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Genetic toxicity in vitro

Description of key information

1. negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2014

2. non-clastogenic and non-aneugenic, in vitro mammalian cell micronucleus (with and without S-9 activation), OECD TG 487, 2018

3. negative, in vitro HPRT locus gene mutation (with and without S-9 activation), OECD TG 476, 2018

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised March 31st, 2011)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: March 2013; signature: May 2013
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Dose range finding study: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 ug/plateExperiment 1: TA1535, TA1537 and TA98: 0.5, 1.7, 5.4, 52, 164, 512 ug/plate
Experiment 2: TA1535, TA1537 and TA100 and TA98 strains: 9,16, 30, 51, 92, 164 ug/plate; WP2uvrA strain: 492, 1568, 2800, 5000 ug/plate
Experiment 3: TA1535 and TA98 strains only: 92, 164, 492 and 878 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was soluble in dimethyl sulfoxide (DMSO).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted. The revertant colonies were counted automatically with a Colony Counter.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (bacterial background lawn) and reduction in the number of revertants

OTHER:
- Dose range finding test on TA100 and WP2urvA with and without 5% (v/v) S9-mix; First mutation assay on TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix.
- To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed on all strains, in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test substance was tested up to the dose level of 164 μg/plate in strains TA1535, TA1537, TA98, TA100 and 5000 μg/plate in strain WP2uvrA.- Since no toxicity and no precipitate on the plates was observed in tester strains TA1535 and TA98 in the presence of S9-mix in experiment 2, an additional mutation experiment was performed in the presence of 10% (v/v) S9-mix. The following dose range was selected for the third mutation assay: 92, 164, 492 and 878 μg/plate.
Evaluation criteria:
See 'Any other information on materials and methods' for details on evaluation of the assay and positive criteria.
Statistics:
No formal hypothesis testing was done. See 'Any other information on materials and methods' for details on the acceptability and evaluation criteria of the assay.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at the top two dose levels dose of 5000 μg/plate in tester strain TA1535 and TA100 (absence and presence of S9-mix) and TA1535 and TA98 (with and without S9-mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

RANGE-FINDING/SCREENING STUDIES:
Test substance was tested in the tester strains TA100 and WP2uvrA with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. The highest concentration the test substance used in the subsequent mutation assay was 512 μg/plate (experiment 1) based on the results of this assay.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1 Dose range finding test: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain

 

 

TA100   

 

WP2uvrA

 

 

Without S9-mix

 

 

 

 

Positive control

853

±

1221

± 76

Solvent control

117

±

30

± 1

1.7

108

±

33

± 5

5.4

123

±

36

± 4

17

121

±

33

± 8

52

111

±

33

± 4

164

 

e MC

27

± 4

512

0

± 0 a

32

± 9

1600

0

± 0 a

24

± 5 n

5000

0

± 0 a NP

15

± 5 s NP

With S9-mix #1

 

 

 

 

Positive control

1249

± 53

141

± 27

Solvent control

116

± 21

46

± 10

1.7

94

± 6

43

± 9

5.4

115

± 9

39

± 9

17

117

± 20

37

± 7

52

114

± 16 n

42

± 5

164

61

± 8 m

37

± 3

512

 

e MC

33

± 9

1600

0

± 15 n

25

± 7

5000

0

± 12 s NP

2

± 2 n NP

#1          Plate incorporation assay (5% S9)

MC        Microcolonies

NP         No precipitate

a            Bacterial lawn absent

e            Bacterial lawn extremely reduced

m           Bacterial lawn moderately reduced

n            Normal bacterial background lawn

s            Bacterial background lawn slightly reduced

 

Table 2 Experiment 1: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium

 

 

TA1535

 

TA1537

 

 

TA98

 

Without S9-mix

 

 

 

 

 

 

Positive control

641

± 35

310

± 11

716

± 33

Solvent control

24

± 2

8

± 2

17

± 4

0.5

22

± 8

7

± 4

21

± 14

1.7

30

± 6

11

± 4

15

± 1

5.4

28

± 2

9

± 7

10

± 2

17

26

± 9 n

8

± 3 n

21

± 8 n

52

13

± 3 s

5

± 3 m

13

± 6 m

164

 

e MC

0

± 0 a

0

± 0 a

512

0

± 0 a NP

0

± 0 a NP

0

± 0 a NP

With S9-mix #1

 

 

 

 

 

 

Positive control

289

± 9

351

±

769

± 59

Solvent control

20

± 9

12

±

25

± 4

0.5

20

± 6

13

±

26

± 4

1.7

13

± 5

11

±

21

± 8

5.4

22

± 9

12

±

24

± 7

17

19

± 1

7

±

24

± 5

52

11

± 3

9

±

20

± 4 n

164

 

e MC

 

e MC

 

e MC

512

 

e NP MC

0

± 0 NP

 

e MC

#1          Plate incorporation assay (5% S9)

MC        Microcolonies

NP         No precipitate

a            Bacterial lawn absent

e            Bacterial lawn extremely reduced

m           Bacterial lawn moderately reduced

n            Normal bacterial background lawn

s            Bacterial background lawn slightly reduced

 

