Aberrations Per Cell: severely damaged cells and pulverizations were counted as 10 aberrations.

- Determination of polyploidy: yes, percent of polyploid cells was evaluated per 100 cells.

- Determination of endoreplication: yes, percent endoreduplicated cells was evaluated per 100 cells.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI) (percentage of cells in mitosis per 500 cells counted)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- The number and types of aberrations per cell, the percentage of structurally and numerically damaged cells (percent aberrant cells), and the frequency of structural aberrations per cell (mean aberrations per cell) in the total population of cells examined was calculated and reported for each treatment group.

- OTHER: Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.
Rationale for test conditions:
Selection of doses for analysis was based on mitotic inhibition (the lowest dose with at least 50% reduction in mitotic index, relative to the solvent control) in the non-activated 20 h exposure group. In the non-activated and S9-activated 4 h treatment groups, due to absence of both test substance precipitation in the treatment medium and at least 50% toxicity, the highest dose level evaluated was 2136 μg/mL (10 mM) (top dose). Two additional lower dose levels were included in the evaluation.
Evaluation criteria:
Evaluation of Results:
The number and types of aberrations per cell, the percentage of structurally and numerically damaged cells (percent aberrant cells), and the frequency of structural aberrations per cell (mean aberrations per cell) in the total population of cells examined was calculated and reported for each treatment group. Chromatid and isochromatid gaps are reported but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell. The test substance was considered to induce a positive response when the percentages of cells with aberrations were increased in a dose-responsive manner with one or more concentrations being statistically elevated relative to the solvent control group (p≤0.05). However, values that are statistically significant but do not exceed the range of historical solvent controls may be judged as not biologically significant. Test substances not demonstrating a statistically significant increase in aberrations will be concluded to be negative.

Criteria for Determination of a Valid Test
The frequency of cells with structural chromosome aberrations in the vehicle control must be within the historical range for vehicle control. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p≤0.05, Fisher's Exact test) relative to the solvent control.
Statistics:
Fisher's Exact test. Fisher's Exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's Exact test at any test substance dose level, the Cochran-Armitage test was used to measure dose-responsiveness.


Key result
Species / strain:
lymphocytes:
Remarks:
human
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
4 h exposure
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
mitotic inhibition was 8%, relative to the solvent control at the highest test concentration (4 h exposure, -S9), no precipitates, tested up to recommended maximum concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes:
Remarks:
human
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
4 h exposure
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes:
Remarks:
human
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
20 h exposure
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
mitotic inhibition was 54% at the highest dose tested, relative to the solvent control (20 h exposure, -S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: approximately 7.0 (highest concentration tested, 2136 μg/mL)
- Data on osmolality: 404 mmol/kg (highest concentration tested, 2136 μg/mL), acceptable because it did not exceed the osmolality of the solvent (DMSO: 409 mmol/kg) by more than 20%.
- Precipitation and time of the determination: precipitation was not observed in any test condition
- Definition of acceptable cells for analysis: yes mitotoc index (500 percentage of cells in mitosis per 500 cells counted), a minimum of 200 metaphases (100 per duplicate treatment condition) were examined and scored for chromatid-type and chromosome-type aberrations

RANGE-FINDING/SCREENING STUDIES: a preliminary toxicity assay with 4 h (+/- S9) and 20 h (-S9) exposure was performed. Precipitates and substantial toxicity (at least 50% reduction in mitotic index relative to the solvent control) were not observed at any dose level in all 3 treatment groups.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
- The frequency of cells with structural chromosome aberrations in the vehicle control was within the historical range for vehicle control.
- The percentage of cells with chromosome aberrations in the positive control was statistically increased (p>0.05, Fisher's Exact test) relative to the solvent control.
- Under the conditions of the assay described in this report, the test substance was concluded to be negative for the induction of structural and numerical chromosome aberrations in human peripheral blood lymphocytes (+/- S9).

Criteria for data analysis and interpretation:
- Concentration-response relationship
- Statistical analysis: *, p ≤ 0.05; **, p ≤ 0.01; using the Fisher's Exact test.
- The frequency of cells with structural chromosome aberrations in the vehicle control must be within the historical range for vehicle control.
- The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p>0.05, Fisher's Exact test) relative to the solvent control.
- The test substance was considered to induce a positive response when the percentages of cells with aberrations were increased in a dose-responsive manner with one or more concentrations being statistically elevated relative to the solvent control group (p≤0.05).
- Values that are statistically significant but do not exceed the range of historical solvent controls may be judged as not biologically significant.
- Test substances not demonstrating a statistically significant increase in aberrations will be concluded to be negative.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic inhibition was 8%, relative to the solvent control at the highest test concentration (4 h exposure, -S9); mitotic inhibition was not reduced relative to the solvent control (4 h exposure, +S9); mitotic inhibition was 54%, relative to the solvent control (20 h exposure, -S9)

