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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The potential of Reaction Mass of 2-Propenoic acid, 2-methyl-, 2- dodecylhexadecyl ester and 2-Propenoic acid, 2- methyl-, 2-tetradecyloctadecyl ester to induce gene mutation in bacteria was assessed using a GLP-compliant study performed in accordance with the OECD Testing Guideline 471.

Experiments were performed with and without metabolic activation using S9-mix. The Bacterial strains used were Salmonella typhimurium, TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA. Plate incorporation method (experiment 1) and pre-incubation method (experiment 2) were used. The maximum dose level of the test item in experiment 1 was 4,700 μg/plate in experiment 2 the maximum dose was 5,000 μg/plate.

In the first experiment (plate incorporation method), no visible reduction in the growth of the bacterial background lawn at any dose level both with or without metabolic activation (S9-mix) were noted. In experiment 2 there was no visible reduction in the growth of the bacterial background lawn noted to any of the tester strains at any dose level, either in the presence or absence of metabolic activation (S9-mix).

 

It is therefore concluded that Reaction Mass of 2-Propenoic acid, 2-methyl-, 2- dodecylhexadecyl ester and 2-Propenoic acid, 2- methyl-, 2-tetradecyloctadecyl ester was not mutagenic under the conditions of the test.

 

 

The potential of Reaction Mass of 2-Propenoic acid, 2-methyl-, 2- dodecyl hexadecyl ester and 2-Propenoic acid, 2- methyl-, 2-tetradecyloctadecyl ester to induce chromosome aberrations in cultured mammalian cells (Human Lymphocytes) was assessed using a GLP-compliant study performed in accordance with the OECD Testing Guideline 473. Experiments were performed with and without metabolic activation using S9-mix. Duplicate cultures of human lymphocytes treated with the test item were assessed for chromosome aberrations at three dose levels, along with positive and vehicle controls.  Dose levels were selected based on the results of a preliminary toxicity test which indicated a limitation on the maximum concentration (483 µg/ml) based on precipitation. Microscopic assessment of the slides prepared from the exposed cultures showed metaphase was present up to 483 µg/ml in all three exposure groups. The test item did not induce toxicity in any of the exposure groups. The maximum dose level for the main experiment was based on the lowest precipitating dose level for all three exposure groups. The dose levels for the main experiment were 0,2,4,8,16,32,64 and 128 µg/ml. A qualitative assessment of the slides showed precipitate similar to that observed in the preliminary toxicity test and that there were metaphases suitable for scoring present at the maximum dose level (128 µg/ml) of the test item in all three exposure groups. Precipitate observations at the end of exposure in blood free cultures was noted at above 32 µg/ml in all three exposure groups. No dose related inhibition of mitotic index was observed. Therefore the maximum dose level selected for metaphase analysis was the lowest precipitating dose level for all three exposure groups which was 32 µg/ml. The test item was found to be non-toxic to human lymphocytes as it did not induce any statistically significant increases in the frequency of cells with aberrations using a dose range that included a dose level based on the lowest precipitating dose level.

 

It is therefore concluded that Reaction Mass of 2-Propenoic acid, 2-methyl-, 2- dodecylhexadecyl ester and 2-Propenoic acid, 2- methyl-, 2-tetradecyloctadecyl ester was non-clastogenic to human lymphocytes under the conditions of this test.

 

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.

 

 

Link to relevant study records

Reference

Reference 1

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

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

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%.

Target gene:

thymidine kinase, TK +/- 3.7.2c.

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.

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.

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.

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.

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.

 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 February 2019 - 08 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: F1-8S006C
- Expiration date of the lot/batch: 31 October 2020
- Purity test date: Not reported


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: No analysis undertaken to determine homogeneity, concentration or stability.
- Solubility and stability of the test substance in the solvent/vehicle: The test item was fully miscible in acetone at 500mg/ml.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Test item formulations, formulated within two hours of being applied and assumed to be stable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prior to each experiment, the test item was accurately weighed, formulated in acetone and appropriate serial dilutions prepared
- Preliminary purification step (if any): None Reported
- Final dilution of a dissolved solid, stock liquid or gel: 483 µg/ml
- Final preparation of a solid: N/A

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: Excessive precipitate observed during the osmolality check and, consequently, the maximum dose level was reduced to 512 µg/mL. This concentration was further adjusted to 483 µg/mL due to the amended purity of 93.3%. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.
- other information: N/A

Target gene:

Metaphase Chromosomes of normal human lymphocytes

Species / strain / cell type:

mammalian cell line, other: Human Peripheral Blood Lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability.
- Suitability of cells: The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- Normal cell cycle time: The calculated average generation time based on 20 years of in house data was found to be approximately 16 hours

For lymphocytes:
- Sex, age and number of blood donors: male and females between 18-35
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: Not reported
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Cells were grown in eagle's minimal essential medium with HEPES buffer (MEM) supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 %
foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Prepared using standardised in house procedures (outside the confines of this study)
- method of preparation of S9 mix : The S9-mix contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM).
- concentration or volume of S9 mix and S9 in the final culture medium : The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test concentrations with justification for top dose:

The dose range of test item used was 0, 1.89, 3.78, 7.55, 15.10, 30.19, 60.38, 120.75, 241.5 and 483 µg/mL.
Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed in 50 µL aliquots. Consequently, the maximum practical concentration was initially set at 2500 µg/mL. However, the presence of excessive precipitate was observed during the osmolality check and, consequently, the maximum dose level was reduced to 512 µg/mL. This concentration was further adjusted to 483 µg/mL due to the amended purity of 93.3%.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: The test item was immiscible in dimethyl sulphoxide at 500 mg/mL but was fully miscible in acetone at the same concentration

- Justification for percentage of solvent in the final culture medium: Due to the sensitivity of human lymphocytes to acetone, formulations were prepared at twice the concentration required in culture and dosed in 50 µL aliquots. Consequently, the maximum practical concentration was initially set at 2500 µg/mL. However, the presence of excessive precipitate was observed during the osmolality check and, consequently, the maximum dose level was reduced to 512 µg/mL. This concentration was further
adjusted to 483 µg/mL due to the amended purity of 93.3%.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments : 3

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: In suspension in parallel flasks containing culture medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4-hour exposure to the test item without S9-mix, 4-hour exposure to the test item with S9-mix (2%), 24-hour continuous exposure to the test item without S9-mix prior to cell harvest.
- Harvest time after the end of treatment (sampling/recovery times): for the 4 hour exposure times with and without S9 metabolic activation a 20 hour culture in treatment free media before cell harvest employed.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 2 replicate cultures, 1000 lymphocyte cell nuclei per replicate were counted
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per replicate)
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated
- Determination of polyploidy:Cells with 69 chromosomes or more scored as polyploid cells
- Determination of endoreplication: Endoreduplicated cells were recorded separately and included in the polyploid cell total number


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI)

Rationale for test conditions:

The dose range for the Preliminary Toxicity Test was 1.89 to 483 µg/mL. The maximum dose was limited from 2500 µg/mL (the maximum practical dose level) to 483 µg/mL because of the presence of excessive precipitate.

Evaluation criteria:

A test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level.

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

mammalian cell line, other: Human Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No significant change in pH (7.33) when the test item was dosed into media : For all the tested concentrations the pH was as follows 9.77µg = pH7.30, 19.53µg = pH7.30, 39.06µg= pH7.30, 78.13µg= pH7.31,156.25µg = pH7.32, 312.5µg= pH7.30, 625µg= pH7.31, 1250µg= pH7.32, 2500µg= pH7.33.
- Data on osmolality:The osmolality(375) did not increase by more than 50 mOsm. For all the tested concentrations the osmolality was as follows 9.77µg = Not performed, 19.53µg = 384mOsm, 39.06µg= 394mOsm, 78.13µg= 386mOsm,156.25µg = 386mOsm, 312.5µg= 376mOsm, 625µg= 366mOsm, 1250µg= 365mOsm, 2500µg= 337mOsm.

- Precipitation and time of the determination: A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 30.19 µg/mL in the 4 (20)-hour exposure groups and at and above 60.38 µg/mL in the continuous exposure group.
- Definition of acceptable cells for analysis: Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range is shown in the results table provided as part of the background documents.