Table 3 Experiment 2: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

 

 

TA1535

 

TA1537

 

 

TA98

 

TA100

 

WP2uvrA

 

Without S9-mix

 

 

 

 

 

 

 

 

 

 

Positive control

713

± 21

445

± 23

823

± 60

981

± 46

1168

± 355

Solvent control

19

± 4

7

± 6

18

± 5

115

± 5

18

± 6

9

22

± 3

7

± 4

19

± 2

114

± 15

-

-

16

27

± 4

9

± 3

22

± 3

114

± 4

-

-

30

23

± 3 n

5

± 1 n

19

± 4 n

123

± 22 n

-

-

51

15

± 5 m

7

± 2 m

17

± 2 s

103

± 5 s

-

-

92

 

e MC

 

e MC

 

e MC

 

e MC

-

-

164

 

e NP MC

0

± 0 a NP

0

± 0 a NP

0

± 0 a NP

-

-

492

-

-

-

-

-

-

-

-

20

± 2

878

-

-

-

-

-

-

-

-

17

± 4

1568

-

-

-

-

-

-

-

-

21

± 9 n

2800

-

-

-

-

-

-

-

-

21

± 3 s

5000

-

-

-

-

-

-

-

-

10

± 3 m NP

With S9-mix #1

 

 

 

 

 

 

 

 

 

 

Positive control

169

± 5

473

± 10

409

± 27

1279

± 57

163

± 10

Solvent control

12

± 6

15

± 6

27

± 4

114

± 23

33

± 5

9

16

± 8

9

± 3

27

± 4

102

± 14

-

-

16

14

± 2

13

± 2

29

± 2

108

± 10

-

-

30

20

± 2

15

± 2

25

± 7

106

± 14

-

-

51

11

± 6

12

± 5

27

± 8

107

± 10

-

-

92

16

± 3

9

± 2 n

17

± 6

115

± 8 n

-

-

164

7

± 4 n NP

0

± 0 a NP

19

± 5 n NP

81

± 5 m NP

-

-

492

-

-

-

-

-

-

-

-

20

± 5

878

-

-

-

-

-

-

-

-

26

± 5

1568

-

-

-

-

-

-

-

-

22

± 8

2800

-

-

-

-

-

-

-

-

20

± 5

5000

-

-

-

-

-

-

-

-

27

± 11 n NP

#1          Plate incorporation assay (10% S9)

MC        Microcolonies

NP         No precipitate

a            Bacterial lawn absent

e            Bacterial lawn extremely reduced

m           Bacterial lawn moderately reduced

n            Normal bacterial background lawn

s            Bacterial background lawn slightly reduced

-             Not tested

 

Table 4 Experiment 3: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium.

 

TA1535

 

TA98

 

 

With S9-mix #1

 

 

 

 

Positive control

176

± 15

472

± 74

Solvent control

16

± 8

22

± 10

92

15

± 7 n

14

± 3

164

10

± 2 m

27

± 4 n

492

 

e MC

 

e MC

878

 

e NP MC

 

e NP MC

 

 

 

 

 

#1          Plate incorporation assay (10% S9)

MC        Microcolonies

NP         No precipitate

e            Bacterial lawn extremely reduced

m           Bacterial lawn moderately reduced

n            Normal bacterial background lawn

Conclusions:
Interpretation of results:
negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation. Negative and strain specific positive control values were within laboratory historical control data.
Executive summary:

The study was performed to OECD 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP; to evaluate the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat). In the dose range finding test, the test substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test substance did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 164 μg/plate and above in the absence and presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at the dose level of 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test the test substance was tested in the first mutation assay at a concentration range of 0.5 to 512 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains. In an independent repeat of the assay with additional parameters, the test substance was tested at a concentration range of 9 to 164 μg/plate in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in tester strain WP2uvrA. In the second mutation assay, in the absence of S9-mix, toxicity was observed in all tester strains. In the presence of S9-mix, toxicity was only observed in tester strains TA1537 and TA100. Since in the second experiment in the presence of S9-mix, insufficient toxicity without precipitate on the plates was observed in tester strains TA1535 and TA98, an additional mutation experiment was performed in the presence of 10% (v/v) S9-mix. The test substance was tested up to the dose level of 878 μg/plate. In the third mutation assay, toxicity was observed in both tester strains. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the conditions of this study it is concluded that that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05-06-2017 to 03-09-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2017; signature: November 2017
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable (in vitro micronucleus test of: clastogenic and aneugenic potential)
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours. Further details on the donors is available in the full study report.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B (4.5 μg/mL)
Metabolic activation:
with and without
Metabolic activation system:
Lot Numbers: PB/βNF S9 31/03/17 and PB/βNF S9 30/06/17
Test concentrations with justification for top dose:
The maximum recommended dose level was 1683 µg/mL, on the basis of the molecular weight of the test item. The test item was immiscible at 16.83 mg/mL in aqueous media but was fully miscible in DMSO at 168.3 mg/mL in solubility checks performed prior to testing. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 1683 μg/mL range (full results recorded in the full study report). The preliminary test used a range of test item concentrations of: 0, 6.57, 13.15, 26.30, 105.19, 210.38, 420.75, 841.5, 1683 μg/mL.