- Genotoxicity results (lymphocytes)
o Definition for chromosome aberrations: Aberrant Cells: numerical includes polyploid and endoreduplicated cells; structural excludes cells with only gaps. Chromatid breaks include chromatid and isochromatid breaks and fragments; chromatid exchange figures (Ex) include quadriradials, triradials and complex rearrangements. Chromosome breaks include breaks and acentric fragments; Dic, dicentric chromosome.
o 200 cells scored for each culture and concentration (number of scored cellswas reduced when the percentage of aberrant cells reached a significant level (at least 10%) before 100 cells were scored), number of cells with chromosomal aberrations and type were given separately for each treated and control culture, including and excludling gaps
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) was reported if seen

HISTORICAL CONTROL DATA
For historical control data please refer to table 8 in the " any other information on results including tables section".

OTHER ANALYTICS:
Test substance dose formulations were analysed for concentration and stability. The results of the analysis indicate that the analyzed concentrations (26.7, 106.8 and 213.6 mg/mL) were 114.0%, 100.2% and 98.5% of their respective targets and were within the protocol specified range of 85 to 115%. No test substance was detected in the vehicle control sample. This indicates that the dose formulations were acceptable for use in the study. The results of the stability analysis indicate that the test substance was determined to be stable at room temperature for at least 14 h at 0.03 and 215 mg/mL.

These results were determined based on the original test substance purity value (92.2%) provided by the Sponsor, the Sponsor later provided a Certificate of Analysis with a purity of 90.5%. This resulted in the dosing solutions being prepared at concentrations that were lower than intended. It concluded that the discrepancy between the two purity values did not have any adverse impact on the integrity of the data or the validity of the study conclusion since 1) the discrepancy in the purity values was minimal (1.7%) and 2) the most concentrated dosing preparations remained within acceptable variation (±15%) from the nominal concentrations, even with the updated purity correction. Therefore, the regulatory-required top dose was achieved in each assay and the results support the validity of the study conclusion.
Remarks on result:
other: no mutagenic potential observed

Table 1: Preliminary toxicity test (4 h exposure without metabolic activation)

4 h Treatment

Mitotic

Percent

(-S9)

Index

Change

μg/mL

(%)

(%)

DMSO

9.8

Test substance

0.2136

10.4

6

0.6408

9.8

0

2.136

9.6

-2

6.408

9.4

-4

21.36

9.2

-6

64.08

8.6

-12

213.6

9.2

-6

640.8

9.4

-4

2136

8.8

-10

Treatment: Human peripheral blood lymphocyte cells were treated in the absence of an exogenous source of metabolic activation for 4 hours at 37±1°C. Metaphase cells were collected following a 16-hour recovery period.

Mitotic Index = (Cells in mitosis/500 cells scored) x 100.

Percent change = (Treatment mitotic index - control mitotic index)/control mitotic index, expressed as a percentage.

Positive control for non-activated studies is mitomycin C (MMC).

Positive control for S9 activated studies is cyclophosphamide (CP).

 

Table 2: Preliminary toxicity test (4 h exposure with metabolic activation)

4 h Treatment

Mitotic

Percent

(+S9)

Index

Change

μg/mL

(%)

(%)

DMSO

9.8

Test substance

0.2136

10.2

4

0.6408

9.6

-2

2.136

9.2

-6

6.408

9.0

-8

21.36

9.4

-4

64.08

9.8

0

213.6

9.0

-8

640.8

8.8

-10

2136

8.6

-12

Treatment: Human peripheral blood lymphocyte cells were treated in the presence of an exogenous source of metabolic activation for 4 h at 37±1°C. Metaphase cells were collected following a 16 h recovery period.

Mitotic Index = (Cells in mitosis/500 cells scored) x 100.

Percent change = (Treatment mitotic index - control mitotic index)/control mitotic index, expressed as a percentage

Positive control for non-activated studies is mitomycin C (MMC).

Positive control for S9 activated studies is cyclophosphamide (CP).

 

Table 3: Preliminary toxicity test (20 h exposure without metabolic activation)

20 Treatment

Mitotic

Percent

(-S9)

Index

Change

μg/mL

(%)

(%)

DMSO

10.4

Test substance

0.2136

9.4

-10

0.6408

10.6

2

2.136

8.8

-15

6.408

9.8

-6

21.36

9.6

-8

64.08

8.6

-17

213.6

8.4

-19

640.8

8.8

-15

2136

8.0

-23

Treatment: Human peripheral blood lymphocyte cells were treated in the absence of an exogenous source of metabolic activation for 20 h at 37±1°C.

