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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 6 May 2004 to January 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Adopted : 21st July 1997
Deviations:
yes
Remarks:
• in the first experiment without and with S9 mix, the vehicle control CE2 value was slightly higher than the range stated in acceptance criteria. These deviations were not considered to have compromised the validity or integrity of the study.
GLP compliance:
yes
Type of assay:
other: in vitro gene mutation in mammalian cells (Mouse lymphoma assay)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methyl-1-phenyl-5-pyrazolone
EC Number:
201-891-0
EC Name:
3-methyl-1-phenyl-5-pyrazolone
Cas Number:
89-25-8
Molecular formula:
C10H10N2O
IUPAC Name:
5-methyl-2-phenyl-2,3-dihydro-1H-pyrazol-3-one
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch No. 62
- Expiration date of the lot/batch: september 2005
- Purity test date: 31 August 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Yellowfish powder stored in glass flask, at +4°C protected from light and under nitrogen gas
- Stability under test conditions: stability known (according to results from solubility assay performed in the CIT/Study No. 26977 AHS)
- Solubility and stability of the test substance in the solvent/vehicle: stability and solubility known (according to results from solubility assay performed in the CIT/Study No. 26977 AHS)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in the vehicle (DMSO) at a concentration of 174.2 mg/mL for the preliminary toxicity test and for both experiments.
- Final dilution of a dissolved solid, stock liquid or gel: 174.2 mg/mL
- Final preparation of a solid: not specified

Method

Target gene:
Thymidine Kinase (TK) locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: L5178Y cells, were obtained from ATCC (American Type Culture Collection, USA), by the intermediate of Biovalley (France).
- Suitability of cells: L5178Y cells are an established cell line recommended by international regulations for use in the in vitro mammalian cell gene mutation test. They have demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a stable spontaneous mutant frequency.
- Cell cycle length, doubling time or proliferation index: The average cell cycle time is 12-14 hours and the TK phenotypic expression time is 2 days
- The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) at -80°C or in liquid nitrogen. Each batch of frozen cells was purged of TK-/- mutants and checked for the absence of mycoplasma.

MEDIA USED
RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin
(100 μg/mL) and sodium pyruvate (200 μg/mL). This medium (RPMI 0) was supplemented by heat inactivated horse serum at 10%, v/v (RPMI 10) or 20% , v/v (RPMI 20). RPMI 10 was diluted with RPMI 0 (1:1, v/v) in order to obtain RPMI 5 which was used for treatment.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
the S9 mix was prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Experiments without S9 mix:
The selected treatment-levels were:
• 0.313, 0.625, 1.25, 2.5, 5 and 10 mM, for the first experiment,
• 0.625, 1.25, 2.5, 5, 7.5 and 10 mM, for the second experiment.

Experiments with S9 mix:
The selected treatment-levels were:
• 0.313, 0.625, 1.25, 2.5, 5 and 10 mM, for the first experiment,
• 0.625, 1.25, 2.5, 5, 7.5 and 10 mM, for the second experiment.

Since the test item was non-severely toxic and freely soluble, the highest dose-level was 10 mM, according to the criteria specified in the international regulations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, test item dissolved at a concentration of 174.2 mg/ml

- Justification for choice of solvent/vehicle: According to the solubility assay performed in the CIT/Study No. 26977 AHS, the vehicle was
dimethylsulfoxide (DMSO), batch Nos. K32136150327 and K32311850346
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Preliminary toxicity test
To assess the cytotoxicity of the test item, at least six dose-levels (one culture/dose-level) were tested both with and without metabolic activation. A treatment of 3 hours (with and without S9 mix) was performed using a final concentration and conditions as described below for the mutagenicity experiment. At the end of treatment, cells were washed and then cell concentrations were adjusted to 2 x 105 cells/mL and cultured for 2 days as for the mutagenicity experiment. Two days after the end of the treatment, cultures were
adjusted in order to seed an average of 1.6 cells per well in the 96-well microtiter plates.