I. Preliminary Test:
0, 6.57, 13.15, 26.30, 105.19, 210.38, 420.75, 841.5, 1683 μg/mL.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 105.19 µg/mL in all three exposure group. Hemolysis was observed following exposure to the test item at and above 26.30 µg/mL in the 4-hour exposure groups and present at and above 52.59 µg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
A reduced cell pellet was observed at and above 26.30 µg/mL in the 4-hour exposure groups and at and above 105.19 µg/ mL indicating that maximum exposure was occurring at these dose levels.
Results from the preliminary toxicity test were used to set the test item dose levels for the main experiments.

II. Main Test:
4(20)-hour without S9: 0*, 4, 8, 12*, 16*, 20*, 24*, 32, 48, MMC 0.2*
4(20)-hour with S9 (2%): 0*, 2, 4, 8, 16, 24*, 32*, 48*, 52, CP 5*
24-hour without S9: 0*, 2, 4, 8, 16, 24*, 32*, 48*, 52*, DC 0.075*
where:
* = dose levels selected for analysis of MN frequency in binucleate cells
MMC= Mitomycin C
CP = Cyclophosphamide
DC = Demecolcine
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The maximum recommended dose level was 1683 µg/mL, on the basis of the molecular weight of the test item. The test item was immiscible at 16.83 mg/mL in aqueous media but was fully miscible in DMSO at 168.3 mg/mL in solubility checks performed prior to testing. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 1683 μg/mL range (full results recorded in the full study report). The preliminary test used a range of test item concentrations of: 0, 6.57, 13.15, 26.30, 105.19, 210.38, 420.75, 841.5, 1683 μg/mL.
Applicant assessment indicates: DMSO is a guideline accepted vehicle with an available laboratory historic control data set.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine
Details on test system and experimental conditions:
METHOD OF APPLICATION: Other:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: 9.05 mL MEM, 10% (FBS); 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin; 0.75 mL heparinized whole blood

DURATION
- Preincubation period: Not reported.
- Exposure duration:
I. With Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL (100 μL) of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of the Main Experiment. The nominal total volume of each culture was 10 mL.
After 4 hours at approximately 37 ºC, 5 % CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

II. Without Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL (100 μL) of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
In the 24-hour exposure in the absence of S9, the exposure was continuous. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours. The tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours.

SELECTION AGENT (mutation assays): Not applicable.

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data. When the slides were dry, they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.

NUMBER OF CELLS EVALUATED: The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI)
The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. It was used to calculate cytostasis and estimate test item cytotoxicity.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable.
- Other: Scoring: Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors. The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are substantially outside the range of the historical negative control data for the test item concentrations.

When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.

The test item l is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
Statistics:
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., (2003), In vitro micronucleus test. In: Encyclopedia of Biopharmaceutical Statistics, 2nd edition. S Chow ed. Marcel Dekker, Inc. New York, NY, pp. 463 – 467.). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.
Species / strain:
lymphocytes: Human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 1 for further information
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media
- Effects of osmolality: There was no significant change osmolality (did not increase by more than 50 mOsm) when the test item was dosed into media
- Evaporation from medium: Not reported.
- Water solubility: Formulated in DMSO.
- Precipitation: In the preliminary test: A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 105.19 µg/mL in all three exposure groups. Main test: No precipitate of the test item was noted at any dose level in any of the exposure groups (4-hour without S9: 48 µg/mL ; 4-hour with S9 (2%): 52 µg/mL and 24-hour without S9: 52 µg/mL, respectively.
- Other confounding effects: In the preliminary test and the main test: Hemolysis was observed following exposure to the test item at and above 26.30 µg/mL in the 4-hour exposure groups and present at and above 52.59 µg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. Additionally, a reduced cell pellet was observed at and above 26.30 µg/mL in the 4-hour exposure groups and at and above 105.19 µg/ mL indicating that maximum exposure was occurring at these dose levels. In the definitive test: Hemolysis was observed following exposure to the test item at and above 20 µg/mL in the 4 hour exposure group in the absence of S9-mix and present at and above 24 µg/mL in the 4 hour exposure group in the presence of S9 and the 24-hour continuous exposure groups.
- Definition of acceptable cells for analysis: The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 1683 μg/mL. The maximum dose was the maximum recommended dose level. The selection of the maximum dose level was based on toxicity for the main test.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: See table 1

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: See table 1
- Indication whether binucleate or mononucleate where appropriate: See table 1

COMPARISON WITH HISTORICAL CONTROL DATA:
- All vehicle (DMSO) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. (Within the Historic Control Data range presented in the full study report).
- All the positive control items induced statistically significant increases in the frequency of cells with micronuclei. (Within the Historic Control Data range presented in the full study report).

