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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Mouse Lymphoma Assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 07 June 2016 and 28 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Qualifier:
according to
Guideline:
other: Japanese Guidline: Kanpoan No. 287 - - Environment Protection Agency
Qualifier:
according to
Guideline:
other: Japanese guidline: Eisei No. 127 - - Ministry of Health and Welfare
Qualifier:
according to
Guideline:
other: Japanese guidline: Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mouse lymphoma assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Physical State / Appearance: Amber colored viscous liquid
Batch: X-19574-00-00
Purity: 100% (UVCB)
Expiry Date: 01 June 2017
Storage Conditions: Room temperature, in the dark

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: Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Cell cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours
- Methods for maintenance in cell culture if applicable: The stocks of cells are stored in liquid nitrogen at approximately -196°C.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at with 5% CO2 in air. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Preliminary toxicity test:
The dose range used in the preliminary toxicity test was set using available information from the corresponding Human Lymphocyte Chromosome Aberration Test study number MF09KY, where high levels of toxicity and precipitate were observed. The dose range was therefore set at 0.61 to 156.25 µg/mL for all three of the exposure groups (4-hour exposure with and without S9 and 24-hour exposure without S9).

Main test:
The dose range of test item used in the main test was selected following the results of the preliminary toxicity test. The test item exhibited marked dose-related toxicity to the cells in each of the three exposure groups of the preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Group:
4-hour without S9: 0.25, 0.5, 1, 2, 4 µg/mL
4-hour with S9 (2%): 1, 2, 4, 8, 10, 12 µg/mL
24-hour without S9: 0.25, 0.5, 1, 2, 4, 6 µg/mL
Vehicle / solvent:
Following solubility checks performed in-house on the corresponding Human Lymphocyte Chromosome Aberration test study number MF09KY the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
absence of S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
presence of S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium, in suspension
- Cell density at seeding (if applicable):
preliminary toxicty tests 5 x 10^5 cells/ml for 4-hour exposure and 1.5 x 10^5 cells/ml for 24-hour exposure. All groups were serially diluted to 2 x 10^5 calls/ml after the exposure period
Main test: 1 x 10^-6 cells/ml for 4-hour exposure and 0.3 x 10^6 cells/ml for 24-hour exposure. All groups were serially diluted to 2 x 10^5 cells.ml after the exposure period. Before plating cells were diluted to 1 x 10^4 cells/ml (or 10 cells/ml for viability assessment)

DURATION
- Preincubation period: Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment.
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-12 days

SELECTION AGENT (mutation assays): 4 µg/mL 5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
Data evaluation:
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
To define positive and negative responses the Global Evaluation Factor (GEF) of 126 x 10^-6 is used. This is the increase in mutation frequency (MF) above the concurrent control.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.


Statistics:
Calculation of Percentage Relative Suspension Growth (%RSG):
The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)
Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

Calculation of Day 2 Viability (%V):
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.
P(0) = number of negative wells / total wells plated
%V = (-ln P(0) x 100) / number of cells per well

Calculation of Relative Total Growth (RTG):
For each culture, the relative cloning efficiency, RCE, was calculated:
RCE = %V / mean solvent control %V
Finally, for each culture RTG is calculated:
RTG = (RCE x RSG)/100%

Calculation of Mutation Frequency (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.


Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
6 ,8, and 10 µg/mL in 4-hr exposure(-S9), 14 and 16 µg/mL (+S9) and 8 and 10 µg/mL in 24-hr exposure were not plated due to excessive toxicity. The dose level 12 µg/mL in the 4-hr exposure (+S9) was plated but later discarded due to excessive toxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Toxicity:
There was evidence of high levels of toxicity following exposure to the test item in all three of the exposure groups. There was no evidence of reductions in viability (%V) in either of the three exposure groups, therefore indicating that residual toxicity had not occurred in these exposure groups. Optimum levels of toxicity were achieved in the 4-hour exposure group in the presence of metabolic activation only. In both of the 4-hour and 24-hour exposures in the absence of metabolic activation the toxicity curve was very steep and optimum toxicity could not be achieved despite using 1-2 µg/mL dose increments. Increments less than this are not achievable and therefore it was considered that the test item was adequately tested to meet the guidelines. Acceptable levels of toxicity were seen with both positive control substances.

Controls:
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

Mutagenicity:
The test item did not induce any toxicologically significant increases in the mutant frequency x 10^-6 per viable cell in either of the three exposure groups. The GEF value of the test item dose levels were not exceeded in any of the three exposure groups including those exhibiting excessive toxicity.

Precipitation:
A precipitate of the test item was observed at and above 12 µg/mL in the 4-hour exposure in the presence of metabolic activation, however this dose level was not included in the analysis due to excessive toxicity.