Mutagenicity experiments
In two independent experiments, at least four dose-levels of the test item
(two cultures/dose-level) were tested both with and without metabolic activation, using a
treatment duration of 3 hours. For the treatment, approximately 0.5 x 106 cells/mL in 20 mL culture medium (RPMI 5) were exposed to the test or control items, at 37°C.
In each experiment, the following controls were included using at least duplicate cultures:
• vehicle controls: cultures treated with the vehicle,
• positive controls: cultures treated with:
- MMS, in the absence of S9 mix,
- CPA, in the presence of S9 mix.
For the treatment, approximately 0.5 x 106 cells/mL in 20 mL culture medium (RPMI 5) were
exposed to the test or control items, at 37°C.
At the end of the treatment periods, the cells were rinsed. After centrifugation and removing of the supernatant, the pellets were suspended in RPMI 10 and the cells were counted using a haemocytometer. Where sufficient cells survived, the cell concentrations were adjusted to 2 x 10 5 cells/mL and to enable the expression of the mutant phenotype, were incubated in RPMI 10 medium for 48 hours, at 37°C in a humidified atmosphere of 5% CO2/95% air. During this expression period, cultures were maintained at the density of approximately 2 x 105 cells/mL, where possible. At the end of this expression period, the cell densities of the cultures were determined using a haemocytometer and seeded after serial dilutions as described below:

Viability plates
An average of 1.6 cells/well (one 96-well plate/culture = two plates/dose-level, except for the vehicle control where two 96-well plates/culture were used = total of four plates) to define the number of viable cells (CE2). After at least 7 days of incubation at 37°C in a humidified atmosphere of 5% CO2/95% air, the clones were counted.

Mutant plates
2000 cells/well (one 96-well plate/culture = two plates/dose-level, except for the vehicle control where two 96-well plates/culture were used = total of four plates) to select the TFTR (trifluorothymidine resistant) mutant cells (for the determination of CEmutant). After 11-12 days of incubation at 37°C in a humidified atmosphere of 5% CO2/95% air in the presence of 4 μg TFT/mL of culture medium, the clones were counted, differentiating small and large colonies:
. size of small colonies: <25% of the diameter of the well,
. size of large colonies: >25% of the diameter of the well.

For scoring of colonies in mutant plates, the following parameters were considered:
. well containing mutant colony (small or large),
. well not containing mutant colony,
. when both small and large colonies are present in the same well both mutant colonies were counted (one small and one large)

DURATION

- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 11-12 cells

SELECTION AGENT (mutation assays): selection of the TFTR (trifluorothymidine resistant) mutant cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

EVALUATION OF THE RESULTS

Data from viability plates (empty wells) were used to calculate CE2 (cloning efficiency at the end of the expression period) and RCE2 (viability relative to vehicle controls at the end of the expression period). These values indicated the viability of the cell populations at the end of the expression period.
RCE2 is calculated as follows : RCE2 = ((CE2 Treated)/(CE2 vehicle control))x100, CE2 was calculated from the zero term of the Poisson distribution.

For evaluation of cytotoxicity, the following parameters were determined: Day cell growth at day 1 (or 2) as SG (suspension growth), Relative SG (RSG), adjusted RSG, Cell count factor and RTG (Relative Total Growth)
Adjusted RTG is calculated with the following parameters as follows : (Adjusted RSG x RCE2)/100

Data from the mutant plates (empty wells) were used to calculate CEmutant (cloning efficiency in selective medium). This value is an indicator of the absolute mutant frequency. CEmutant is calculated from the zero term of the Poisson distribution (only mutant clones are able to grow in TFT containing medium). The relative mutant frequency (MF) was calculated as : MF = (CEmutant x 10E6)/CE2


Evaluation criteria:
A reproducible noteworthy (at least two-fold) increased in the mutant frequency when compared with the vehicle controls, at any dose-level and/or evidence a dose-relationship was considered a positive result. Reference to historical data, or other considerations of biological relevance were also taken into account in the evaluation of the data obtained. Positive responses observed only at high levels of cytotoxicity (survival lower than 10%) were not considered.

Since no equivocal result was observed in the first experiment and since all the acceptance criteria were obtained in the second experiment with and without S9 mix, these deviations were
not considered to have compromised the validity or integrity of the study.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST
The test item was freely soluble in the vehicle (DMSO) at 174.2 mg/mL.
In the culture medium, the dose-level of 10 mM (corresponding to 1742 μg/mL) showed no precipitate. At this dose-level, the pH was 7.4 (7.1 for the vehicle control) and the osmolality equal to 443 mOsm/kg H2O (482 for the vehicle control).
Consequently, with a dose volume of 200 μL/20 mL culture medium, the treatment-levels for the preliminary toxicity test were: 0.02, 0.2, 1, 2, 5 and 10 mM.
Following the 3-hour treatment without S9 mix (table 1), a moderate toxicity was noted at 10 mM, as shown by 57% decrease in adjusted relative suspension growth (Adj. RSG) and 47% decrease in adjusted relative total growth (Adj. RTG)).
Following the 3-hour treatment with S9 mix (table 1), a marked toxicity was observed at 10 mM,
as shown by 90% decrease in Adj. RSG and 83% decrease in Adj. RTG.