- Other confounding effects:
RANGE-FINDING/SCREENING STUDIES:
Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed in 50 µL aliquots. Consequently, the maximum practical concentration was initially set at 2500 µg/mL. However, the presence of excessive precipitate was observed during the osmolality check and, consequently, the maximum dose level was reduced to 512 µg/mL. This concentration was further adjusted to 483 µg/mL due to the amended purity of 93.3%. Preliminary toxicity test was performed using all three of the exposure conditions as for the main experiment. The dose range of test item used was 0, 1.89, 3.78, 7.55, 15.10, 30.19, 60.38, 120.75, 241.5 and 483µg/mL.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
: All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis: The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytes in primary cultures: A total of 1000 lymphocyte cell nuclei per replicate were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

- Genotoxicity results (lymphocytes)
o Definition for chromosome aberrations, including gaps:

Gaps are small areas of the chromosome that are unstained. The chromatids remain alignedas normal and the gap does not extend along the chromatid for a distance greater than the width of a chromatid. If the gap occurs on one chromatid only it is a chromatid gap (g). If a gap appears in both chromatids at the same position it is a chromosome gap (G).

Chromatid breaks (ct) vary in appearance. The chromatid may remain aligned but show agap which is too large to classify as a gap. Alternatively, the chromatid may be broken sothat the broken fragment is displaced. In some cases, the fragment is not seen at all. A chromatid fragment (f) should be scored if the chromosome of origin cannot be identified. Very small fragments are scored as minutes (m).

Chromosome breaks (CS) are breaks in both chromatids of the chromosome. A fragmentwith two chromatids is formed and may be displaced by varying degrees. Breaks are distinguished from gaps by the size of the unstained region. A chromosome break is scored if the fragment is associated with a chromosome from which it was probably derived. However, fragments are often seen in isolation and are then scored as chromosome fragments (F). Very small fragments are scored as minutes (M).

Exchanges are formed by faulty rejoining of broken chromosomes and may be of the chromosome or chromatid type. Chromatid exchanges (c/c,r) have numerous different forms but are generally not further classified. Where multiple exchanges have occurred each exchange point is counted as one chromatid exchange. Chromosome exchanges generally appear as either a dicentric (D) or a ring (R) form, either of which can be associated with a fragment, which if possible should be scored as part of the exchange.

If many aberrations are present in one metaphase, the exact details may not be scorable. This is particularly the case when chromosome pulverisation occurs. If the number of aberrations is 10 or more then the cell is classified as X.

If the chromosome (centromere) number is 44-48 then it is classified as a diploid cell and scored for aberrations. If less than 44 chromosomes are counted then the cell is ignored under the assumption that some chromosomes may have been lost for technical reasons. If greater than 44 chromosomes are scored and the number is a multiple of the haploid count then the cell is classified as a polyploid cell. If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated (E).

o Number of cells scored for each culture and concentration: Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per replicate).

number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excluding gaps
: The test item did not induce toxicity in any of the exposure groups.The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen
: The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:

-4(20)-hour exposure without s9: %cells with aberrations(-gaps) = Minimum 6.1 Maximum: 63.5, Mean 25.13, Standard deviation:13.14,Number: 96
-4(20)-hour exposure without s9: %cells with polyploids = Minimum 0.0 Maximum: 0.4,Mean 0.01, Standard deviation: 0.06, Number: 94
-4(20)-hour exposure with s9 (2%):%cells with aberrations(-gaps) = Minimum 5.7 Maximum: 37.1,Mean 16.22, Standard deviation: 7.00, Number: 96
-4(20)-hour exposure with s9 (2%):%cells with polyploids = Minimum 0.0 Maximum: 0.7,Mean 0.03, Standard deviation: 0.12, Number: 95
-24-hour exposure without s9 :%cells with aberrations (-gaps) = Minimum 6.3 Maximum: 65.2,Mean 26.81, Standard deviation: 12.28, Number: 96
-24-hour exposure without s9 :%cells with polyploids = Minimum 0.0 Maximum: 0.9,Mean 0.02, Standard deviation: 0.11,Number: 95

- Vehicle historical control data:
-4(20)-hour exposure without s9: %cells with aberrations(-gaps) = Minimum 0.0 Maximum: 1.7,Mean 0.48, Standard deviation: 0.40, Control Limits: 0-1.68 Number: 96
-4(20)-hour exposure without s9: %cells with polyploids = Minimum 0.0 Maximum: 0.7,Mean 0.04, Standard deviation: 0.13, Control Limits: 0-0.43 Number: 96
-4(20)-hour exposure with s9 (2%):%cells with aberrations(-gaps) = Minimum 0.0 Maximum: 2.0,Mean 0.54, Standard deviation: 0.53, Control Limits: 0-2.13 Number: 96
-4(20)-hour exposure with s9 (2%):%cells with polyploids = Minimum 0.0 Maximum: 0.7,Mean 0.03, Standard deviation: 0.10, Control Limits: 0-0.33 Number: 96
-24-hour exposure without s9 :%cells with aberrations (-gaps) = Minimum 0.0 Maximum: 2.7,Mean 0.36, Standard deviation: 0.43, Control Limits: 0-1.65 Number: 96
-24-hour exposure without s9 :%cells with polyploids = Minimum 0.0 Maximum: 0.3,Mean 0.02, Standard deviation: 0.07, Control Limits: 0-0.23 Number: 95

Conclusions:

No statistically significant increase in the number of cells with chromosome aberrations, in either the presence or absence of a liver enzyme metabolizing system were recorded. Reaction Mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-,2-tetradecyloctadecyl ester was therefore considered to be non-clastogenic to human lymphocytes under the conditions of this test.

Executive summary:

The potential of Reaction Mass of 2-Propenoic acid, 2-methyl-, 2- dodecyl hexadecyl ester and 2-Propenoic acid, 2- methyl-, 2-tetradecyloctadecyl ester to induce chromosome aberrations in cultured mammalian cells (Human Lymphocytes) was assessed using a GLP-compliant study performed in accordance with the OECD Testing Guideline 473. Experiments were performed with and without metabolic activation using S9-mix. Duplicate cultures of human lymphocytes treated with the test item were assessed for chromosome aberrations at three dose levels, along with positive and vehicle controls. Three exposure conditions namely 4 hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation(s9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation were investigated. Dose levels were selected based on the results of a preliminary toxicity test which indicated a limitation on the maximum concentration (483 µg/ml) based on precipitation. Microscopic assessment of the slides prepared from the exposed cultures showed metaphase was present up to 483 µg/ml in all three exposure groups. The test item did not induce toxicity in any of the exposure groups. The maximum dose level for the main experiment was based on the lowest precipitating dose level for all three exposure groups. The dose levels for the main experiment were 0,2,4,8,16,32,64 and 128 µg/ml. A qualitative assessment of the slides showed precipitate similar to that observed in the preliminary toxicity test and that there were metaphases suitable for scoring present at the maximum dose level (128 µg/ml) of the test item in all three exposure groups. Precipitate observations at the end of exposure in blood free cultures was noted at above 32 µg/ml in all three exposure groups. No dose related inhibition of mitotic index was observed. Therefore the maximum dose level selected for metaphase analysis was the lowest precipitating dose level for all three exposure groups which was 32 µg/ml. Vehicle controls had frequencies of cells with aberrations within the normal expected range for lymphocytes. All the positive controls induced substantial increases in the frequency of cells with aberrations thereby validating the sensitivity of the assay and efficiency of the S9 mix. The test item was found to be non-toxic to human lymphocytes as it did not induce any statistically significant increases in the frequency of cells with aberrations using a dose range that included a dose level based on the lowest precipitating dose level.

 

It is therefore concluded that Reaction Mass of 2-Propenoic acid, 2-methyl-, 2- dodecylhexadecyl ester and 2-Propenoic acid, 2- methyl-, 2-tetradecyloctadecyl ester was non-clastogenic to human lymphocytes under the conditions of this test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 February 2019 - 25 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

See 'Principles of method if other than guideline'

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

See 'Principles of method if other than guideline'

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

yes

Remarks:

See 'Principles of method if other than guideline'

Principles of method if other than guideline:

The following deviations from study plan occurred:

Testing for Experiment 1 was performed using the originally supplied test item purity value of 100%. This was accounted for in all formulations of the test item. Upon completion of Experiment 1, the sponsor supplied a new purity value of 93.3% and it was agreed that the second experiment should be performed using the corrected purity value. As a consequence, the test item has been slightly under tested in the first experiment by 6.7%. A correction factor has therefore been applied to the stated test item concentrations for Experiment 1 and the solubility test. The corrected values are therefore reported. This is considered a minor deviation as the overall result was negative even after accounting for purity allowance in the second mutation test (the more sensitive pre-incubation method).

All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore,not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus in the genome of Salmonella typhimurium and tryptophan locus in the genome of Escherichia coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The S9 Microsomal fractions (CD Sprague-Dawley) were pre-prepared using standardized inhouse procedures.
Lot No. PB/βNF S9 28 October 2018 was used in this study. A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Test concentrations with justification for top dose:

Experiment 1: 1.4, 4.7, 14.1, 47, 141, 470, 1,410 and 4,700 μg/plate
Experiment 2: 5, 15, 50, 150, 500, 1,500, 5,000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile water at 47 mg/mL and in dimethyl sulphoxide at 45 mg/mL but was fully miscible in acetone at 92mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
- Justification for percentage of solvent in the final culture medium:Acetone is toxic to the bacterial cells at 0.1 mL (100 µL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 µL) aliquots.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Details on test system and experimental conditions
The test item was immiscible in sterile distilled water at 47mg/ml and dimethyl sulphoxide at 45 mg/mL but was fully miscible in acetone at 92 mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
The test item was accurately weighed and, on the day of each experiment, approximate half-log
dilutions prepared in pre-dried acetone by mixing on a vortex mixer. Originally, the formulated concentrations were not adjusted to allow for purity content of the test item. However, following information received from the Sponsor, a new purity value of 93.3% was implemented. Acetone is toxic to the bacterial cells at 0.1 mL (100 µL) after employing the pre-incubation modification; therefore, all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 µL) aliquots. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with
regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
The test item was tested using the following method. The maximum concentration was
5,000 μg/plate (the maximum recommended dose level). However, following consultation with the Sponsor, the purity value was amended at the end of Experiment 1 from 100% to 93.3% resulting in the test item being slightly under tested at a maximum dose of 4700 µg/plate. Eight concentrations of the test item (1.4, 4.7, 14.1, 47, 141, 470, 1,410 and 4,700 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. 0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added together with 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer to 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates. With metabolic activation, the procedure was the same except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.All of the plates were incubated at 37 ± 3°C for approximately 48 hours and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A single manual count was performed due to spreading colonies which prevented an accurate automated count.


Test for Mutagenicity: Experiment 2 – Pre- Incubation Method
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the
pre-incubation method in the presence and absence of metabolic activation (S9-mix). The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500,
1500 and 5000 ug/plate. A test item purity allowance of 93.3% was employed for the second experiment and therefore, the maximum dose concentration was achieved. 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the appropriate concentration of test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated)
controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxicity of the test item following the change in test methodology from plate incorporation to pre-incubation With Metabolic Activation: The procedure was the same except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate. All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity)

Rationale for test conditions:

In accordance with the OECD Testing Guideline 471.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

No significant increases in the frequency of revertant colonies for any of the bacterial strains were recorded. Reaction Mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The potential of Reaction Mass of 2-Propenoic acid, 2-methyl-, 2- dodecylhexadecyl ester and 2-Propenoic acid, 2- methyl-, 2-tetradecyloctadecyl ester to induce gene mutation in bacteria was assessed using a GLP-compliant study performed in accordance with the OECD Testing Guideline 471.

Experiments were performed with and without metabolic activation using S9-mix. The Bacterial strains used were Salmonella typhimurium, TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA. Acetone was selected as a vehicle and used for negative control, yielding counts of revertant colonies within the normal range. All the positive controls used induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Therefore, the sensitivity of the assay and efficacy of the S9-mix were considered as valid. Plate incorporation method (experiment 1) and pre-incubation method (experiment 2) were used. The maximum dose level of the test item in experiment 1 was 4,700 μg/plate in experiment 2 the maximum dose was 5,000 μg/plate.

In the first experiment (plate incorporation method), the maximum dose level of the test item was initially selected as the OECD TG 471 recommended dose of 5,000 μg/plate. However following consultation with the sponsor, the purity value was amended after experiment 1 from 100% to 93.3% resulting in the test item being slightly undertested at a maximum dose of 4,700 μg/plate. This was considered as a minor deviation as the overall result was negative even after accounting for purity allowance in the second mutation test (the more sensitive pre-incubation method). No visible reduction in the growth of the bacterial background lawn at any dose level both with or without metabolic activation (S9-mix) in the first experiment were noted. In experiment 2 there was no visible reduction in the growth of the bacterial background lawn noted to any of the tester strains at any dose level, either in the presence or absence of metabolic activation (S9-mix).

A test item precipitate (light and globular in appearance) was noted both in the presence and absence of metabolic activation (S9-mix) at 4,700 μg/plate in experiment 1 and at 5,000 μg/plate in experiment 2. This observation did not prevent the scoring of revertant colonies. There were no toxicologically meaningful increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

It is therefore concluded that Reaction Mass of 2-Propenoic acid, 2-methyl-, 2- dodecylhexadecyl ester and 2-Propenoic acid, 2- methyl-, 2-tetradecyloctadecyl ester was not mutagenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)