ADDITIONAL INFORMATION ON CYTOTOXICITY: See ‘other confounding effects’ listed above.
- Measurement of cytotoxicity used: CBPI
- Other observations when applicable: frequency of binucleated cells

Table 1. Definitive test: 4-hour exposure without and with S9 (2%) and 24-hour exposure without S9: CBPI and micronucleus data

Exposure

Dose Level

Notes

Replicate

Nucleate cells

/500 cells

CBPI

Mean CBPI

% cytostasis

MN per 1000 Binucleate cells

% Binucleate Cells with MN

Mean % Binucleate Cells with MN

Mono

Bi

Multi

1 MN

2 MN

> 2 MN

4-hours (without S9)

0

 

A

321

150

29

1.42

 

 

1

0

0

0.10

 

 

 

B

350

126

24

1.35

1.39

0

5

0

0

0.50

0.30

4

 

A

-

-

-

-

-

-

-

-

-

-

-

 

 

B

-

-

-

-

-

-

-

-

-

-

-

8

 

A

-

-

-

-

-

-

-

-

-

-

-

 

 

B

-

-

-

-

-

-

-

-

-

-

-

12

 

A

337

153

20

1.39

 

 

2

0

0

0.20

 

 

 

B

311

159

30

1.44

1.42

0 ‡

3

0

0

0.30

0.25

16

 

A

330

150

20

1.38

 

 

3

0

0

0.30

 

 

 

B

314

163

23

1.42

1.40

0 ‡

4

0

0

0.40

0.35

20

H

A

333

141

26

1.39

 

 

7

1

0

0.80

 

 

 

B

321

158

21

1.40

1.40

0 ‡

2

2

0

0.40

0.60

24

H

A

372

122

6

1.27

 

 

2

0

0

0.20

 

 

 

B

350

139

11

1.32

1.30

23

2

1

0

0.50

0.35

32

H

A

488

12

0

1.02

 

 

-

-

-

-

-

 

 

B

480

20

0

1.04

1.03

92

-

-

-

-

-

48

H

A

NB

NB

NB

-

-

-

-

-

-

-

-

 

 

B

NB

NB

NB

-

-

-

-

-

-

-

-

MMC

 

A

366

127

7

1.28

 

 

57

1

0

5.80

 

0.2

 

B

354

141

5

1.30

1.29

25

46

1

0

4.60

5.20 ***

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4-hours (with S9 2%)

0

 

A

352

137

11

1.32

 

 

2

0

0

0.20

 

 

 

B

335

135

30

1.39

1.36

0

2

0

0

0.20

0.20

2

 

A

-

-

-

-

-

-

-

-

-

-

-

 

 

B

-

-

-

-

-

-

-

-

-

-

-

4

 

A

-

-

-

-

-

-

-

-

-

-

-

 

 

B

-

-

-

-

-

-

-

-

-

-

-

8

 

A

-

-

-

-

-

-

-

-

-

-

-

 

 

B

-

-

-

-

-

-

-

-

-

-

-

16

A

346

142

12

1.33

 

 

-

-

-

-

-

 

 

B

326

153

21

1.39

1.36

0 ‡

-

-

-

-

-

24

H

A

335

146

19

1.37

 

 

4

0

0

0.40

 

 

 

B

346

145

9

1.33

1.35

1

1

0

0

0.10

0.25

32

H

A

325

151

24

1.40

 

 

8

0

0

0.80

 

 

 

B

289

192

19

1.46

1.43

0 ‡

0

0

0

0.00

0.40

48

H

A

425

75

0

1.15

 

 

0

0

0

0.00

 

 

 

B

424

73

3

1.16

1.16

55

2

0

0

0.20

0.10

52

H

A

-

-

-

-

-

-

-

-

-

-

-

 

 

B

-

-

-

-

-

-

-

-

-

-

-

CP

 

A

447

53

0

1.11

 

 

26

2

0

2.80

5

 

B

408

90

2

1.19

1.15

58

34

4

1

3.90

3.35 ***

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

24-hours (without S9)

0

 

A

205

266

29

1.65

 

 

4

0

0

0.40

 

 

 

B

146

318

36

1.78

1.72

0

0

0

0

0.00

0.20

2

 

A

-

-

-

-

-

-

-

-

-

-

-

 

 

B

-

-

-

-

-

-

-

-

-

-

-

4

 

A

-

-

-

-

-

-

-

-

-

-

-

 

 

B

-

-

-

-

-

-

-

-

-

-

-

8

 

A

-

-

-

-

-

-

-

-

-

-

-

 

 

B

-

-

-

-

-

-

-

-

-

-

-

16

A

-

-

-

-

-

-

-

-

-

-

-

 

 

B

-

-

-

-

-

-

-

-

-

-

-

24

H

A

133

330

37

1.81

 

 

1

0

0

0.10

 

 

 

B

161

314

25

1.73

1.77

0 ‡

4

0

0

0.40

0.25

32

H

A

147

314

39

1.78

 

 

2

1

0

0.30

 

 

 

B

145

325

30

1.77

1.78

0 ‡

5

0

0

0.50

0.40

48

H

A

330

166

4

1.35

 

 

0

0

0

0.00

 

 

 

B

301

194

5

1.41

1.38

47

4

0

0

0.40

0.20

52

H

A

257

230

13

1.51

 

 

1

0

0

0.10

 

 

 

B

365

130

5

1.28

1.40

45

6

0

0

0.60

0.35

DC

 

A

241

226

33

1.58

 

 

17

5

8

3.00

 

0.075

 