Any other information on results incl. tables

Preliminary cytotoxicity test:

In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The toxicity curve was very steep in all exposure groups. A precipitate of the test item was observed at and above 39.06 µg/mL in the 4-hour exposure in the absence of metabolic activation, 19.53 in the 4-hour exposure in the presence of metabolic activation and 156.25 µg/mL in the 24-hour exposure group in the absence of metabolic activation. In the subsequent mutagenicity experiments the maximum dose was limited by test item induced toxicity.

The dose range of the test item used in the preliminary toxicity test was 0.61 to 156.25 µg/mL.  The results for the Relative Suspension Growth (%RSG) were as follows:

Dose

(mg/mL)

% RSG (-S9)

4-Hour Exposure

% RSG (+S9)

4-Hour Exposure

% RSG (-S9)

24-Hour Exposure

0

100

100

100

0.61

100

112

95

1.22

103

106

101

2.44

91

97

92

4.88

58

98

55

9.77

0

67

0

19.53

0

0

0

39.06

0

0

0

78.13

0

0

0

156.25

0

0

0

A summary of the results in the main test is shown in the tables below

Treatment

(µg/mL)

4-hours-S-9

Treatment

(µg/mL)

4-hours+S-9

 

%RSG

RTG

MF§

 

%RSG

RTG

MF§

0

 

100

1.00

170.49

 

0

 

100

1.00

138.89

 

0.25

 

95

1.03

131.16

 

1

 

100

0.94

133.57

 

0.5

 

89

1.43

120.42

 

2

 

95

0.87

141.48

 

1

 

93

1.06

159.76

 

4

 

103

0.98

145.83

 

2

 

91

1.21

131.94

 

8

 

50

0.58

110.86

 

4

 

65

0.95

121.27

 

10

 

17

0.13

118.40

 

6

Ø

1

 

 

 

12

X

6

0.04

144.39

 

8

Ø

0

 

 

 

14

Ø

1

 

 

 

10

Ø

0

 

 

 

16

Ø

0

 

 

 

MF threshold for a positive response = 296.49

MF threshold for a positive response = 264.89

EMS

 

 

 

 

 

CP

 

 

 

 

 

400

 

55

0.51

697.79

 

1.5

 

61

0.34

992.90

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment

(µg/mL)

24-hours-S-9

 

%RSG

RTG

MF§

0

 

100

1.00

149.24

 

0.25

 

82

0.84

170.01

 

0.5

 

96

1.04

171.70

 

1

 

104

1.08

153.26

 

2

 

100

1.10

144.75

 

4

 

76

0.92

122.80

 

6

 

24

0.54

133.46

 

8

Ø

0

 

 

 

10

Ø

0

 

 

 

MF threshold for a positive response = 275.24

EMS

 

 

 

 

 

150

 

48

0.39

1104.43

 

A summary of the analysis for mutagenicity test of each exposure group is displayed in the tables below

(-S9) 4 -hour exposure

Treatment

(µg/mL)

 

SG

%RSG

%V

RTG

MF§

0

 

11.34

100

86.93

1.00

170.49

0.25

 

10.36

95

95.38

1.03

131.16

0.5

 

10.68

89

138.63

1.43

120.42

1

 

11.16

93

99.97

1.06

159.76

2

 

10.85

91

115.65

1.21

131.94

4

 

8.53

65

125.83

0.95

121.27

6

Ø

0.57

1

 

 

 

8

Ø

0.08

0

 

 

 

10

Ø

0.03

0

 

 

 

Positive Control EMS

Treatment

(µg/mL)

Survival

%RSG

%V

RTG

MF§

400

 

6.90

55

81.00

0.51

697.79

GEF = 126, therefore MF threshold for positive response = 296.49

(+S9) 4 -hour exposure

Treatment

(µg/mL)

 

SG

%RSG

%V

RTG

MF§

0

 

10.49

100

100.45

1.00

138.89

1

 

11.56

100

93.66

0.94

133.57

2

 

10.87

95

91.99

0.87

141.48

4

 

12.00

103

96.26

0.98

145.83

8

 

6.40

50

114.35

0.58

110.86

10

 

2.58

17

94.51

0.13

118.40

12

X

1.77

6

62.06

0.04

144.39

14

Ø

0.31

1

 

 

 

16

Ø

0.23

0

 

 

 

Positive Control CP

Treatment

(µg/mL)

SG

%RSG

%V

RTG

MF§

1.5

 

7.19

61

56.12

0.34

992.90

GEF = 126, therefore MF threshold for a positive response = 264.89

(-S9) 24 -hour exposure

Treatment

(µg/mL)

 

SG

%RSG

%V

RTG

MF§

0

 

64.49

100

70.10

1.00

149.24

0.25

 

56.44

82

66.78

0.84

170.01

0.5

 

62.22

96

74.81

1.04

171.70

1

 

69.26

104

69.84

1.08

153.26

2

 

63.12

100

78.43

1.10

144.75

4

 

55.32

76

74.24

0.92

122.80

6

 

31.41

24

76.59

0.54

133.46

8

Ø

0.54

0

 

 

 

10

Ø

0.00

0

 

 

 