MUTAGENICITY EXPERIMENTS
The cloning efficiencies CE2 and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. Since the test item was non-severely toxic and freely soluble, the highest dose-level was 10 mM, according to the criteria specified in the international regulations.

Experiments without S9 mix:
The selected treatment-levels were:
• 0.313, 0.625, 1.25, 2.5, 5 and 10 mM, for the first experiment,
• 0.625, 1.25, 2.5, 5, 7.5 and 10 mM, for the second experiment.

Cytotoxicity:
A moderate toxicity was noted at 10 mM in the first experiment, as shown by 47% decrease in adjusted RSG and 35% decrease in adjusted RTG (tables 3 and 5).
Mutagenicity:
The test item did not induce any noteworthy increase in the mutation frequency, following the 3-hour treatment (tables 2 and 4).

Experiments with S9 mix:

The selected treatment-levels were:
• 0.313, 0.625, 1.25, 2.5, 5 and 10 mM, for the first experiment,
• 0.625, 1.25, 2.5, 5, 7.5 and 10 mM, for the second experiment.

Cytotoxicity:
A slight to marked toxicity was noted at all dose-levels in the first experiment, as shown by 27-76% decrease in adjusted RSG and 37-77% decrease in adjusted RTG (table 7).
A moderate toxicity was noted at 10 mM in the second experiment, as shown by 57% decrease in adjusted RSG and 68% decrease in adjusted RTG (table 9).
Mutagenicity:
Noteworthy dose-related increases in the mutation frequency (up to 3.9-fold the vehicle control value) were observed following the 3-hour treatment in both experiments (tables 6 and 8).

Any other information on results incl. tables

Table 1: Preliminary toxicity test

 

 

 

 

 

Doses mM

Post-treat. cell count*

Day 0

 

Adj. Conc.*

Cell count*

Day 1

 

Adj. Conc.*

Cell count*

Day 2

Cell count

factor

 

 

SG

Adj. RSG

%

 

Empty wells

 

 

CE2

 

RCE2

%

Adj. RTG

%

 

 

 

 

 

 

 

 

 

 

36

 

 

 

 

0

3.1

2.0

6.4

2.0

8.6

1.0

13.6

100

 

0.5

100

100

 

 

 

 

 

 

 

 

 

 

46

 

 

 

 

0.02

2.9

2.0

5.7

2.0

6.5

0.9

9.1

63

26

0.8

154

97

without

0.2

3.4

2.0

4.8

2.0

9.2

1.1

10.9

90

34

0.6

122

110

S9 mix

1

2.9

2.0

5.2

2.0

8.9

1.0

11.6

82

32

0.7

129

106

(3-hour)

2

3.5

2.0

5.4

2.0

6.3

1.1

8.4

71

10

1.4

266

188

 

5

2.6

2.0

5.4

2.0

6.6

0.9

8.8

55

11

1.4

255

140

 

10

2.0

2.0

4.6

2.0

7.9

0.6

9.1

43

34

0.6

122

53

 

 

 

 

 

 

 

 

 

 

29

 

 

 

 

0

3.8

2.0

5.5

2.0

11.0

1.0

15.0

100

 

0.7

100

100

 

 

 

 

 

 

 

 

 

 

34

 

 

 

 

0.02

2.9

2.0

6.4

2.0

10.5

0.8

16.7

86

23

0.9

128

110

with

0.2

4.2

2.0

5.8

2.0

8.7

1.1

12.6

94

24

0.9

124

117

S9 mix

1

3.9

2.0

6.2

2.0

8.4

1.0

13.0

89

36

0.6

88

78

(3-hour)

2

2.7

2.0

7.8

2.0

6.4

0.7

12.3

59

21

0.9

136

81

 

5

3.2

2.0

5.7

2.0

8.8

0.9

12.4

71

11

1.4

194

137

 