B

271

201

28

1.51

1.55

24

27

7

2

3.60

3.30 ***

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MMC = Mitomycin C

CP = Cyclophosphamide

DC = Demecolcine

***= P<0.001

- = Not selected for scoring

NB = No binucleate cells or insufficient binucleate cells for scoring

H = Hemolysis observed at the end of exposure

= Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control

Conclusions:
Interpretation of results:
negative
Under the conditions of this study the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in the presence and absence of S9 activation.
Executive summary:

The study was performed to the requirements of OECD TG 487 under GLP conditions to assess the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4 hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test with a dose range of 6.57 to 1683 µg/mL in DMSO vehicle which indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Test were as follows: 4-hour without S9-Mix (2%): 0, 4, 8, 12, 16, 20, 24, 32, 48 μg/mL, with S9-Mix (2%): 0, 2, 4, 8, 16, 24, 32, 48, 52 μg/mL and 24-hour without S9: 0, 2, 4, 8, 16, 24, 32, 48, 52 μg/mL, respectively. All vehicle (DMSO)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with micronuclei, indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that included a dose level that generally approached 50% cytostasis. It was considered that the test item had been adequately tested given step cytotoxicity observed and reduced cell pellet observed in preliminary testing. Under the conditions of this study, the test item was considered to be non-clastogenic and non- aneugenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-12-2017 to 16-02-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2017; signature: November 2017
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the Chinese hamster V79 cell line
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type.
- Cell cycle length, doubling time or proliferation index: Doubling time 12 - 16 h in stock cultures.
- Sex, age and number of blood donors if applicable: Not applicable.
- Whether whole blood or separated lymphocytes were used if applicable: Not applicable.
- Number of passages if applicable: Not applicable.
- Methods for maintenance in cell culture if applicable: Laboratory stock cell cultures will be periodically checked for stability and absence of mycoplasma contamination. The stock of cells is stored in liquid nitrogen. Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM). Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.
- Modal number of chromosomes: The cells have a stable karyotype with a modal chromosome number of 22 (Reference: Howard-Flanders, 1981).
- Normal (negative control) cell cycle time: The average absolute cloning efficiency of the Day 7 negative controls should exceed 50%.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: For use, a sample of cells was removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10 % fetal bovine serum (FBS)) at approximately 37 °C with 5 % CO2 in humidified air.
- Properly maintained: Yes.
- Periodically checked for Mycoplasma contamination: Yes.
- Periodically checked for karyotype stability: Yes.
- Periodically 'cleansed' against high spontaneous background: Yes.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix: Prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G-6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration.
Test concentrations with justification for top dose:
The maximum recommended dose level was 1683 µg/mL, on the basis of the molecular weight of the test item. The test item was immiscible at 16.83 mg/mL in aqueous media but was fully miscible in DMSO at 168.3 mg/mL in solubility checks performed prior to testing. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 1683 μg/mL range (full results recorded in the full study report). The preliminary test used a range of test item concentrations of: 0, 6.57, 13.15, 26.30, 105.19, 210.38, 420.75, 841.5, 1683 μg/mL.

I. Preliminary Test:
A concentration range of 2.0 to 80 µg/mL was used in the preliminary cytotoxicity test. The maximum concentration tested was selected to avoid too many precipitating dose levels and excessive toxicity.
0 (control), 2, 4, 8, 16, 32, 40, 48, 64, 80 μg/mL (limited by test item precipitation and excessive toxicity, demonstrated in a previous Micronucleus in vitro assay - full details provided in the full study report).

II. Main Test:
Main Test (with S9-mix): 0 (control)*, 2, 4*, 8*, 16*, 20*, 24*, 28*, 32, DMBA: 1.0* and 2.0* µg/mL
Main Test (without S9-mix): 0 (control)*, 0.5, 1, 2*, 4*, 5*, 6*, 7*, 8*, EMS: 500* µg/mL
* concentration levels plated for cloning efficiency and mutant frequency
Plated concentrations were based on cytotoxicity, as applicable.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The maximum recommended dose level was 1683 µg/mL, on the basis of the molecular weight of the test item. The test item was immiscible at 16.83 mg/mL in aqueous media but was fully miscible in DMSO at 168.3 mg/mL in solubility checks performed prior to testing. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 1683 μg/mL range (full results recorded in the full study report). The preliminary test used a range of test item concentrations of: 0, 6.57, 13.15, 26.30, 105.19, 210.38, 420.75, 841.5, 1683 μg/mL.
Applicant assessment indicates: DMSO is a guideline accepted vehicle with an available laboratory historic control data set.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: Pre-incubation with test item
- Cell density at seeding (if applicable): For mutation expression = 2,000,000 cells per 225 cm2 flask; for cloning efficiency = 200 cells per 25 cm2 flask.

DURATION
- Preincubation period: Cells were seeded at 1 x 10^7 cells/225 cm2 flask approximately 24 hours being exposed to the test or control items.
- Exposure duration: Treatment was for 4 hours in serum free media (MEM) at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

SELECTION AGENT (mutation assays): Not applicable.

SPINDLE INHIBITOR (cytogenetic assays): Not applicable.

STAIN (for cytogenetic assays): Not applicable. See ‘Methods of Slide Preparation and Staining Technique Used’ for further information on staining technique.

