2,3-dihydroxypropyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:

Gene mutation in bacteria (Reverse Mutation Assay, OECD 471): negative

Gene mutation in mammalian cells (Mouse Lymphoma Assay, OECD 490): positive in the 24 -hour group without metabolic activation

Cytotoxicity in mammalian cells (Micronucleus Test in human lymphocytes, OECD 487): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from male Wistar Rats

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 2500 and 5000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqua destillata

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene

Details on test system and experimental conditions:

For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, preculture of the strain),
2000 µL Overlay agar.
For the pre-incubation method 100 µL of the test item preparation was preincubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 minutes at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Evaluation criteria:

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Experiment II, only in tester strain TA 1537 at concentration of 5000 µg/plate (with metabolic activation)

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 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

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 102

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

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I. In experiment II toxic effects of the test item were noted only in tester strain TA 1537 at a concentration of 5000 µg/plate (with metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with C-SAT 090022 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and Il.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, 2,3-dihydroxypropyl methacrylate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, 2,3-dihydroxypropyl methacrylate is considered to be non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 02 January 2018 Experimental completion date: 23 January 2018.

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from 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 Cultures
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 subcultured accordingly. 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. 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:

liver S9 from male Sprague-Dawley rats treated with Phenobarbital / B-Naphtha flavone

Test concentrations with justification for top dose:

Preliminary Cytotoxicity test
0, 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1600 µg/mL
The test item had a molecular weight of 160 therefore the maximum recommended dose level was set at 1600 µg/mL.

Mutagenicity Test
4-hour without S9: 0, 25, 50, 100, 200, 400, 800, 1200, 1600, Ethylmethanesulphonate (EMS) 400
4-hour with S9 (2%): 0, 12.5, 25, 50, 100, 200, 400, 800, 1200, Cyclophosphamide (CP) 1.5
24-hour without S9: 0, 50, 100, 200, 400, 600, 700, 800, 1000, EMS 150
The dose levels selected for the main test in the 4-hour –S9 exposure group was the 10mM maximum recommended dose level of 1600 µg/mL. The dose levels selected for the main test in the 4-hour +S9 exposure group were based on precipitate. The dose levels selected for the main test in the 24-hour –S9 exposure group were based on test item induced toxicity.

Vehicle / solvent:

Following solubility checks performed in-house, the test item was accurately weighed and formulated in R0 culture media prior to serial dilutions being prepared.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (R0)

True negative controls:

no

Positive controls:

yes

Remarks:

400 μg/ml in 4-hour exposure group and 150 μg/ml in 24-hour exposure group

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (R0)

True negative controls:

no

Positive controls:

yes

Remarks:

1.5 μg/ul in 4-hour exposure group

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 12.5 to 1600 µg/mL for all three of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.
The cultures were incubated at 37°C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

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 10^6 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 10^6 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 eight dose levels of the test item, vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 2.0 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).
The exposure vessels were incubated at 37°C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Evaluation criteria:

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.
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, which is based on the analysis of the distribution of the vehicle 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 this test system.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

4 hour exposure

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

24 hour exposure

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Test
There was evidence of dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in the 4-hour and 24-hour –S9 exposure groups. However toxicity was the most prominent in the 24-hour exposure group. Precipitate of the test item was only observed in the presence of metabolic activation at and above 400 µg/mL. The dose levels selected for the main test in the 4-hour –S9 exposure group was the 10mM maximum recommended dose level of 1600 µg/mL. The dose levels selected for the main test in the 4-hour +S9 exposure group were based on precipitate. The dose levels selected for the main test in the 24-hour –S9 exposure group were based on test item induced toxicity.

Mutagenicity Test
As was seen previously, there was evidence of dose related toxicity following exposure to the test item in all of the three exposure groups. The toxicity was more prominent in the absence of metabolic activation exposures, as indicated by the %RSG and RTG values (Tables 3, 6, and 9). In the 4-hour –S9 exposure group the test item was exposed up to the MRD (10 mM) of 1600 µg/mL. In the 4-hour +S9 exposure group the test item was exposed up to 1200 µg/mL where precipitate can be observed. In the 24-hour –S9 exposure group optimum toxicity was achieved at 600 µg/mL. All three exposure groups satisfy the requirements of the OECD 490 guideline.
There was evidence of slight reductions in viability (%V) in all three exposure groups, indicating that residual toxicity had occurred. Acceptable levels of toxicity were seen with the positive control substances. The concentrations of 700, 800 and 900 µg/mL in the 24-hour –S9 exposure group were not plated out for 5-TFT resistance and viability due to excessive toxicity.
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.
In the 24-hour –S9 exposure group the test item induced toxicologically significant increases in the mutant frequency x 10-6 per viable cell at 400 and 600 µg/mL. The GEF value was exceeded at both concentrations. There was an indication of heterogeneity of colony counts at these two concentrations however; this was considered to be due to the high levels of toxicity observed. Therefore this was still considered to be a valid response. The colony formation counts were predominantly small at 600 µg/mL, which indicates a possible contribution to a clastogenic response.

