Reaction mass of 2-dodecylhexadecyl methacrylate and 2-Tetradecyloctadecyl methacrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 April- 12 May 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

2-Dodecylhexadecyl methacrylate

Cas Number:

536972-42-6

IUPAC Name:

2-Dodecylhexadecyl methacrylate

Constituent 2

Reference substance name:

2-Tetradecyloctadecyl methacrylate

Cas Number:

1046154-63-5

IUPAC Name:

2-Tetradecyloctadecyl methacrylate

impurity 1

Reference substance name:

2-Decyltetradecyl methacrylate

IUPAC Name:

2-Decyltetradecyl methacrylate

impurity 2

Reference substance name:

Dodecyl methacrylate

EC Number:

205-570-6

EC Name:

Dodecyl methacrylate

Cas Number:

142-90-5

Molecular formula:

C16H30O2

IUPAC Name:

dodecyl methacrylate

impurity 3

Reference substance name:

Tetradecyl methacrylate

EC Number:

219-835-9

EC Name:

Tetradecyl methacrylate

Cas Number:

2549-53-3

Molecular formula:

C18H34O2

IUPAC Name:

tetradecyl methacrylate

impurity 4

Reference substance name:

Pentadecyl methacrylate

EC Number:

228-126-3

EC Name:

Pentadecyl methacrylate

Cas Number:

6140-74-5

Molecular formula:

C19H36O2

IUPAC Name:

pentadecyl methacrylate

impurity 5

Reference substance name:

Tridecyl methacrylate

EC Number:

219-671-8

EC Name:

Tridecyl methacrylate

Cas Number:

2495-25-2

Molecular formula:

C17H32O2

IUPAC Name:

tridecyl methacrylate

impurity 6

Reference substance name:

Hexadecyl methacrylate

EC Number:

219-672-3

EC Name:

Hexadecyl methacrylate

Cas Number:

2495-27-4

Molecular formula:

C20H38O2

IUPAC Name:

hexadecyl methacrylate

impurity 7

Reference substance name:

13-Methylheptacosane

IUPAC Name:

13-Methylheptacosane

impurity 8

Reference substance name:

Unknown Impurities

IUPAC Name:

Unknown Impurities

Test material form:

liquid

Specific details on test material used for the study:

The test material Reaction Mass of 2-Propenoic acid, 2-methyl-, 2-
dodecylhexadecyl ester and 2-Propenoic acid, 2- methyl-, 2-tetradecyloctadecyl
ester was stored at room temperature in the dark.
The batch number of the test item was F1-9K038C. The purity of the test item was 93.3%.

Method

Target gene:

thymidine kinase, TK +/- 3.7.2c.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
L5178Y TK+/- 3.7.2c mouse lymphoma cell line were obtained frozen in liquid nitrogen at the University of Sussex, Brighton, UK.

CELL LINE
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in 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 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), were used during the course of the study. All donor horse serum was purchased heat inactivated from the supplier. Master stocks of cells were tested and found to be free of mycoplasma.

CELL CLEANSING
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal Enzyme Fraction was purchased from Moltox and Lot no PB/BNF S9 4127 (Expiry 25 July 2021). The S9 was pre-tested for acceptability by the supplier prior to purchase and was supplied with a relevant “Quality Control & Production Certificate”. The protein content was adjusted to 20 mg/ml prior to use. The S9 mix was prepared by mixing S9 with 100 mM phosphate buffer containing NADP (5 mM), G6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% S9-mix concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.

Test concentrations with justification for top dose:

CONCENTRATIONS IN PRELIMINARY TEST
The concentrations used in the preliminary test were: 0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320 μg/mL. The dose levels were selected based on the excessive precipitate observed in the solubility test. Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments.

CONCENTRATIONS IN MUTAGENICITY TEST
The concentrations used in the mutagenicity test were:
2.5, 5, 10, 20, 40 μg/mL on a 4 hour exposure without S9.
1.25, 2.5, 5, 10, 20 μg/mL on a 4 hour exposure with S9 (2%).
2.5, 5, 10, 20, 40 μg/mL on a 24 hour exposure without S9.

The maximum dose level used was limited by the presence of precipitate effectively reducing exposure of the test item to the cells. A precipitate of test item was observed at and above 40 μg/mL at the end of the exposure period in the absence of metabolic activation and at and above 20 μg/mL at the end of exposure in the presence of metabolic activation.

Vehicle / solvent:

The solvent used was acetone. The test item was soluble in acetone at 500 mg/mL according to solubility checks performed in-house. Therefore, the test item was formulated at 500 mg/ml and dosed at 0.5% to give a maximum achievable dose level of 2500 μg/mL. The concentration was selected as acetone is toxic to L5178Y cells at dose volumes greater than 0.5% of the total culture volume.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

The solvent controls used in the Main Test were Acetone, supplied by Acros Organics, batch number 1882926, purity of 99.97% and with expiry date on 20 November 2020.