Mitotic Index = (Cells in mitosis/500 cells scored) x 100.

Percent change = (Treatment mitotic index - control mitotic index)/control mitotic index, expressed as a percentage.

Positive control for non-activated studies is mitomycin C (MMC).

Positive control for S9 activated studies is cyclophosphamide (CP).

 

Table 4: Results of the main experiment (4 h exposure without metabolic activation)

 

 

Mitotic

index

Cells scored

% Aberrant cells

Gaps

Chromatid

Chromosome

Severely damaged

cells

Average

aberrations per cell

Treatment

Flask

Numerical

Structural

Numerical

Structural

 

Br

Ex

Br

Dic

Ring

(µg/mL)

 

(%)

 

 

 

 

 

 

 

 

 

 

 

 

DMSO

A

10.8

100

100

0

0

0

0

0

0

0

0

0

0.000

 

B

9.8

100

100

0

0

0

0

0

0

0

0

0

0.000

Test substance

 

 

 

 

 

 

 

 

 

 

 

 

 

 

267

A

11.0

Not

Scored

 

 

 

 

 

 

 

 

 

 

 

B

10.6

Not

Scored

 

 

 

 

 

 

 

 

 

 

534

A

8.2

100

100

0

0

0

0

0

0

0

0

0

0.000

 

B

8.4

100

100

0

0

0

0

0

0

0

0

0

0.000

1068

A

8.0

100

100

0

0

0

0

0

0

0

0

0

0.000

 

B

7.6

100

100

0

0

0

0

0

0

0

0

0

0.000

2136

A

9.2

100

100

0

0

0

0

0

0

0

0

0

0.000

 

B

9.8

100

100

0

0

0

0

0

0

0

0

0

0.000

MMC

A

5.6

100

50

0

18

0

7

4

0

0

0

0

0.220

0.6

B

5.8

100

50

0

20

0

10

3

0

0

0

0

0.260

Treatment: Human peripheral blood lymphocytes were treated for 4 hours at 37± 1°C in the absence of an exogenous source of metabolic activation. An additional dose level of 267 μg/mL was tested as a safeguard against excessive toxicity at higher dose levels but was not required for microscopic examination.

Mitotic index = number mitotic figures x 100/500 cells counted.

% Aberrant Cells: numerical includes polyploid and endoreduplicated cells; structural excludes cells with only gaps.

Chromatid breaks include chromatid and isochromatid breaks and fragments; chromatid exchange figures (Ex) include quadriradials, triradials and complex rearrangements.

Chromosome breaks include breaks and acentric fragments; Dic, dicentric chromosome.

Severely damaged cells includes cells with one or more pulverized chromosomes and cells with 10 or more aberrations.

Average Aberrations Per Cell: severely damaged cells and pulverizations were counted as 10 aberrations.

Positive control for non-activated studies is mitomycin C (MMC).

Positive control for S9 activated studies is cyclophosphamide (CP).

 

Table 5: Results of the main experiment (4 h exposure with metabolic activation)

 

 

Mitotic

sndex

Cells scored

% Aberrant cells

Gaps

Chromatid

Chromosome

Severely damaged

cells

Average

aberrations per cell

Treatment

Flask

Numerical

Structural

Numerical

Structural

 

Br

Ex

Br

Dic

Ring

(µg/mL)

 

(%)

 

 

 

 

 

 

 

 

 

 

 

 

DMSO

A

9.2

100

100

0

0

0

0

0

0

0

0

0

0.000

 

B

9.8

100

100

0

0

0

0

0

0

0

0

0

0.000

Test substance

 

 

 

 

 

 

 

 

 

 

 

 

 

 

267

A

9.4

Not

Scored

 

 

 

 

 

 

 

 

 

 

 

B

10.0

Not

Scorded

 

 

 

 

 

 

 

 

 

 

534

A

9.8

100

100

0

1

0

1

0

0

0

0

0

0.010

 

B

9.0

100

100

0

0

0

0

0

0

0

0

0

0.000

1068

A

10.2

100

100

0

0

0

0

0

0

0

0

0

0.000

 

B

9.8

100

100

0

0

0

0

0

0

0

0

0

0.000

2136

A

10.0

100

100

0

0

0

0

0

0

0

0

0

0.000

 

B

10.6

100

100

0

0

0

0

0

0

0

0

0

0.000

CP

A

5.2

100

50

0

22

0

8

3

0

0

0

0

0.220

20

B

5.8

100

50

0

22

0

7

5

0

0

0

0

0.240

Treatment: Human peripheral blood lymphocytes were treated for 4 hours at 37 ± 1°C in the presence of an exogenous source of metabolic activation. An additional dose level of 267 μg/mL was tested as a safeguard against excessive toxicity at higher dose levels but was not required for microscopic examination.

Mitotic index = number mitotic figures x 100/500 cells counted.

% Aberrant Cells: numerical includes polyploid and endoreduplicated cells; structural excludes cells with only gaps.

Chromatid breaks include chromatid and isochromatid breaks and fragments; chromatid exchange figures (Ex) include quadriradials, triradials and complex rearrangements.

Chromosome breaks include breaks and acentric fragments; Dic, dicentric chromosome.

Severely damaged cells includes cells with one or more pulverized chromosomes and cells with 10 or more aberrations.

Average Aberrations Per Cell: severely damaged cells and pulverizations were counted as 10 aberrations.

Positive control for non-activated studies is mitomycin C (MMC).

Positive control for S9 activated studies is cyclophosphamide (CP).

 

Table 6: Results of the main experiment (20 h exposure without metabolic activation)

 

 

Mitotic

index

Cells scored

% Aberrant cells

Gaps

Chromatid

Chromosome

Severely damaged

cells

Average

aberrations per cell

Treatment

Flask

Numerical

Structural

Numerical

Structural

 

Br

Ex

Br

Dic

Ring

(µg/mL)

 

(%)

 

 

 

 

 

 

 

 

 

 

 

 

DMSO

A

10.6

100

100

0

0

0

0

0

0

0

0

0

0.000

 

B

10.0

100

100

0

1

0

1

0

0

0

0

0

0.010

Test substance

 

 

 

 

 

 

 

 

 

 

 

 

 

 

267

A

10.8

Not

Scored

 

 

 

 

 

 

 

 

 

 

 

B

10.4

Not

Scored

 

 

 

 

 

 

 

 

 

 

534

A

8.6

100

100

0

0

0

0

0

0

0

0

0

0.000

 

B

7.8

100

100

0

0

0

0

0

0

0

0

0

0.000

1068

A

8.6

100

100

0

0

0

0

0

0

0

0

0

0.000

 

B

8.6

100

100

0

0

0

0

0

0

0

0

0

0.000

2136

A

5.0

100

100

0

0

0

0

0

0

0

0

0

0.000

 

B

4.4

100

100

0

1

0

1

0

0

0

0

0

0.010

MMC

A

5.8

100

50

0

22

0

10

1

0

0

0

0

0.220

0.3

B

6.0

100

50

0

20

0

9

1

0

0

0

0

0.200

Treatment: Human peripheral blood lymphocytes were treated for 20 hours at 37 ± 1°C in the absence of an exogenous source of metabolic activation. An additional dose level of 267 μg/mL was tested as a safeguard against excessive toxicity at higher dose levels but was not required for microscopic examination.

Mitotic index = number mitotic figures x 100/500 cells counted.

% Aberrant Cells: numerical includes polyploid and endoreduplicated cells; structural excludes cells with only gaps.

Chromatid breaks include chromatid and isochromatid breaks and fragments; chromatid exchange figures (Ex) include quadriradials, triradials and complex rearrangements.

Chromosome breaks include breaks and acentric fragments; Dic, dicentric chromosome.

Severely damaged cells includes cells with one or more pulverized chromosomes and cells with 10 or more aberrations.

Average Aberrations Per Cell: severely damaged cells and pulverizations were counted as 10 aberrations.

Positive control for non-activated studies is mitomycin C (MMC).

Positive control for S9 activated studies is cyclophosphamide (CP).

 

Table 7: Summary of results

 

 

 

Mean

mitotic

index

Cells scored

Aberrations

per cell

(Mean +/- SD)

Cells with aberrations

 

Treatment

µg/mL

 

S9

Activation

 

Treatment

time

Numerical

Structural

Numerical

(%)

Structural

(%)

DMSO

-S9

4

10.3

200

200

0.000

±0.000

0.0

0.0

Test substance

534

-S9

4

8.3

200

200

0.000

±0.000

0.0

0.0

1068

-S9

4

7.8

200

200

0.000

±0.000

0.0

0.0

2136

-S9

4

9.5

200

200

0.000

±0.000

0.0

0.0

MMC

-S9

4

5.7

200

100

0.240

±0.534

0.0

19.0**

0.6

 

 

 

 

 

 

 

 

 

 

DMSO

 

+S9

 

4

 

9.5

 

200

 

200

 

0.000

 

±0.000

 

0.0

 

0.0

Test substance

534

+S9

4

9.4

200

200

0.005

±0.071

0.0

0.5

1068

+S9

4

10.0

200

200

0.000

±0.000

0.0

0.0

2136

+S9

4

10.3

200

200

0.000

±0.000

0.0

0.0

CP

+S9

4

5.5

200

100

0.230

±0.446

0.0

22.0**

20

 

 

 

 

 

 

 

 

 

 

DMSO

 

-S9

 

20

 

10.3

 

200

 

200

 

0.005

 

±0.071

 

0.0

 

0.5

Test substance

534

-S9

20

8.2

200

200

0.000

±0.000

0.0

0.0

1068

-S9

20

8.6

200

200

0.000

±0.000

0.0

0.0

2136

-S9

20

4.7

200

200

0.005

±0.071

0.0

0.5

MMC,

0.3

-S9

20

5.9

200

100

0.210

±0.409

0.0

21.0**

 

 

 

 

 

 

 

 

 

Treatment: Cells from all treatment conditions were harvested at 20 hours after the initiation of the treatments.

Aberrations per Cell: Severely damaged cells were counted as 10 aberrations.

Percent Aberrant Cells: *, p ≤ 0.05; **, p ≤ 0.01; using the Fisher's Exact test.

Positive control for non-activated studies is mitomycin C (MMC).

Positive control for S9 activated studies is cyclophosphamide (CP).

 

Table 8: Historical control data

 

Without Metabolic Activation

 

Historical Values

Percent Aberrant Cells (%)

 

 

Solvent Control1

Positive Control2

Mean

0.0

17.5

Standard Deviation

±0.1

±5.0

Range

0.0-1.0

6.0-40.0

 

With Metabolic Activation

 

Historical Values

Percent Aberrant Cells (%)

 

 

Solvent Control1

Positive Control3

Mean

0.0

16.4

Standard Deviation

±0.1

±3.3

Range

0.0-0.5

9.0-30.0

1 Solvents include water, saline, DMSO, ethanol, acetone, and other non-standard and

Sponsor-supplied vehicles.

2 Positive control for non-activated studies is mitomycin C (MMC).

3 Positive control for S9 activated studies is cyclophosphamide (CP).

Conclusions:
Under the conditions of the conducted test the substance did not induce chromosomal aberrations in peripheral human lymphocytes with and without metabolic activation up to an dose of 2136 µg/mL.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 May - 09 August 2007
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 using the Hprt and xprt genes)
Version / remarks:
adopted 21 July 1997
(according to the study report 1998)
Deviations:
yes
Remarks:
The highest test dose selected did not induce cytotoxicity of at least 10%.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HGPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHO-K1 cells, (American Type Culture Collection in Manassas, VA, USA)

For cell lines:
- Number of passages: 4
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: F12FBS5-Hx is Ham's F12 medium, without hypoxanthine supplemented with 5% dialyzed FBS, 100 units penicillin/mL, 100 μg streptomycin/mL and 2mM L-glutamine/mL, 5 ± 1% CO2, humidified atmosphere, 37 ± 1°C
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Aroclor-induced rat liver S9, lot 2088 (Moltox, Boone, NC, USA)
- method of preparation of S9 mix: the S9 reaction mixture was prepared by mixing S9 and 10 mM calcium chloride (CaCl2) with a filter-sterilized cofactor pool to contain 100 μL S9/mL cofactor pool, 4 mM nicotinamide adenine dinucleotide phosphate (NADP), 5 mM glucose-6-phosphate, 30 mM potassium chloride (KCl), 10 mM magnesium chloride (MgCl2), and 50 mM sodium phosphate buffer, pH 8.0, immidiately prior to use.
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL S9 reaction mixture/ 4 mL F12FBS5-Hx medium
- quality controls of S9: assayed for sterility and its ability to metabolize at least 2 pro-mutagens.
Test concentrations with justification for top dose:
Preliminary toxicity test (+/- S9): 0.5, 1.5, 5, 15, 50, 140, 460, 1370 and 4000 µg/mL (+/- S9, visible precipitates at 4000 µg/mL, relative cloning efficiency at 4000 µg/mL: 72% (-S9) and 108% (+S9), at 1370 µg/mL: 115% (-S9) and 89% (+S9))
Main experiment (+/- S9): 500, 750, 1000, 1500, 1750 and 2150 µL/mL (concentrations selected based on results of the preliminary toxicity test)
Vehicle / solvent:
- Vehicle used: DMSO

- Justification for choice of solvent/vehicle: different vehicles were tested in the order of preference as listed: distilled water, dimethyl sulfoxide, ethanol, and acetone. DMSO was determined to be the solvent of choice based on solubility of the test substance and compatibility with the target cells. The test substance was soluble in DMSO at a maximum concentration of 400 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5x105 cells/25 cm²
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 18 - 24 h
- Exposure duration/duration of treatment: 5 h
- Harvest time after the end of treatment: 18 - 24 h

FOR GENE MUTATION:
- Expression time: 7 - 9 days
- Selection time (if incubation with a selective agent): 7 - 10 days
- If a selective agent is used: 6-thioguanine (TG, 2-amino-6-mercaptopurine), 10 µM, 7 - 10 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2x10E5 cells/100 mm dish. After 7-10 days of incubation, the colonies were fixed, stained and counted for both cloning efficiency and mutant selection.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: the replicates from each treatment condition were detached using trypsin and subcultured independently in F12FBS5-Hx, in triplicate, at a density of 100 cells/60 mm dish. After incubation for 7-10 days, the colonies were rinsed with HBSS, fixed with methanol, stained with 10% aqueous Giemsa, counted and cloning efficiency was determined.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Determination of mutant frequency (division of the total number of mutant colonies by the number of cells selected (usually 2x10E6 cells: 10 plates at 2x10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection. Expressed as TG-resistant mutants per 10E6 clonable cells. For experimental conditions in which no mutant colonies were observed, mutant frequencies were expressed as less than the frequency obtained with one mutant colony.
Evaluation criteria:
Evaluation criteria
- The test substance was considered to induce a positive response if there was a concentration-related increase in mutant frequencies with at least 2 consecutive doses showing mutant frequencies of >40 mutants per 10E6 clonable cells.
- If a single point above 40 mutants per 106 clonable cells was observed at the highest dose, the assay was considered suspect.
- If no culture exhibited a mutant frequency of >40 mutants per 10E6 clonable cells, the test substance was considered negative.

Criteria for a valid test:
- The cloning efficiency of the solvent control must be greater than 50%.
- The spontaneous mutant frequency in the solvent control must fall within the range of 0-25 mutants per 10E6 clonable cells.
- The positive control must induce a mutant frequency at least 3 times that of the solvent control and must exceed 40 mutants per 10E6 clonable cells.
- There must be at least 4 analyzable test substance concentrations with mutant frequency data.
Statistics:
Calculation of Cloning efficiency and mutant frequency:

Cloning efficiency = average colonies / 100 cells/dish

Mutants/10E6 clonable cells = (average mutant colonies / cloning efficiency X 2 x 10E5 cells) x 10E6
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cloning efficiency was 93% in the highest test dose
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS :
- Precipitation and time of the determination: precipitate observed at a concentration of 4000 µg/mL (Preliminary toxicity test), no precipitate was observed at any concentration (main test)
- Other: cultures treated with 500 μg/mL were terminated prior to cloning because there were sufficient higher concentrations.

RANGE-FINDING/SCREENING STUDIES: CHO cells were exposed to solvent alone and 9 concentrations of test substance ranging from 0.5 to 4000 μg/mL (+/- S9). There was visible precipitate in the treatment medium at 4000 μg/mL. Cloning efficiency relative to the solvent controls (relative cloning efficiency) at 4000 μg/mL was 72% without activation and 108% with S9 activation. Based on these findings and the molecular weight of the test substance, the doses chosen for the mutagenesis assay ranged from 500 to 2150 μg/mL (10 mM) (+/- S9 10).

STUDY RESULTS
- None of the treated cultures exhibited mutant frequencies > 40 mutants per 10E6 clonable cells.
- Positive controls (+/- S9) induced mutant frequencies that were at least 3 times that of the solvent control and > 40 mutants per 10E6 clonable cells.
- None of the vehicle treated cultures exhibited mutant frequencies > 40 mutants per 10E6 clonable cells.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship was analyzed
- Any other criteria: mutant frequency > 40 mutants per 10E6 clonable cells (test considered positive)

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o relative cloning efficiency was 93% (- S9) and 107% (+ S9) at the highest dose cloned for mutant selection.