10

2.1

2.0

2.1

2.0

4.9

0.6

2.6

10

13

1.2

179

17

*:x105cells/mL                                                         0: vehiclecontrol(DMSO) Adj. conc.: adjusted cellconcentration

SG: suspension growth

Adj. RSG: adjusted relative suspension growth

Empty wells are counted on a total number of 96 wells CE2: cloning efficiency

RCE2: relative cloning efficiency

Adj. RTG: adjusted relative total growth


 

Table 2: First experiment without S9 mix, 3-hour treatment: mutagenicity results

 

 

Cytotoxicity       Mutation frequency:MF

DosesmM

Adj. RTG

%

 

CE2

Empty wells*

 

LC

 

SC

MF10-6

 

R

 

 

 

81

6

10

 

 

0

100

1.7

73

9

14

66

1.0

 

 

 

74

10

13

 

 

 

 

 

79

7

10

 

 

 

 

 

80

5

11

 

 

0.313

111

2.2

 

 

 

35

0.5

 

 

 

85

5

6

 

 

 

 

 

 

83

 

5

 

8

 

 

0.625

86

2.1

 

 

 

32

0.5

 

 

 

85

5

6

 

 

 

 

 

 

90

 

2

 

4

 

 

1.25

59

1.2

 

 

 

53

0.8

 

 

 

78

11

7

 

 

 

 

 

 

79

 

6

 

12

 

 

2.5

123

2.4

 

 

 

35

0.5

 

 

 

83

5

8

 

 

 

 

 

 

77

 

8

 

11

 

 

5

107

2.2

 

 

 

46

0.7

 

 

 

80

6

11

 

 

 

 

 

 

65

 

10

 

21

 

 

10

65

2.1

 

 

 

91

1.4

 

 

 

67

6

23

 

 

 

 

 

 

45

 

17

 

36

 

 

MMS

61

1.2

 

 

 

371

5.6

25 µg/mL

 

 

35

19

45

 

 

0: vehicle control (DMSO)

MMS:methyl methanes sulfonate   LC: large colonies CE2:cloning efficiency                                              SC:small colonies Adj. RTG: adjusted relative totalgrowth

R: ratio between Mutation Frequency of treated cells/Mutation Frequency ofcontrol cells

*: empty wells are counted on a total number of 96 wells


 

 

 

Table 3: First experiment without S9 mix, 3-hour treatment: cytotoxicity

 

 

 

 

Doses mM

Post-treat.

cell count*

Day 0

 

Adj. Conc.*

Cell

count*

Day 1

 

Adj. Conc.*

Cell

count*

Day 2

Cell

count factor

 

 

SG

Adj.

RSG

%

 

Empty wells

RCE2

CE2

%

Adj.

RTG

%

 

 

 

 

 

 

 

 

 

0

 

 

 

 

2.7

2.0

6.1

2.0

6.5

 

9.8

 

6

 

 

 

0

 

 

 

 

 

1.0

 

100

 

1.7

100

100

 

2.7

2.0

5.9

2.0

6.9

 

10.1

 

11

 

 

 

 

 

 

 

 

 

 

 

 

9

 

 

 

 

3.2

2.0

4.1

2.0

8.8

 

9.0

 

2

 

 

 

0.313

 

 

 

 

 

1.1

 

86

 

2.2

129

111

 

2.7

2.0

4.0

2.0

6.7

 

6.7

 

4

 

 

 

 

2.6

2.0

4.0

2.0

7.7

 

7.7

 

3

 

 

 

0.625

 

 

 

 

 

1.0

 

70

 

2.1

123

86

 

2.5

2.0

3.7

2.0

7.5

 

6.9

 

4

 

 

 

 

2.8

2.0

3.9

2.0

7.2

 

6.9

 

8

 

 

 

1.25

 

 

 

 

 

1.0

 

79

 

1.2

74

59

 

2.4

2.0

4.9

2.0

7.7

 

9.4

 

18

 

 

 

 

2.8

2.0

4.2

2.0

8.2

 

8.6

 

3

 

 

 

2.5

 

 

 

 

 

1.0

 

86

 

2.4

144

123

 

2.6

2.0

4.5

2.0

7.6

 

8.5

 

1

 

 

 

 

2.8

2.0

4.5

2.0

7.7

 

8.5

 

0

 

 

 

5

 

 

 

 

 

1.0

 

84

 

2.2

129

107

 