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

PRELIMINARY TEST
A preliminary test was conducted to set the test item dose levels for the following mutagenicity experiment. Cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL. The dose range used in the preliminary toxicity test was 1.25 to 320 μg/mL. The dose levels were selected based on the excessive precipitate observed in the solubility test.

MUTAGENICITY TEST
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. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at 6 dose levels of the test item and solvent and positive controls. To each universal was added 2 mL of S9-mix if required, 0.1 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix. The dose levels of test item used were 2.5 to 80 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%). The dose levels of test item used were 1.25 to 40 μg/mL.
iii) 24-hour exposure to the test item without S9-mix. The dose levels of test item used were 2.5 to 80μg/mL.
The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/mL.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for cloning efficiency (%V) in non-selective medium.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the solvent control, and when combined with the cloning efficiency (%V) data, a Relative Total Growth (RTG) value.

OTHER SUPPLEMENTARY INFORMATION
PLATE SCORING
Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutant plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et a.l, 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutant plates. The plates were incubated for approximately two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

Evaluation criteria:

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. Optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Hence, 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.
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, i.e. the mutant frequency of the concurrent solvent control plus 126, which is based on the analysis of the distribution of the solvent control MF data from participating laboratories.
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 the test system.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

ACCEPTABILITY OF THE ASSAY
This mutation assay is considered acceptable as it meets the following acceptability criteria, the current recommendations of the IWGT will be considered (Moore et al., 2002; Moore et al., 2003; Moore et al., 2006; Moore et al., 2007):
1. For non-toxic test items the upper test item concentrations will be 10mM, 2 mg/mL or 2μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCBs) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL. Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point. In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) and/or percentage Relative Suspension Growth (%RSG) to approximately 10 to 20 % of survival.
2. The absolute cloning efficiency (%V) at the time of mutant selection of the solvent controls is 65 to 120 %.
3. The total suspension growth of the solvent control following 4 hour exposure, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24 hour exposure the total suspension growth should be in the range 32 to 180.
4. The in-house historical solvent control mutant frequency is in the range of approximately 50 – 170 x 10-6 cells. Solvent control results should ideally be within this range, although minor errors in cell counting and dilution, or exposure to the metabolic activation system, may cause this to be slightly elevated.
5. Every test should also be evaluated as to whether the positive controls (EMS and CP) meet at least one of the following two acceptance criteria developed by the IWGT workgroup:
• The positive control should demonstrate an absolute increase in total MF, that is, an increase above the spontaneous background MF [an induced MF (IMF)] of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF.
• The positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent untreated/solvent control (a small colony IMF of 150 x 10-6).
6. The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures i.e. the Relative Total Growth (RTG) and percentage Relative Suspension Growth (%RSG) should be greater than approximately 10 % of the concurrent selective control group.
7. A minimum of four analyzed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.

RESULTS OF THE MUTAGENICITY TEST
There was no evidence of marked toxicity following exposure to the test item in all three of the exposure groups, as indicated by the %RSG and RTG values (Tables 1, 2, 3). There was no evidence of any marked reductions in viability (%V), therefore indicating that no residual toxicity occurred in any of the three exposure groups (Tables 1, 2, 3). Acceptable levels of toxicity were seen with both positive control substances (Tables 1, 2, 3). A precipitate of test item was observed at and above 40 μg/mL at the end of the exposure period in the 4-hour and 24-hour exposure in the absence of metabolic activation. In the 4-hour exposure in the presence of metabolic activation a precipitate of the test item was observed at and above 20 μg/mL at the end of exposure. The solvent 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.
The test item did not induce any increases in the mutant frequency at any of the dose levels in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating dose level in all exposure groups, and at least four analysable dose levels in each exposure group, as recommended by the OECD 490 guideline.

Any other information on results incl. tables

Table 1. Cell and 96-Well Plate Counts and Summary Analysis: Mutagenicity Test (-S9) 4-Hour Exposure

Concentration (µg/mL)		Cell counts $			Viability § after day 2 (2 cells per well)				Resistant mutants § after day 2 (2000 cells per well)				SG	Day 0 Factor	%RSG	%V	RTG	MF§
		0h	24h	48h
0	A	8.09	5.95	6.74	76	75	84	71	12	22	14	14	10.56	1	100	72.54	1	131.07
	B	7.08	6.85	6.46	67	76	71	68	17	22	14	18
2.5	A	7.76	6.12	6.59	72	76			22	18			11.03	0.99	103	78.43	1.11	148.93
	B	7.21	7.05	6.81	80	76			17	23
5	A	6.89	7.62	6.03	78	73			18	18			10.62	0.91	92	80.34	1.01	131.22
	B	6.92	6.16	6.3	78	78			17	20
10	A	7.3	6.25	6.67	81	78			22	22			10.7	0.95	97	92.81	1.24	140.22
	B	7.18	6.43	6.83	82	83			24	20
20	A	7.44	5.99	6.09	79	81			24	22			10.83	0.95	97	91.18	1.22	133.55
	B	6.94	6.64	7.63	83	79			19	18
40P	A	7.52	6.54	6.52	85	84			11	19			10.64	0.89	89	90.38	1.11	109.58
	B	5.94	6.5	6.54	82	70			19	20
80P	A	7.1	6.23	7.43	NP	NP			NP	NP			11.45	1.14	124	0	0
	B	6.74	6.68	6.76	NP	NP			NP	NP
Positve Control EMS (µg/mL)
400	A	7.95	5.06	6.05	64	64			62	67			8.58	0.97	79	0.66	0.66	830.83
	B	6.83	5.29	7.2	71	71			59	56

GEF = 126 therefore MF threshold for a positive response = 257.07.

Table 2. Cell and 96-Well Plate Counts and Summary Analysis: Mutagenicity Test (+S9) 4-Hour Exposure

Concentration (µg/mL)		Cell counts $			Viability § after day 2 (2 cells per well)				Resistant mutants § after day 2 (2000 cells per well)				SG	Day 0 Factor	%RSG	%V	RTG	MF§
		0h	24h	48h
0	A	6.26	7.84	8.12	72	78	76	70	16	19	20	21	68.38	1	100	73.38	1	140.39
	B	6.53	6.56	9.7	75	73	71	76	19	18	12	18
1.25	A	5.93	7.03	8.93	83	69			18	29			61.18	0.96	86	79.7	0.93	136.33
	B	6.34	6.26	9.08	76	78			19	9
2.5	A	6.27	6.46	9.54	75	72			25	25			62.36	1	91	76.59	0.95	176.57
	B	6.46	5.25	10.54	81	73			24	17
5	A	7.16	5.46	10.51	81	78			18	14			64.62	1.06	100	90.38	1.23	99.14
	B	6.4	5.34	10.67	79	83			13	18
10	A	7.18	6.21	9.59	78	81			22	19			71.78	1.07	112	85.83	1.31	126.59
	B	6.48	5.94	11.17	76	80			17	17
20P	A	6.62	6.06	9.13	80	79			17	26			61.1	1.01	91	88.05	1.09	151.66
	B	6.35	5.76	10	81	78			26	21
40P	A	6.67	6.07	9.6	NP	NP			NP	NP			65.54	1.02	98	0	0
	B	6.41	5.95	10.44	NP	NP			NP	NP
Positve Control EMS (µg/mL)
400	A	5.58	4.88	10.4	74								45.41	0.81	54	62.96	0.46	978.44
	B	4.84	6.8	7.51	69

GEF = 126 therefore MF threshold for a positive response = 274.81.

Table 3. Cell and 96-Well Plate Counts and Summary Analysis: Mutagenicity Test 24-Hour Exposure

Concentration (µg/mL)		Cell counts $			Viability § after day 2 (2 cells per well)				Resistant mutants § after day 2 (2000 cells per well)				SG	Day 0 Factor	%RSG	%V	RTG	MF§
		0h	24h	48h
0	A	7.95	6.45	6.82	70	72	78	66	14	22	22	20	11.61	1	100	76.29	1	148.81
	B	7.66	6.02	8.08	76	75	87	67	15	20	21	22
1.25	A	8.11	6.54	5.47	72	83			19	24			9.77	0.99	84	88.81	0.97	154.19
	B	7.38	6.47	6.55	85	79			28	21
2.5	A	6.96	7.27	7.25	75	73			22	18			12.02	0.99	102	78.43	1.05	142.67
	B	8.43	5.81	7.45	77	79			19	18
5	A	8.4	6.04	6.85	81	75			18	26			11.62	1.02	102	85.11	1.14	148.98
	B	7.52	6.56	7.91	77	81			23	19
10	A	8.67	6.2	6.68	85	83			26	19			11.53	1.04	103	90.38	1.22	129.25
	B	7.56	7.29	7	77	76			19	16
20P	A	6.79	7.62	6.76	81	81			19	23			13.77	0.91	108	79.06	1.12	133.35
	B	7.48	7.81	7.52	74	69			14	17
40P	A	7.59	6.8	6.41	NP	NP			NP	NP			10.63	1.03	95	0	0
	B	8.54	6.19	6.68	NP	NP			NP	NP
Positve Control EMS (µg/mL)
400	A	7.43	5.1	8.25	68	65			56	54			10.74	0.91	84	0.71	0.71	575.75
	B	6.83	6.62	6.41	77	67			42	48

GEF = 126 therefore MF threshold for a positive response = 266.39.

Applicant's summary and conclusion

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

The study was conducted according to the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, 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. 

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 6 dose levels in duplicate, together with solvent (Acetone), 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 maximum dose level used was limited by the presence of precipitate effectively reducing exposure of the test item to the cells. A precipitate of test item was observed at and above 40 μg/mL at the end of the exposure period in the absence of metabolic activation and at and above 20 μg/mL at the end of exposure in the presence of metabolic activation.
The solvent 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 increases in the mutant frequency at any of the dose levels in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating dose level in all exposure groups, and at least four analysable dose levels in each exposure group, as recommended by the OECD 490 guideline.

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.