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 12.5 to 1600 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
6.25	110	95	107
12.5	100	99	88
25	95	105	84
50	106	100	107
100	96	95	99
200	89	100	51
400	76	95 p	3
800	48	101 p	0
1600	32	81 p	0

Mutagenicity Test summary of results

Treatment (µg/ml)		4-hours-S-9				Treatment (µg/ml)		4-hours+S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	150.04		0		100	1.00	134.66
25	Ø	107				12.5	Ø	93
50	Ø	102				25	Ø	101
100		117	1.28	115.61		50		99	1.12	159.60
200		103	1.34	122.61		100		100	1.03	144.53
400		89	1.01	135.28		200		92	0.89	154.45
800		70	0.71	171.31		400		99	0.96	171.19
1200		42	0.43	172.31		800		92	0.98	158.62
1600		33	0.30	208.86		1200		68	0.63	205.26
MF threshold for a positive response 276.04						MF threshold for a positive response 262.66
EMS						CP
400		75	0.69	1242.43		1.5		72	0.52	1972.34

Treatment (µg/ml)		24-hours-S-9
		%RSG	RTG	MF§
0		100	1.00	143.67
50		108	0.95	151.41
100		90	0.86	147.02
200		76	0.66	157.79
400		32	0.43	288.37
600		9	0.15	690.42
700	Ø	1
800	Ø	0
1000	Ø	0
MF threshold for a positive response 269.67
EMS
150		38	0.36	1832.91

Key

$ = Cell counts (x10⁵cells/ml). Set up on previous day to 2 x 10⁵cells/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/10⁶viable cells 2 days after exposure

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

Ø  = Not plated surplus to requirements

NP = Not plated for viability or 5-TFT resistance

P = Precipitate present at the end of the exposure period

Conclusions:

The test item induced toxicologically significant 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 mutagenic in the 24-hour –S9 exposure group of 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 VitroMammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

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 eight dose levels in duplicate, together with vehicle (R0 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 dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test

Group	Concentration 2,3-dihydroxypropyl methacrylate (µg/mL)  plated for viability and mutant frequency
4-hour without S9	100, 200, 400, 800, 1200, 1600
4-hour with S9 (2%)	50, 100, 200, 400, 800, 1200
24-hour without S9	50, 100, 200, 400, 600

Results

The maximum dose level used in the Mutagenicity Test was limited by a combination of test item induced toxicity/precipitate and the maximum recommended 10 mM dose level. 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 induced toxicologically increases in the 24-hour –S9 (absence of metabolic activation) exposure group. The GEF value was exceeded at two test item concentrations (400 and 600 µg/mL).

Conclusion

The test item induced toxicologically significant 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 mutagenic in the 24-hour –S9 exposure group of this assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 05 September 2017. Experimental completion date 23 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

primary culture, other: whole blood

Details on mammalian cell type (if applicable):

Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a
non-smoking volunteer (18-35) who had been previously screened for suitability. 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. Based on over 20 years
in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine)
incorporation to assess the number of first, second and third division metaphase cells to
calculate the average generation time (AGT) for human lymphocytes it is considered to be
approximately 16 hours. Therefore using this average the in-house exposure time for the
experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 31 years
Main Experiment: male, aged 26 years

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES
buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin,
amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in
humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide
by the addition of phytohaemagglutinin (PHA).

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/B-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test
0, 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1600 μg/mL
The molecular weight of the test item was given as 160, therefore, the maximum dose level was 1600 μg/mL, which was calculated to be equivalent to 10mM, the maximum recommended dose level.

Main Experiment
4-hour without S9 100, 200, 400, 800, 1200, 1600 μg/mL
4-hour with S9 (2%) 100, 200, 400, 800, 1200, 1600 μg/mL
24-hour without S9 100, 200, 300, 400, 600, 800, 1600 μg/mL
The selection of the maximum dose level for the Main Experiment was based on the maximum recommended dose level but with toxicity being taken into account in the selection of the dose range for the 24-hour exposure group.

Vehicle / solvent:

The test item was soluble in Minimal Essential Medium (MEM) at 16 mg/mL in solubility checks performed in-house. Prior to each experiment, the test item was accurately weighed, dissolved in MEM and serial dilutions prepared.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium (MEM)

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 μg/mL for 4-hour exposure

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium (MEM)

True negative controls:

no

Positive controls:

yes

Remarks:

0.075 μg/mL for 24-hour continuous exposure

Positive control substance:

other: Demecolcine (DC)

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium (MEM)

True negative controls:

no

Positive controls:

yes

Remarks:

5 μg/mL for 4-hour exposure

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours.
The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.
The preliminary toxicity test was performed using the exposure conditions as described for the Main Experiment but using single cultures only, whereas the Main Experiment used replicate cultures.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used was 6.25 to 1600 μg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the experiments of the main test.

Main Experiment
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 100, 200, 400, 800, 1200 and 1600 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 100, 200, 400, 800, 1200 and 1600 μg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 100, 200, 300, 400, 600, 800 and 1600 μg/mL.

Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

Preparation of Microscope Slides
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
Test items that induce micronuclei in the MNvit test may do so because they induce chromosome breakage, chromosome loss, or a combination of the two. Further analysis using anti-kinetechore antibodies, centromere specific in situ probes, or other methods can be used to determine whether the mechanism of micronucleus induction is due to clastogenic and/or aneugenic activity.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.

Species / strain:

primary culture, other: whole blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 6.25, 12.5, 25, 50, 100, 200, 400, 800 and 1600 μg/mL. The maximum dose was the maximum recommended dose level, the 10 mM concentration.
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in any of the three exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 1600 μg/mL in the 4-hour exposure groups, both in the presence and absence of metabolic activation (S9). The maximum dose with binucleate cells present in the 24-hour continuous exposure was 800 μg/mL. The test item induced very modest toxicity in the 4-hour exposure groups and more
marked toxicity in the 24-hour exposure group.
The selection of the maximum dose level for the Main Experiment was based on the maximum recommended dose level but with toxicity being taken into account in the selection of the dose range for the 24-hour exposure group.

Micronucleus Test – Main Experiment
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test. There was very modest toxicity seen in the 4-hour exposure groups with binucleate cells suitable for scoring at the maximum dose level of test item, 1600 μg/mL. In the 24-hour exposure group the toxicity was more marked and there were binucleate cells up to 800 μg/mL.
No precipitate of test item was observed at the end of exposure in any of the three exposure groups.
The CBPI data for the short exposure groups and for the 24-hour exposure group confirm the qualitative observations in that a doserelated inhibition of CBPI was observed. In the 4-hour exposure groups, modest toxicity was observed at the maximum recommended dose level, 1600 μg/mL with 25% and 16% cytostasis in the absence and presence of S9, respectively. The 24-hour exposure group achieved cytostasis of 47% and 69% at 400 μg/mL and 600 μg/mL, respectively. The higher dose level of 800 μg/mL in the 24-hour exposure group achieved 83% cytostasis and was considered too toxic for scoring. The maximum dose level selected for scoring of the binucleate cells was the maximum recommended dose level, 1600μg/mL for the 4-hour exposure groups and 600 μg/mL for the 24-hour exposure group. The 600 μg/mL dose level in the 24-hour exposure group marginally exceeded acceptable toxicity but was selected since it was an intermediate dose level in the toxicity curve.
The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation using a dose range which achieved acceptable toxicity. The 24-hour exposure group did demonstrate a small but statistically significant response at a dose level which exceeded acceptable toxicity and can therefore be discounted. The response is considered to be likely due to a cytotoxic rather than a mutagenic mechanism since there are no indications of a response at the lower dose levels.

The dose levels of the controls and the test item are given in the table below:

Exposure Group	Final concentration of test item 2,3-dihydroxypropyl methacrylate (μg/mL)
4-hour without S9	0*, 100, 200, 400, 800*, 1200*, 1600*, MMC 0.2*
4-hour with S9 (2%)	0*, 100, 200, 400, 800*, 1200*, 1600*, CP 5*
24-hour without S9	0*, 100, 200*, 300*, 400*, 600*, 800, 1600, DC 0.075*

* = Dose levels selected for analysis of micronucleus frequency in binucleate cells

MMC = Mitomycin C

CP = Cyclophosphamide

DC = Demecolcine

Conclusions:

The test item, 2,3-dihydroxypropyl methacrylate, did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system using a dose range which achieved acceptable toxicity. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

Introduction

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that there was modest toxicity and the maximum recommended dose level was selected as the maximum dose level. The dose levels selected for the Main Test were as follows:

Exposure Group	Final concentration of test item 2,3-dihydroxypropyl methacrylate (μg/mL)
4-hour without S9	100, 200, 400, 800, 1200, 1600
4-hour with S9 (2%)	100, 200, 400, 800, 1200, 1600
24-hour without S9	100, 200, 300, 400, 600, 800, 1600

Results

All vehicle (Minimal Essential Medium) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was modestly toxic but did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the maximum recommended dose level for the 4-hour exposure groups in the absence and presence of S9 and a dose level which induced near optimum toxicity in the 24-hour exposure group.

Conclusion

The test item, 2,3-dihydroxypropyl methacrylate was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo:

Genotoxicity in somatic mammalian cells (Alkaline Comet Assay, OECD 489): negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 June 2018 - 23 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

The method was designed to be compatible with the procedures indicated in the OECD 489 Guideline (2016)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

other: Wistar Han™ (HsdRCCHan™WIST)

Details on species / strain selection:

A range-finding test was performed to find suitable dose levels of the test item and the most appropriate sex. The Comet assay main test was conducted in male animals.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo (UK)
- Age at study initiation: approximately eight to ten weeks old
- Weight at study initiation: 187.3 to 215.5 g
- Assigned to test groups randomly: yes
- Fasting period before study: no information
- Housing: in groups of up to five by sex in solid-floor polypropylene cages with woodflake bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): set to achieve limits of 19 to 25 ºC . Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study
- Humidity (%): set to achieve limits of 30 to 70%. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


The groups of rats from each dose level were killed by humane euthanasia (carbon dioxide asphyxiation) approximately 4 hours following the second administration, 28 hours after the start of the test.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: distilled water
- Lot/batch no. (if required): 17K13BA1A
- Storage Conditions: Room temperature

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was freshly prepared as required as a solution at the appropriate concentration in distilled water. The test item was formulated within 2 hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.

The positive control item was freshly prepared as required as a solution at the appropriate concentration in distilled water.

The Vehicle control (distilled water) was used as supplied.

Duration of treatment / exposure:

28 hours

Frequency of treatment:

Animals were dosed twice with a 24-hour interval

Post exposure period:

4 hours

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Range -Finding Toxicity Test /2 Males and 2 Females

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Range-finding Toxicity Test/ 2 Males/0 Females

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Comet Test (Animal Numbers 9-15)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Comet Test (Animal Numbers 16-20)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Comet Test (Animal Numbers 21-25)

No. of animals per sex per dose:

Comet Test:
2000 mg/kg bw/day (seven rats),
1000 mg/kg bw/day (five rats) and
500 mg/kg bw/day (five rats)

Control animals:

yes, concurrent vehicle

Positive control(s):

N-Nitroso-N-methylurea (MNU)
- Route of administration: oral
- Doses / concentrations: 25 mg/kg bw/day

Tissues and cell types examined:

The primary target tissues of this assay were liver and glandular stomach.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION AND METHOD OF ANALYSIS:
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. The slides were labelled for animal number, study number and tissue type prior to use for the comet assay.
Approximately 30 μL of the cell suspension was added to 270 μL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 μL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension mix was immediately covered with a glass cover slip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.
Once the LMP agarose had set, the cover slips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.
Following lysis, the slides were removed from the solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis bath, which was filled with chilled electrophoresis buffer (pH>13), until the slide surface was just covered. The slides were then left for 20 minutes to allow the DNA to unwind, after which they were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis to ensure the electrophoresis solution was maintained at low temperature (2-10 °C).
At the end of the electrophoresis period, the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralization buffer and left for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer was performed twice. The slides were further drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry.
Once dry the slides were stored prior to scoring. Two of the four processed slides were scored and the remaining slides were stored as backup slides.

Evaluation criteria:

Each slide was also assessed for the incidence of ‘hedgehog’ cells to give an indication of cell integrity. Hedgehogs are cells that exhibit a microscopic image consisting of a small or non-existent head, and large diffuse tails and are considered to be heavily damaged cells, although the etiology of the hedgehogs is uncertain.

Statistics:

Statistical analysis was considered un-necessary since there were no marked increases over the vehicle control values for the test item dose groups for the liver or glandular stomach. The positive control group demonstrated marked increases over the vehicle control group comparable with the current historical range for the liver and glandular stomach.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Mortality data from the range-finding test is presented below:

Dose Level (mg/kg bw/day)	Sex	Number of Animals Treated	Route	Deaths on Day		Total Deaths
				0	1
2000	Male	2	oral	0	0	0/2
2000	Female	2	oral	0	0	0/2
2000	Male	2	oral	0	0	0/2

There was no noticeable difference in clinical signs between the male and female animals and therefore only male animals were used for the main test.

Conclusions:

The test item did not induce any significant increases in the percentage tail intensity or median percentage tail intensity compared to the vehicle control in the liver or glandular stomach. Therefore the test item was considered to be unable to induce DNA strand breakage to these tissues in vivo under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Gene mutation in bacteria (Ames Test):

In order to investigate the potential of 2,3-dihydroxypropyl methacrylate for its ability to induce gene mutations the plate incorporation test (experiment 1) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 in accordance with OECD 471 and GLP (BSL Bioservice, 2009b). In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations were applied: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose (with and without metabolic activation) in experiment I. In experiment II toxic effects of the test item were noted only in tester strain TA 1537 at a concentration of 5000 µg/plate (with metabolic activation). No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 2,3 -dihydroxypropyl methacrylate at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and Il. The reference mutagens induced a distinct increase in revertant colonies indicating the validity of the experiment.

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, 2,3-dihydroxypropyl methacrylate is considered to be non-mutagenic in this bacterial reverse mutation assay.

Gene mutation in mammalian cells (Mouse Lymphoma Assay):

An in vitro mammalian cell gene mutation assay with 2,3-dihydroxypropyl methacrylate was performed in mouse-lymphoma L5178Y cells according to OECD guideline 490 and GLP (Envigo Research Limited, 2018a). In a preliminary cytotoxicity test with concentrations ranging from 6.25 to 1600 µg/mL, with the maximum recommended dose level (MRD) of 1600 µg/mL based on the molecular weight of 160 g/mol. There was evidence of dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in the 4-hour and 24-hour –S9 exposure groups. However, toxicity was most prominent in the 24-hour exposure group. Precipitates of the test item were only observed in the presence of metabolic activation at and above 400 µg/mL. Based on those results, doses ranging from 25 – 1600 µg/mL were selected for the main test in the 4-hour –S9 exposure group to reach the MRD. The dose levels selected for the main test in the 4-hour +S9 exposure group were based on the precipitating properties of the test item and ranged from 12.5 – 1200 µg/mL. The dose levels selected for the main test in the 24-hour –S9 exposure group were based on test item induced toxicity (50 – 1000 µg/mL).

As was seen previously, there was evidence of dose related toxicity following exposure to the test item in all of the three exposure groups. The toxicity was more prominent in the absence of metabolic activation exposures, as indicated by the RSG and RTG values. In the 4-hour –S9 exposure group the test item was exposed up to the MRD (10 mM) of 1600 µg/mL. In the 4-hour +S9 exposure group the test item was exposed up to 1200 µg/mL where precipitates were observed. In the 24-hour –S9 exposure group optimum toxicity was achieved at 600 µg/mL. All three exposure groups satisfy the requirements of the OECD 490 guideline.

There was evidence of slight reductions in viability (%V) in all three exposure groups, indicating that residual toxicity had occurred. Acceptable levels of toxicity were seen with the positive control substances. The concentrations of 700, 800 and 900 µg/mL in the 24-hour –S9 exposure group were not plated for 5-TFT resistance and viability due to excessive toxicity. 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, thereby validating the study.

In the 24-hour –S9 exposure group the test item induced toxicologically significant increases in the mutant frequency x 10^-6per viable cell at 400 and 600 µg/mL. The Global Evaluation Factor (GEF) value of 126 x 10^-6was exceeded at both concentrations. There was an indication of heterogeneity of colony counts at these two concentrations. However, this was considered to be due to the high levels of toxicity observed. Therefore, this was still considered to be a valid response. The colony formation counts were predominantly small at 600 µg/mL, which indicates a possible contribution to a clastogenic response. Therefore, 2,3-dihydroxypropyl methacrylate is considered to be mutagenic in the Mouse Lymphoma Assay under the conditions chosen (24-hour –S9 exposure).

 

Cytotoxicity in mammalian cells (Micronucleus Test):

An in vitro micronucleus test was performed with 2,3-dihydroxypropyl methacrylate in primary cells from peripheral human blood according to OECD guideline 487 and in compliance with GLP (Envigo Research Limited, 2018b). Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The dose levels used in the main experiment were selected based on a preliminary toxicity test in which modest toxicity was observed and hence, the maximum recommended dose level was selected as the highest dose level. The dose levels selected for the main test were as follows: in the 4-hour –S9 and +S9 exposure groups, cells were exposed to the test item at 100, 200, 400, 800, 1200 and 1600 µg/mL, respectively, and in the 24-hour –S9 exposure group cells were exposed at 100, 200, 300, 400, 600, 800 and 1600 µg/mL.

All vehicle (minimal essential medium) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item was modestly toxic but did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the maximum recommended dose level for the 4-hour exposure groups in the absence and presence of S9 and a dose level which induced near optimum toxicity in the 24-hour exposure group. Therefore, under the conditions of this assay, 2,3-dihydroxypropyl methacrylate did not exhibit clastogenic or aneugenic activity in primary human lymphocytes in the presence and absence of metabolic activation under the conditions of this assay.

 

In vivo

As 2,3-dihydroxypropyl methacrylate was considered to be mutagenic in the Mouse Lymphoma Assay, an in vivo genotoxicity assay in somatic mammalian cells according to OECD 489 and GLP was conducted (Envigo Research Limited, 2018c). According to the Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a (ECHA; version 6.0, 2017), the in vivo Comet Assay is an appropriate test for investigating potentially reactive substances that may express their mutagenic potential at the initial site of contact in the body. The Comet Assay is one of the study protocols referred to as a reliable in vivo study capable of detecting gene mutations. It is therefore a suitable in vivo test method to further investigate the mutagenic potential of the test item in vivo as follow-up to the positive Mouse Lymphoma Assay.

The in vivo Mammalian Alkaline Comet Assay was conducted in Wistar Han^TM(HsdRCCHan™WIST) rats. In order to find suitable dose levels for the main experiment and the most appropriate sex, a range-finding test was performed in male and female animals at a dose level of 2000 mg/kg bw/day. The animals were administered the test substance by gavage twice with a 24-hour interval. Since no mortality was observed in the range-finding study and there was no noticeable difference in clinical signs between the sexes, the main experiment was performed in male rats only. In the main experiment, a group of seven male rats was administered 2000 mg/kg bw/day and two groups of five male rats were administered 1000 and 500 mg/kg bw/day, respectively. A control group was treated with the vehicle distilled water, whereas the positive control group animals received 25 mg/kg bw/day N-Nitroso-N-methylurea (MNU). The liver and especially the glandular stomach as initial site of contact were identified as primary target tissues of this assay.

The vehicle control group induced percentage tail intensities which were consistent with the laboratory’s historical control range. The positive control item (MNU) produced a marked increase in the percentage tail intensity and median percentage tail intensity in the liver and glandular stomach, comparable with the laboratory’s historical control range for these tissues. The test method itself was therefore operating as expected and was considered to be valid under the conditions of the test.

There were no marked increases in percentage tail intensity over the vehicle control for any of the test item dose levels in the glandular stomach or liver tissues. The values were consistent with the current historical control range for a vehicle, confirming the test item did not induce DNA damage in the liver or glandular stomach. There was no marked increase in hedgehog (ghost cell) frequency for any of the test item dose levels in either of the tissues investigated. Therefore, 2,3-dihydroxypropyl methacrylate was considered to be unable to induce DNA strand breakage to these tissues in vivo under the conditions of the test.

Based on the available in vitro and in vivo data, the overall assessment is that 2,3-dihydroxypropyl methacrylate does not exhibit a clastogenic or aneugenic activity in vitro nor a mutagenic activity in vivo.

 

Justification for classification or non-classification

The available data on the genetic toxicity of 2,3-dihydroxypropyl methacrylate (CAS 5919 -74 -4) do not meet the criteria for classification according to Regulation (EC) No 1272/2008 and are therefore conclusive but not sufficient for classification.