Positive Control EMS

Treatment

(µg/mL)

SG

%RSG

%V

RTG

MF§

150

 

37.78

48

44.72

0.39

1104.43

GEF = 126, therefore MF threshold for a positive response = 275.24

A summary of mutation frequencies for each exposure group is displayed in the following tables:

(-S9) 4 -hour exposure

Treatment

(µg/mL)

 

Small colonies

Large Colonies

Proportion

small

colony

mutants

 

Viable

Mutants

 

Mutants

 

 

 

Yv

Nv

Ym

Nm

MF§

Ym

Nm

MF§

 

0

 

135

768

712

768

43.5

627

768

116.7

0.28

0.25

 

57

384

360

384

33.8

323

384

90.7

0.28

0.5

 

24

384

356

384

27.3

303

384

85.4

0.26

1

 

52

384

349

384

47.8

314

384

100.7

0.33

2

 

38

384

360

384

27.9

307

384

96.8

0.24

4

 

31

384

354

384

32.3

313

384

81.2

0.30

400 EMS

 

76

384

278

384

199.4

230

384

316.4

0.41

(+S9) 4 -hour exposure

Treatment

(µg/mL)

 

Small colonies

Large Colonies

Proportion

small

colony

mutants

 

Viable

Mutants

 

Mutants

 

 

 

Yv

Nv

Ym

Nm

MF§

Ym

Nm

MF§

 

0

 

103

768

705

768

42.6

644

768

87.6

0.34

1

 

59

384

349

384

51.0

334

384

74.5

0.41

2

 

61

384

364

384

29.1

316

384

105.9

0.23

4

 

56

384

348

384

51.1

326

384

85.0

0.38

8

 

39

384

360

384

28.2

322

384

77.0

0.28

10

 

58

384

359

384

35.6

332

384

77.0

0.32

12

 

111

384

367

384

36.5

338

384

102.8

0.27

1.5 CP

 

125

384

210

384

537.8

300

384

220.0

0.67

(-S9) 24 -hour exposure

Treatment

(µg/mL)

 

Small colonies

Large Colonies

Proportion

small

colony

mutants

 

Viable

Mutants

 

Mutants

 

 

 

Yv

Nv

Ym

Nm

MF§

Ym

Nm

MF§

 

0

 

189

768

709

768

57.0

682

768

84.7

0.41

0.25

 

101

384

364

384

40.1

326

384

122.6

0.26

0.5

 

86

384

351

384

60.1

330

384

101.3

0.38

1

 

95

384

358

384

50.2

336

384

95.6

0.35

2

 

80

384

361

384

39.4

329

384

98.5

0.29

4

 

87

384

363

384

37.9

341

384

80.0

0.33

6

 

83

384

354

384

53.1

343

384

73.7

0.42

150 EMS

 

157

384

297

384

287.2

230

384

573.1

0.36

KEY TO TABLES 1 TO 10

$       =       Cell counts (x10^5 cells/mL).  Set up on previous day to 2 x 10^5 cells/mL unless otherwise stated in parenthesis.

%RSG       =       Relative Suspension Growth

RTG       =       Relative Total Growth

%V       =       Viability Day 2

§ or #       =       Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis

A,B       =       Replicate cultures

CP       =       Cyclophosphamide

EMS       =       Ethylmethanesulphonate

MF§       =       5-TFT resistant mutants/10^6 viable cells 2 days after treatment

NP       =       Not plated, surplus to requirments

Ø       =       Not plated for viability or 5-TFT resistance

Nv       =       Number of wells scored, viability plates

Yv       =       Number of wells without colonies, viability plates

Ym       =       Number of wells without colonies, mutation plates

Nm       =       Number of wells scored, mutation plates

X       =       Excluded due to excessive toxicity

Applicant's summary and conclusion

Conclusions:
The test item, X-19574 Amines, C16-18 and C18-unsatd. alkyl, O,O-di-Bu phosphorothioates, CASRN 97808-07-6 did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic in this assay.
Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.  The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 28 July 2015, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods

One main Mutagenicity Test was performed.  In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 8 dose levels in duplicate, together with vehicle (DMSO media), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test.  The test item exhibited marked dose-related toxicity to the cells in each of the three exposure groups of the preliminary toxicity test.  

Results

The maximum dose level used was limited by test item induced toxicity.  A precipitate of the test item was noted at and above 12 µg/mL in the 4-hour exposure in the presence of metabolic activation.  The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus.  The 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.

The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.  The results were limited by the high levels of toxicity. During the main experiment 1-2 µg/mL test item dose increments were used to try to achieve optimum toxicity, however the toxicity curve observed was very steep, particularly in the absence of

metabolic activation.  Increments less than this are not achievable and therefore it was considered that the test item was adequately tested to meet the guidelines.

Conclusion

The test item X-19574 Amines, C16-18 and C18-unsatd. alkyl, O,O-di-Bu phosphorothioates, CASRN 97808-07-6, did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic in this assay.