2.8

2.0

4.0

2.0

7.6

 

7.6

 

6

 

 

 

 

2.1

2.0

3.6

2.0

6.9

 

6.3

 

4

 

 

 

10

 

 

 

 

 

0.8

 

53

 

2.1

123

65

 

2.0

2.0

5.1

2.0

5.9

 

7.4

 

3

 

 

 

 

2.8

2.0

3.3

2.0

9.5

 

7.9

 

11

 

 

 

MMS

 

 

 

 

 

1.0

 

87

 

1.2

70

61

25 µg/mL

2.6

2.0

5.3

2.0

7.0

 

9.2

 

18

 

 

 

*: x105cells/mL                                                                               0:vehiclecontrol(DMSO)

Adj. conc.: adjustedcellconcentration                                              MMS:methylmethanesulfonate SG:suspensiongrowth

Adj. RSG: adjusted relative suspension growth

Empty wells are counted on a total number of 96 wells CE2: cloning efficiency

RCE2: relative cloning efficiency

Adj. RTG: adjusted relative total growth


 

 

 

Table 4: Second experiment without S9 mix, 3-hour treatment: mutagenicity results

 

 

Cytotoxicity         Mutation frequency:MF

Doses mM

Adj. RTG

 

CE2

Empty wells*

 

LC

 

SC

MF10-6

 

R

 

 

 

72

11

14

 

 

0

100

1.1

80

5

12

87

1.0

 

 

 

84

2

10

 

 

 

 

 

80

3

13

 

 

 

 

 

76

7

13

 

 

0.625

208

1.9

 

 

 

65

0.7

 

 

 

74

5

17

 

 

 

 

 

 

72

 

6

 

18

 

 

1.25

215

2.2

 

 

 

55

0.6

 

 

 

79

3

14

 

 

 

 

 

 

73

 

8

 

15

 

 

2.5

312

3.3

 

 

 

40

0.5

 

 

 

75

4

17

 

 

 

 

 

 

75

 

7

 

14

 

 

5

179

2.3

 

 

 

50

0.6

 

 

 

78

2

16

 

 

 

 

 

 

74

 

5

 

17

 

 

7.5

148

1.8

 

 

 

87

1.0

 

 

 

65

10

21

 

 

 

 

 

 

65

 

6

 

26

 

 

10

141

2.2

 

 

 

86

1.0

 

 

 

67

7

23

 

 

 

 

 

 

56

 

8

 

35

 

 

MMS

83

1.0

 

 

 

397

4.6

25 µg/mL

 

 

31

21

47

 

 

0: vehicle control (DMSO)

MMS:methylmethanesulfonate           LC:large colonies CE2:cloningefficiency           SC:smallcoloniesAdj. RTG: adjusted relative totalgrowth

R: ratio between Mutation Frequency of treated cells/MutationFrequency ofcontrol cells

*: empty wells are counted on a total number of 96 wells


 

 

 

Table 5: Second experiment without S9 mix, 3-hour treatment: cytotoxicity

 

 

 

Doses mM

Post-treat.

cell count*

Day 0

 

Adj. Conc.*

Cell

count*

Day 1

 

Adj. Conc.*

Cell

count*

Day 2

Cell

count factor

 

 

SG

Adj.

RSG

%

 

Empty wells

RCE2

CE2

%

Adj.

RTG

%

 

 

 

 

 

 

 

 

 

3

 

 

 

 

4.2

2.0

5.5

2.0

7.5

 

10.2

 

20

 

 

 

0

 

 

 

 

 

1.0

 

100

 

1.1

100

100

 

3.3

2.0

8.4

2.0

7.4

 

15.3

 

4

 

 

 

 

 

 

 

 

 

 

 

 

37

 

 

 

 

3.5

2.0

7.9

2.0

8.5

 

16.7

 

6

 

 

 

0.625

 

 

 

 

 

1.0

 

122

 

1.9

171

208

 

3.8

2.0

7.3

2.0

8.4

 

15.1

 

3

 

 

 

 

3.2

2.0

7.4

2.0

10.3

 

19.1

 

4

 

 

 

1.25

 

 

 

 

 

0.9

 

111

 

2.2

193

215

 

3.5

2.0

6.5

2.0

8.0

 

12.9

 

2

 

 

 

 

3.5

2.0

7.4

2.0

8.1

 

15.0

 

1

 

 

 

2.5

 

 

 

 