NUMBER OF REPLICATIONS: 1 for growth and mutation expression and 3 for cloning efficiency.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes

NUMBER OF CELLS EVALUATED: 2 x 10^5 cells/petri dish (ten replicates per group) to determine mutant frequency; triplicate at 200 cells/25 cm2 flask to determine cloning efficiency

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Not applicable.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Not applicable.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning Efficiency (CE)
- Any supplementary information relevant to cytotoxicity: Cloning Efficiency (CE), % control, mutant plate counts, mutant frequency/10^6 (MF10^-6) and mutant frequency/10^6 survival rate (MFSV) were calculated.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable.
- Determination of endoreplication: Not applicable.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): Not applicable.

- OTHER: Not applicable.
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.

When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
When there is an indication of any increases in mutant frequency at any concentration, comparisons are made between the appropriate vehicle control value and each individual concentration, using Student’s t-test.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 1 for further information
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects determined.
- Effects of osmolality: No effect determined.
- Evaporation from medium: Tissue culture flasks were sealed due to the potential volatility of the test item
- Water solubility: Formulated in DMSO
- Precipitation: Yes, above cytotoxic dose levels. Not plated when observed (see Table 1 for more information)
- Definition of acceptable cells for analysis: Reported and within specification. Fresh cultures used for each test.
- Other confounding effects: Not applicable.

RANGE-FINDING/SCREENING STUDIES: A range finding (preliminary test was performed and was used to determine concentrations, employed in the main test).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: Not applicable.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: Not applicable.
- Indication whether binucleate or mononucleate where appropriate: Not applicable.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: All Positive Controls gave marked increases in mutant frequency in the current HCD. This indicates the test and the metabolic activation system were operating as expected. The purpose of the PC is to verify the functioning of the test system. Full details are provided in the full study report on the PC HCD.
- Negative (solvent/vehicle) historical control data: All vehicle controls indicated background mutant frequencies per survivor (MFS) in the current HCD range. Full details are provided in the full study report on the VC HCD.): Not applicable.

Table 1. Main test: 4-hour exposure without and with Metabolic Activation (S9)

4-hour exposure without S9

 

 

 

Day 0 Viability

Day 7 Viability

Day 7 Mutant

Dose

(µg/mL)

 

 

Colonies / flask

(200 cells in plated flask)

% CE

% Control

Mean % Control

Colonies / flask

(200 cells in plated flask)

% CE

% Control

Mean % Control

Colonies / flask

(2x10^5 cells plated/flask)

MF

MSF 10^-6

SD

Group MFS 10^-6

0

 

A

175

160

175

85.0

100

 

17

143

172

80.3

100

 

3

0

3

1

1

1

1

2

0

2

7

8.7

 

 

 

 

B

194

187

199

96.7

100

100

146

159

157

77.0

100

100

3

4

4

4

3

1

1

1

1

1

11.5

14.9

1.31

12

0.5

 

A

182

188

177

91.2

107.3

 

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

 

 

B

208

212

194

102.3

105.9

107

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1

 

A

198

206

154

93.0

109.4

 

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

 

 

B

216

195

208

103.2

106.7

108

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

2

 

A

187

182

189

93.0

109.4

 

161

150

158

78.2

97.3

 

1

3

1

0

0

1

1

1

1

1

5

6.4

 

 

 

 

B

198

214

194

101.0

104.5

107

156

173

172

83.5

108.4

103

2

0

0

3

1

0

1

1

0

0

4

4.8

0.91

6

4

 

A

198

160

172

88.3

103.9

 

153

154

156

77.2

96.1

 

2

4

1

2

2

2

1

2

5

2

11.5

14.9

 

 

 

 

B

187

162

182

88.5

91.6

98

152

145

160

76.2

98.9

97

4

0

2

1

1

1

1

2

0

0

6

7.9

1.33

11

5

A

179

190

201

95.0

111.8

 

160

165

167

82.0

102.1

 

2

1

4

0

0

1

1

1

2

0

6

7.3

 

 

 

 

B

173

169

182

87.3

90.3

101

166

135

157

76.3

99.1

101

1

2

4

4

1

2

2

1

2

0

9.5

12.4

1.28

10

6

 

A

181

185

184

91.7

107.8

 

139

123

140

67.0

83.4

 

1

4

3

5

6

2

1

1

1

0

12

17.9

 

 

 

 

B

176

162

169

84.5

87.4

98

166

170

166

83.7

108.7

96

2

2

2

1

2

1

1

2

1

3

8.5

10.2

1.50

14

7

 

A

104

103

102

51.5

60.6

 

185

158

154

82.8

103.1

 

1

1

1

1

0

1

1

1

0

0

3.5

4.2

 

 

 

 

B

139

131

129

66.0

68.3

64

175

163

169

84.5

109.7

106

3

2

3

1

1

3

2

1

1

3

10

11.8

0.99

8

8

 

A

19

29

26

12.3

14.5

 

160

155

138

75.5

94.0

 

1

1

2

3

1

1

1

1

1

0

6

7.9

 

 

 

 