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures : 5x10E5 cells/25 cm² flask
o Number of cells plated in selective and non-selective medium : for selection of the TG-resistant phenotype, the replicates from each treatment condition were trypsinized and replated, in quintuplicate, at a density of 2x10E5 cells/100 mm dish. For cloning efficiency
determinations at the time of selection, 100 cells/60 mm dishes were plated in triplicate
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency (please refer to table 2, 3 and 4 in the "Any other information incl. tables" section)

HISTORICAL CONTROL DATA (Please refer to table 5 in the "Any other information icl. tables" section)
- Positive historical control data: valid (within historical control data range)
- Negative (vehicle) historical control data: valid (within historical control data range)

DOSE SOLUTION ANALYSIS:
Two 0.5 mL aliquots of the 50 mg/mL dosing formulation and vehicle, three 0.5 mL aliquots from the top, middle, and bottom of the 100 and 215 mg/mL formulations from the mutagenesis assay analysed at BioReliance chemistry lab for concentration and homogeneity. An additional 0.5 mL sample of the middle and low concentrations and the vehicle was retained for stability analysis. No test article was found in the vehicle samples.

Results: All formulations met the acceptance criteria, except for 50 mg/mL which was 115.3% of target. Though this is technically above the limit, the study director has determined that it had no effect on the integrity or conclusion of the study. The formulations were found to be stable for at least 14 hours at room temperature.

These results were determined based on the original test substance purity value (92.2%) provided by the Sponsor in the characterization. The Sponsor later provided a Certificate of Analysis with a purity of 90.5%, following reanalysis of the test substance. This resulted in the dosing solutions being prepared at concentrations that were lower than intended. The Study Director has concluded that the discrepancy between the two purity values did not have any adverse impact on the integrity of
the data or the validity of the study conclusion since 1) the discrepancy in the purity values was minimal (1.7%) and 2) the most concentrated dosing preparations remained within acceptable variation (±15%) from the nominal concentrations, even with the updated purity correction. Therefore, the regulatory-required top dose was achieved in and the results support the validity
of the study conclusion.
Conclusions:
negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Several genotoxicity tests were conducted with the test substance to evaluate its genotoxic potential. In detail, the following studies have been performed with the test substance:

 

- Genetic toxicity in bacteria (Ames)

The in-vitro genetic toxicity of the test substance was assessed in a bacterial reverse mutation assay (Ames test) according to OECD TG 471and GLP criteria (2011j). The mutagenic potential of the test substance dissolved in Dimethyl sulfoxide (DMSO) was assessed in S. typhimurium tester strains TA 98, 100, 1535, 1537 and E.coli WP2 uvra at concentrations up to 5000 µg/plate in two independent experiments (plate incorporation method). In the first experiment (part of the dose-range finding experiment) concentrations of 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate were used. In the second experiment 50, 150, 500, 1500 and 5000 μg/plate were tested. In both experiments, concentrations were tested with and without metabolic activation. Cofactor supplemented post-mitochondrial fraction (S9 mix) from liver of male Sprague-Dawley rats treated with Aroclor 1254 was used for metabolic activation at 2% in the final medium. The test substance did not induce an increase in reversions in any of the tested strains with or without metabolic activation. Cytotoxicity or precipitates were not observed. The vehicle and positive controls proved validity of the experiment. Under the conditions of the conducted test, the test substance was not mutagenic in any of the five tester strains (TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA) with and without metabolic activation up to the maximum recommended concentration of 5000 µg/plate.

 

- Cytogenicity/chromosome aberration in mammalian cells

A GLP conform chromosome aberration study was performed according to OECD guideline 473 (2011 k). Clastogenicity of the test material was investigated in peripheral human lymphocytes stimulated with 1% Phytohemagglutinin (PHA). Cells were treated with the test material for 4 h both with and without metabolic activation and for 20 h without metabolic activation. The used cofactor supplemented post-mitochondrial fraction (S9 mix) was prepared from livers of rats treated with Aroclor 1254 and was used at concentrations of 20 µl/ml medium. The test material was suspended in Dimethyl sulfoxide (DMSO), due to the limited solubility of the test substance in water.

A pre-test was performed at concentrations of 0.2136, 0.6408, 2.136, 6.408, 21.36, 64.08, 213.6, 640.8 and 2136 µg/mL, in which the cells were exposed for 4 h with and without S9 and for 20 h only without S9. The highest test dose was the maximum recommended concentration of 2 mg/mL. Precipitation of the test substance or relevant cytotoxicity (at least 50% reduction in mitotic index relative to the solvent control) measured by determination of the mitotic index were not observed at any concentration tested.

Based on the results of the pre-test, the following concentrations were tested in the main study: 534, 1068 and 2136 μg/mL. Cultures of all concentrations were exposed to the test substance for 4 h/20 h followed by harvesting after 20 h. Colcemid-solution (0.1 µg/mL) was added to each culture 2 h prior to the end of the incubation period. The positive controls used were mitomycin C in the absence and cyclophosphamide in the presence of metabolic activation. Duplicate cultures were tested for every test and positive, solvent and vehicle controls (DMSO) were set up and handled in parallel.

Cytotoxicity was determined by calculation of the mitotic index (percentage of cells in mitosis per 500 cells counted). From each culture 200 metaphases (100 per culture) were examined for chromosome aberrations. The number of metaphases examined, were only reduced when the percentage of aberrant cells reached a significant level (at least 10%) before 100 cells were scored. At the 4 h exposure without metabolic activation, the mitotic index was reduced to 8% in cultures treated with 2136 µg/mL, with metabolic activation the mitotic index was not reduced. At the 4 h exposure without metabolic activation, the mitotic index was reduced in cultures treated with 2136 µg/mL to 54%. Cultures treated with mitomycin C or cylophosphamid showed reduced mitotic indices compared to the negative control.

There were no biologically relevant and statistically significant increases in the incidence of metaphases with aberrations in any culture examined, neither in the presence nor in the absence of S9.

Positive controls with and without metabolic activation showed a clear and statistically significant increase of metaphases with aberrations. Vehicle controls were within the historical control data range. Based on the results of the conducted study, the test substance was evaluated as not clastogenic under the conditions of this test.

 

- Gene mutation in mammalian cells:

A GLP guideline study on gene mutation in mammalian cells was performed according to OECD Guideline 476 (2011 l). In this study the ability of the test material to induce reverse mutations at the HGPRT locus was investigated in Chinese hamster ovary cells (CHO). Cells were treated with the test material solved in Dimethyl sulfoxide (DMSO) both with and without the addition of cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of rats treated with Aroclor 1254.

Preliminary tests for evaluation of solubility and cytotoxicity were performed at concentrations of 0.5, 1.5, 5, 15, 50, 140, 460, 1370 and 4000 µg/mL with and without S9 mix. The test substance was soluble in DMSO up to concentrations of 400 mg/mL. When administered to the culture medium precipitates were observed at a concentration of 4000 µg/mL and above. Cytotoxicity was determined by comparison of colonies in treated cultures versus vehicle control cultures (relative cloning efficiency). The relative cloning efficiency at 4000 µg/mL was 72% (-S9) and 108% (+S9). Based on these findings concentrations used in the main test were 500, 750, 1000, 1500, 1750 and 2150 µL/mL with and without S9 mix.

In the main test, duplicate cultures were treated with the test substance for 5 h. Subsequently cells were washed and cultured for an additional 18 – 24 h. At this time, cells were subcultured to assess cytotoxicity and to initiate the phenotypic expression period.For expression of the mutant phenotype, the replicates from each treatment condition weretrypsinized and subcultured independently at 2-4 day intervalsfor an expression period of7-9days. Atthe end of the expression period, selection for the mutant phenotype wasperformed.For mutagenicity testing replicates from each treatment condition were trypsinized and replated, in quintuplicate, at a density of 2x105 cells/100 mm dish and incubated with selective medium containing 6-thioguanidine (6-TG) for 7 to 10 days before 6-TG resistant colonies were counted. For cloning efficiency determinations at the time of selection, 100 cells/60 mm dishes were plated in triplicate. The corresponding vehicle and positive controls (0.2 µL/mL ethylmethanesulfonate (EMS) (without metabolic activation) and 4 µL/mL benz(a)pyrene B(a)P (with metabolic activation)) were treated and cultured under the same conditions.

In the main test, no precipitates were observed at any test condition. With metabolic activation no cytotoxic effects were observed as indicated by a relative cloning efficiency of 107% at the highest dose tested, but the test substance was tested up to the limit concentration of 2 mg/mL. Without metabolic activation slight toxicity, indicated by relative cloning efficiency of 93% was observed at the highest dose tested and the test substance was tested up to the limit concentration. There was no increase in mutation frequency (> 40 mutants per 106clonable cells) observed at any tested concentration with or without metabolic activation. The vehicle control was valid and did not show an increased mutation frequency. Both positive controls, EMS and B(a)P showed a clear mutagenic effect. Therefore, the test system was considered sensitive to detect mutagens and the test material was evaluated as not mutagenic under the conditions of this test.

 

Conclusion on genetic toxicity

Overall, the available in vitro studies on genetic toxicity do not indicate that the test substance exhibits genotoxic properties. The test substance did not induce gene mutations in bacteria or mammalian cells and did not increase the incidence of metaphases with chromosomal aberrations in peripheral human lymphocytes.

Justification for classification or non-classification

The available genotoxicity data obtained for the test substance are conclusive but not sufficient for classification according to Regulation (EC) No. 1272/2008 (CLP).