 

0.9

 

106

 

3.3

293

312

 

2.9

2.0

8.3

2.0

8.1

 

16.7

 

0

 

 

 

 

2.7

2.0

7.1

2.0

8.0

 

14.2

 

1

 

 

 

5

 

 

 

 

 

0.8

 

88

 

2.3

204

179

 

3.4

2.0

7.1

2.0

7.6

 

13.5

 

4

 

 

 

 

3.4

2.0

5.4

2.0

8.7

 

11.7

 

7

 

 

 

7.5

 

 

 

 

 

0.8

 

90

 

1.8

165

148

 

2.7

2.0

8.8

2.0

7.4

 

16.3

 

3

 

 

 

 

3.4

2.0

5.4

2.0

7.5

 

10.0

 

2

 

 

 

10

 

 

 

 

 

0.8

 

73

 

2.2

193

141

 

2.5

2.0

6.6

2.0

8.3

 

13.6

 

4

 

 

 

 

3.1

2.0

6.4

2.0

8.7

 

13.8

 

12

 

 

 

MMS

 

 

 

 

 

0.9

 

93

 

1.0

89

83

25 µg/mL

4.0

2.0

5.4

2.0

8.5

 

11.4

 

27

 

 

 

*: x105cells/mL                                                                           0:vehiclecontrol(DMSO)

Adj. conc.: adjustedcellconcentration                                          MMS: methylmethane sulfonate SG:suspensiongrowth

Adj. RSG: adjusted relative suspension growth

Emptywellsarecountedonatotalnumberof96wells CE2:cloningefficiency

RCE2: relative cloning efficiency

Adj. RTG: adjusted relative total growth


 

 

 

Table 6: First experiment with S9 mix, 3-hour treatment: mutagenicity results

 

 

 

Cytotoxicity          Mutation frequency:MF

 

DosesmM

 

Adj. RTG

 

CE2

 

Empty wells*

 

LC

 

SC

MF10-6

 

R

 

 

 

81

7

8

 

 

0

100

1.5

81

5

10

59

1.0

 

 

 

82

8

6

 

 

 

 

 

76

3

17

 

 

 

 

 

69

6

21

 

 

0.313

57

1.3

 

 

 

121

2.0

 

 

 

72

5

19

 

 

 

 

 

 

65

 

12

 

19

 

 

0.625

63

1.3

 

 

 

150

2.5

 

 

 

64

11

21

 

 

 

 

 

 

71

 

9

 

16

 

 

1.25

41

1.4

 

 

 

158

2.7

 

 

 

54

11

32

 

 

 

 

 

 

73

 

11

 

12

 

 

2.5

34

1.2

 

 

 

168

2.8

 

 

 

55

16

24

 

 

 

 

 

 

71

 

9

 

17

 

 

5

50

1.2

 

 

 

165

2.8

 

 

 

56

13

28

 

 

 

 

 

 

43

 

18

 

39

 

 

10

23

1.5

 

 

 

232

3.9

 

 

 

52

13

35

 

 

 

 

 

 

15

 

28

 

70

 

 

CPA

26

0.9

 

 

 

1003

16.9

3 µg/mL

 

 

17

24

66

 

 

0: vehicle control (DMSO)

CPA:cyclophosphamide                        LC: large colonies

CE2:cloningefficiency                           SC:small colonies Adj. RTG: adjusted relative totalgrowth

R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells

*: empty wells are counted on a total number of 96 wells


 

 

 

Table 7: First experiment with S9 mix, 3-hour treatment: cytotoxicity

 

 

 

 

Doses mM

Post-treat.

cell count*

Day 0

 

Adj. Conc.*

Cell

count*

Day 1

 

Adj. Conc.*

Cell

count*

Day 2

Cell

count factor

 

 

SG

Adj.

RSG

%

 

Empty wells

RCE2

CE2

%

Adj.

RTG

%

 

 

 

 

 

 

 

 

 

2

 

 

 

 

3.1

2.0

6.6

2.0

11.1

 

18.3

 

16

 

 

 

0

 

 

 

 

 

1.0

 

100

 

1.5

100

100

 

2.6

2.0

7.3

2.0

11.0

 

20.1

 

6

 

 

 

 

 

 

 

 

 

 

 

 

9

 

 

 

 

3.0

2.0

7.4

2.0

7.5

 

13.8

 

24

 

 

 

0.313

 

 

 

 

 

1.0

 

69

 

1.3

83

57

 

2.7

2.0

5.7

2.0

8.8

 

12.6

 

1

 

 

 

 

3.3

2.0

6.5

2.0

7.5

 

12.0

 

19

 

 

 

0.625

 

 

 

 

 

1.1

 

73

 

1.3

86

63

 

3.1

2.0

6.1

2.0

8.5

 

12.9

 

4

 

 

 

 

2.8

2.0

5.3

2.0

8.2

 

10.8

 

20

 

 

 

1.25

 

 

 

 

 

0.8

 

46

 

1.4

88

41

 

2.0

2.0

5.4

2.0

7.5

 

10.4

 

2

 

 

 

 

2.6

2.0

6.6

2.0

7.5

 

12.3

 

26

 

 

 

2.5

 

 

 

 

 

0.8

 

43

 

1.2

78

34

 

2.0

2.0

5.1

2.0

6.4

 

8.1

 

2

 

 

 

 

2.5

2.0

8.1

2.0

8.8

 

17.8

 

24

 

 

 

5

 

 

 

 

 

0.8

 

62

 

1.2

81

50

 

2.1

2.0

6.1

2.0

7.7

 

11.7

 

2

 

 

 

 

2.0

2.0

3.5

2.0

6.5

 

5.6

 

15

 

 

 

10

 

 

 

 

 

0.6

 

24

 

1.5

99

23

 

1.6

1.6

3.6

2.0

7.8

 

8.6

 

2

 

 

 

 

2.7

2.0

5.1

2.0

6.8

 

8.6

 

29

 

 

 

CPA

 

 

 

 

 

0.9

 

44

 

0.9

58

26

3 µg/mL

2.6

2.0

5.2

2.0

7.4

 

9.5

 

17

 

 

 

*: x105cells/mL                                                                            0:vehiclecontrol(DMSO)

Adj. conc.: adjustedcellconcentration                                           CPA:cyclophosphamideSG:suspensiongrowth

Adj. RSG: adjusted relative suspension growth

Emptywellsarecountedonatotalnumberof96wells CE2:cloningefficiency

RCE2: relative cloning efficiency

Adj. RTG: adjusted relative total growth


 

 

 

Table 8: Second experiment with S9 mix, 3-hour treatment: mutagenicity results

 

 

Cytotoxicity         Mutation frequency:MF

Doses mM

Adj. RTG

 

CE2

Empy wells*

 

LC

 

SC

MF10-6

 

R

 

 

 

73

7

16

 

 

0

100

0.7

71

8

19

179

1.0

 

 

 

75

11

10

 

 

 

 

 

76

7

13

 

 

 

 

 

73

7

17

 

 

0.625

89

0.6

 

 

 

225

1.3

 

 

 

71

6

19

 

 

 

 

 

 

76

 

5

 

16

 

 

1.25

86

0.7

 

 

 

179

1.0

 

 

 

73

6

18

 

 

 

 

 

 

78

 

3

 

16

 

 

2.5

80

0.7

 

 

 

192

1.1

 

 

 

70

8

20

 

 

 

 

 

 

66

 

8

 

24

 

 

5

96

0.7

 

 

 

269

1.5

 

 

 

68

6

24

 

 

 

 

 

 

55

 

13

 

28

 

 

7.5

86

0.6

 

 

 

473

2.6

 

 

 

56

13

30

 

 

 

 

 

 

47

 

16

 

38

 

 

10

32

0.5

 

 

 

605

3.4

 

 

 

52

12

34

 

 

 

 

 

 

20

 

19

 

64

 

 

CPA

21

0.3

 

 

 

2439

13.6

3 µg/mL

 

 

27

17

59

 

 

0: vehicle control (DMSO)

CPA:cyclophosphamide                       LC: largecolonies

CE2:cloningefficiency                          SC:smallcolonies Adj. RTG: adjusted relativetotal growth

R: ratio between Mutation Frequency of treated cells/MutationFrequency ofcontrol  cells

*: empty wells are counted on a total number of96 wells


 

 

 

Table 9: Second experiment with S9 mix, 3-hour treatment: cytotoxicity

 

 

 

DosesmM

Post-treat.

cell count*

Day 0

 

Adj. Conc.*

Cell

count*

Day 1

 

Adj. Conc.*

Cell

count*

Day 2

Cell

count factor

 

 

SG

Adj.