B

77

34

28

23.2

24.0

19

159

157

156

78.7

102.2

98

4

3

3

1

2

3

4

1

2

1

12

15.3

1.15

12

EMS

 

A

150

178

176

84.0

98.8

 

192

190

170

92.0

114.5

 

57

43

38

53

29

52

44

59

57

51

241.5

262.5

 

 

500

 

B

194

188

199

98.7

102.1

100

162

179

173

85.7

113.3

113

44

48

29

37

29

31

55

35

34

42

192

224.1

10.26

243

 

 

4-hour exposure with S9

 

 

Day 0 Viability

Day 7 Viability

Day 7 Mutant

Dose

(µg/mL)

 

 

Colonies / flask

(200 cells in plated flask)

% CE

% Control

Mean % Control

Colonies / flask

(200 cells in plated flask)

% CE

% Control

Mean % Control

Colonies / flask

(2x10^5 cells plated/flask)

MF

MSF 10^-6

SD

Group MFS 10^-6

0

 

A

191

181

181

92.2

100

 

124

122

126

62.0

100

 

1

0

1

1

0

2

2

2

0

0

4.5

7.3

 

 

 

 

B

151

161

163

79.2

100

100

151

162

124

72.8

100

100

1

3

1

3

2

1

1

0

1

1

7

9.6

0.93

8

2

 

A

200

210

185

99.2

107.6

 

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

 

 

B

194

192

192

96.3

121.7

115

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

4

 

A

138

135

158

71.8

77.9

 

138

130

111

63.2

101.9

 

2

1

1

0

0

4

3

1

1

1

7

11.1

 

 

 

 

B

179

169

175

87.2

110.1

94

112

127

126

60.8

83.5

93

1

1

1

0

1

2

1

1

3

3

7

11.5

1.10

11

8

 

A

171

179

193

90.5

98.2

 

142

138

146

71.0

114.5

 

2

3

2

1

1

2

1

1

0

1

7

9.9

 

 

 

 

B

172

169

167

84.7

106.9

103

159

141

153

75.5

103.7

109

2

2

3

1

1

3

1

3

2

3

10.5

13.9

0.91

12

16

 

A

178

154

157

81.5

88.4

 

139

124

133

66.0

106.5

 

3

1

2

1

0

5

3

1

2

0

9

13.6

 

 

 

 

B

155

151

149

75.8

95.8

92

183

163

171

86.2

118.3

112

3

1

3

2

1

1

0

2

2

2

8.5

9.9

1.25

12

20

 

A

174

166

157

82.8

89.9

 

127

137

132

66.0

106.5

 

2

0

3

3

2

3

3

1

2

2

10.5

15.9

 

 

 

 

B

174

176

177

87.8

110.9

100

151

164

156

78.5

107.8

107

2

0

1

0

0

1

1

1

4

2

6

7.6

1.18

12

24

 

A

116

135

124

62.5

67.8

 

159

159

164

80.3

129.6

 

1

0

3

2

1

0

1

0

0

1

4.5

5.6

 

 

 

 

B

138

129

136

67.2

84.8

76

163

155

155

78.8

108.2

199

0

1

2

3

0

2

0

1

0

0

4.5

5.7

1.02

6

28

P

A

64

51

54

28.2

30.6

 

140

137

181

76.7

123.7

 

1

1

1

2

0

2

1

3

3

0

7

9.1

 

 

 

 

B

113

104

103

53.3

67.4

49

180

170

172

87.0

119.5

122

0

1

0

0

2

2

0

1

2

1

4.5

5.2

0.99

7

32

P

A

0

0

0

0.0

0.0

 

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

 

 

B

0

0

0

0.0

0.0

0

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

DMBA

 

A

120

125

100

57.5

62.4

 

127

109

110

57.7

93.0

 

78

80

67

98

86

93

79

61

69

78

394.5

684.1

 

 

2

 

B

133

113

115

60.2

76.0

69

149

168

151

78.0

107.1

100

100

84

104

109

94

85

98

77

79

82

456

584.6

12.71

634

EMS = Ethyl methane sulphonate

DMBA = Dimethyl benzanthracene

CE = Cloning efficiency

MF = Mutant frequency

MFS = Mutant frequency per survivor

SD = Standard deviation

P = Precipitate present at the end of exposure period

Conclusions:
Interpretation of results:
negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation.
Executive summary:

The study was performed to the requirements of OECD TG 476 under GLP conditions to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. Following a check for solubility which indicated precipitation above 625 μg/mL in DMSO solvent, a preliminary test for cytotoxicity within the range 2.0 to 80 μg/mL concentration was performed. Subsequently, Chinese hamster (V79) cells were treated in a main test with the test item at eight concentrations, in duplicate, together with vehicle (DMSO) and positive controls in both the absence and presence of metabolic activation. The dose levels were selected from using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by a combination of toxicity and the onset of test item precipitate in the absence of metabolic activation, and toxicity in the presence of metabolic activity. The concentrations employed including vehicle and positive controls were, respectively: (i) with S9-mix (2%): 0 (control), 0 (control), 2, 4, 8, 16, 20, 24, 28, 32, DMBA: 1.0 and 2.0 µg/mL and (ii) without S9-mix: 0 (control), 0.5, 1, 2, 4, 5, 6, 7, 8, EMS: 500* µg/mL. The test item exhibited dose-related cytotoxicity to the cells. The dose levels plated for cloning efficiency and expression of mutant colonies were: 4-hour with S9 (2%): 4, 8, 16, 20, 24, 28 μg/mL and 4-hour without S9: 2, 4, 5, 6, 7, 8 μg/mL, respectively. Concurrent vehicle (DMSO) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The concurrent positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. Optimum toxicity was achieved in the 4-hour –S9 (absence of metabolic activation) exposure group. One precipitating dose level was achieved and analyzed in the 4-hour +S9 exposure (presence of metabolic activation). Both exposure groups met the requirements of the OECD 476 guideline. The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test using a dose range that included the lowest precipitating dose level, and achieved optimum levels of toxicity. Under the conditions of this study, the test item was considered to be non-mutagenic on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line in vitro.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable.

Additional information

Key study: OECD TG 471, 2014 - The study was performed to the requirements of OECD Guideline 471, EU Method B13/14 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. A preliminary test was performed to determine the toxicity of the test material. A range-finding study was performed to determine the doses used for the main test. In the main test, the plate incorporation method was used and was evaluated at a concentration of up to 5000 µg/plate. Positive controls appropriate for each strain, in the presence and absence of S9 -mix, were included. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The vehicle (DMSO) control plates gave counts of revertant colonies within the historic laboratory control range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. Cytotoxicity, as evidenced by a decrease of the bacterial background lawn, in tester strain TA100, toxicity was observed at dose levels of 164 μg/plate and above in the absence and presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at the dose level of 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test the test substance was tested in the first mutation assay at a concentration range of 0.5 to 512 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains. In an independent repeat of the assay with additional parameters, the test substance was tested at a concentration range of 9 to 164 μg/plate in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in tester strain WP2uvrA. In the second mutation assay, in the absence of S9-mix, toxicity was observed in all tester strains. In the presence of S9-mix, toxicity was only observed in tester strains TA1537 and TA100. Since in the second experiment in the presence of S9-mix, insufficient toxicity without precipitate on the plates was observed in tester strains TA1535 and TA98, an additional mutation experiment was performed in the presence of 10% (v/v) S9-mix. The test substance was tested up to the dose level of 878 μg/plate. In the third mutation assay, toxicity was observed in both tester strains. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiment. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

 

Key study: OECD TG 487, 2018 - The study was performed to the requirements of OECD TG 487 under GLP conditions to assess the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4 hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test with a dose range of 6.57 to 1683 µg/mL in DMSO vehicle which indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Test were as follows: 4-hour without S9-Mix (2%): 0, 4, 8, 12, 16, 20, 24, 32, 48 μg/mL, with S9-Mix (2%): 0, 2, 4, 8, 16, 24, 32, 48, 52 μg/mL and 24-hour without S9: 0, 2, 4, 8, 16, 24, 32, 48, 52 μg/mL, respectively. All vehicle (DMSO)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with micronuclei, indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that included a dose level that generally approached 50% cytostasis. It was considered that the test item had been adequately tested given step cytotoxicity observed and reduced cell pellet observed in preliminary testing. Under the conditions of this study, the test item was considered to be non-clastogenic and non- aneugenic to human lymphocytes in vitro.

 

Key study: OECD TG 476, 2018 - The study was performed to the requirements of OECD TG 476 under GLP conditions to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. Following a check for solubility which indicated precipitation above 625 μg/mL in DMSO solvent, a preliminary test for cytotoxicity within the range 2.0 to 80 μg/mL concentration was performed. Subsequently, Chinese hamster (V79) cells were treated in a main test with the test item at eight concentrations, in duplicate, together with vehicle (DMSO) and positive controls in both the absence and presence of metabolic activation. The dose levels were selected from using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by a combination of toxicity and the onset of test item precipitate in the absence of metabolic activation, and toxicity in the presence of metabolic activity. The concentrations employed including vehicle and positive controls were, respectively: (i) with S9-mix (2%): 0 (control), 0 (control), 2, 4, 8, 16, 20, 24, 28, 32, DMBA: 1.0 and 2.0 µg/mL and (ii) without S9-mix: 0 (control), 0.5, 1, 2, 4, 5, 6, 7, 8, EMS: 500* µg/mL. The test item exhibited dose-related cytotoxicity to the cells. The dose levels plated for cloning efficiency and expression of mutant colonies were: 4-hour with S9 (2%): 4, 8, 16, 20, 24, 28 μg/mL and 4-hour without S9: 2, 4, 5, 6, 7, 8 μg/mL, respectively. Concurrent vehicle (DMSO) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The concurrent positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. Optimum toxicity was achieved in the 4-hour –S9 (absence of metabolic activation) exposure group. One precipitating dose level was achieved and analyzed in the 4-hour +S9 exposure (presence of metabolic activation). Both exposure groups met the requirements of the OECD 476 guideline. The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test using a dose range that included the lowest precipitating dose level, and achieved optimum levels of toxicity. Under the conditions of this study, the test item was considered to be non-mutagenic on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line in vitro.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity