Amines, C16-18-(even numbered, saturated and unsaturated) alkyl, O,O-di-Bu phosphorothioates

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Guidline: Kanpoan No. 287 - - Environment Protection Agency

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Japanese guidline: Eisei No. 127 - - Ministry of Health and Welfare

Test guideline 6

Qualifier:

according to guideline

Guideline:

other: Japanese guidline: Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mouse lymphoma assay

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C16-18-(even numbered, saturated and unsaturated) alkyl, O,O-di-Bu phosphorothioates

Molecular formula:

Not applicable - UVCB

IUPAC Name:

Amines, C16-18-(even numbered, saturated and unsaturated) alkyl, O,O-di-Bu phosphorothioates

Constituent 2

Reference substance name:

C16-18-(even numbered, saturated and unsaturated)-alkylamines

EC Number:

627-034-4

Cas Number:

1213789-63-9

Molecular formula:

R-NH2 R = alkyl mainly C16-18-(even numbered, saturated and unsaturated)-alkyl

IUPAC Name:

C16-18-(even numbered, saturated and unsaturated)-alkylamines

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.

Controls

Controls 1

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

Controls 2

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.