RSG

%

 

Empty wells

RCE2

CE2

%

Adj.

RTG

%

 

 

 

 

 

 

 

 

 

32

 

 

 

 

3.7

2.0

6.8

2.0

9.2

 

15.5

 

35

 

 

 

0

 

 

 

 

 

1.0

 

100

 

0.7

100

100

 

3.1

2.0

6.3

2.0

8.5

 

13.4

 

28

 

 

 

 

 

 

 

 

 

 

 

 

23

 

 

 

 

2.9

2.0

7.6

2.0

8.8

 

16.5

 

41

 

 

 

0.625

 

 

 

 

 

0.9

 

102

 

0.6

87

89

 

3.0

2.0

8.6

2.0

8.2

 

17.5

 

28

 

 

 

 

3.6

2.0

6.1

2.0

9.3

 

14.1

 

39

 

 

 

1.25

 

 

 

 

 

1.1

 

90

 

0.7

96

86

 

3.7

2.0

5.4

2.0

7.7

 

10.3

 

23

 

 

 

 

3.1

2.0

6.0

2.0

9.1

 

13.6

 

32

 

 

 

2.5

 

 

 

 

 

1.0

 

87

 

0.7

92

80

 

3.4

2.0

7.4

2.0

7.0

 

13.0

 

33

 

 

 

 

3.3

2.0

7.7

2.0

8.0

 

15.3

 

32

 

 

 

5

 

 

 

 

 

1.0

 

106

 

0.7

90

96

 

3.5

2.0

7.3

2.0

8.4

 

15.3

 

34

 

 

 

 

2.6

2.0

8.1

2.0

9.2

 

18.5

 

37

 

 

 

7.5

 

 

 

 

 

0.8

 

109

 

0.6

79

86

 

3.2

2.0

7.1

2.0

10.5

 

18.6

 

39

 

 

 

 

2.7

2.0

2.8

2.0

10.1

 

7.1

 

33

 

 

 

10

 

 

 

 

 

0.8

 

43

 

0.5

74

32

 

2.5

2.0

4.0

2.0

9.2

 

9.1

 

47

 

 

 

 

3.5

2.0

5.2

2.0

5.6

 

7.1

 

63

 

 

 

CPA

 

 

 

 

 

1.1

 

55

 

0.3

39

21

3 µg/mL

4.1

2.0

4.4

2.0

6.4

 

6.9

 

58

 

 

 

*: x105cells/mL                                                                          0: vehicle control(DMSO)

Adj. conc.: adjustedcellconcentration                                        CPA:cyclophosphamide SG: suspensiongrowth

Adj. RSG: adjusted relativesuspension growth

Empty wells are counted on a total number of 96 wells CE2: cloning efficiency

RCE2: relative cloning efficiency

Adj. RTG: adjusted relativetotal growth

 

Applicant's summary and conclusion

Conclusions:
In the study report, the test item (Phenylmethyl pyrazolone) induced significant increases in the mutant frequencies after the 3 hours treatment in presence of S9 mix. Under the experimental conditions of the study, the test item was mutagenic in mammalian cells L5178Y (mouse lymphoma cells).
Executive summary:

This GLP-compliant study report was assessed to determine potential mutagenic effect of the registered item (phenylmethyl pyrazolone) on mammalian cell line (L5178Y mouse lymphoma cells), precisely to induce mutation at the thymidine kinase locus. This study was according to OECD Guideline 476 method for In vitro Mammalian Cell Gene Mutation Test.

When tested up to 10 mM, there were no increases in mutant frequency in the absence of S9 mix. In the presence of S9 mix, dose-related increases in mutant frequency were observed at all concentrations tested in experiment 1 and at the three highest concentrations tested in experiment 2 (up to 3.9-fold the mean control value). It should be noted that in experiment 2 no excessive cytotoxicity was observed. Mutation frequencies in solvent negative controls remained within normal ranges for all experiment. However, in experiment 2 in the presence of S9 mix, the mutant frequency observed for control cultures

was high (179), and above the upper limit of the laboratory historical negative control range (56 – 146). Treatment with positive controls CPA and MMS yielded distinct increases in mutant frequency.

Under the conditions of this study, phenylmethyl pyrazolone was considered to be genotoxic (mutagenic or clastogenic) in the mouse lymphoma assay (tk locus) in the presence of metabolic activation