Reaction mass of 2-hydroxy-1,3-propanediyl bismethacrylate and 101525-90-0

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17.08.2017 to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Version July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 29 June 2015.

Version / remarks:

Version 29.06.2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-hydroxy-1,3-propanediyl bismethacrylate and 101525-90-0

EC Number:

931-227-1

Cas Number:

28497-59-8

Molecular formula:

C11H16O5

IUPAC Name:

Reaction mass of 2-hydroxy-1,3-propanediyl bismethacrylate and 101525-90-0

impurity 1

Reference substance name:

2,3-dihydroxypropyl methacrylate

EC Number:

227-642-6

EC Name:

2,3-dihydroxypropyl methacrylate

Cas Number:

5919-74-4

Molecular formula:

C7H12O4

IUPAC Name:

2,3-dihydroxypropyl methacrylate

impurity 2

Reference substance name:

Glycerol trimethacrylate

EC Number:

231-004-2

EC Name:

Glycerol trimethacrylate

Cas Number:

7401-88-9

Molecular formula:

C15H20O6

IUPAC Name:

propane-1,2,3-triyl tris(2-methylacrylate)

impurity 3

Reference substance name:

oxybis(propane-2,1,3-triyl) tetrakis(2-methylacrylate)

Molecular formula:

C22H30O9

IUPAC Name:

oxybis(propane-2,1,3-triyl) tetrakis(2-methylacrylate)

impurity 4

Reference substance name:

Benzyl methacrylate

EC Number:

219-674-4

EC Name:

Benzyl methacrylate

Cas Number:

2495-37-6

Molecular formula:

C11H12O2

IUPAC Name:

benzyl methacrylate

impurity 5

Reference substance name:

Methacrylic acid

EC Number:

201-204-4

EC Name:

Methacrylic acid

Cas Number:

79-41-4

Molecular formula:

C4H6O2

IUPAC Name:

2-methylprop-2-enoic acid

additive 1

Reference substance name:

Mequinol

EC Number:

205-769-8

EC Name:

Mequinol

Cas Number:

150-76-5

Molecular formula:

C7H8O2

IUPAC Name:

4-methoxyphenol

Test material form:

liquid

Specific details on test material used for the study:

Batch: 1270300006
Purity: Reactiveester content: 95.3%
Diester content (isomer mix): 86.4%
Methacrylic acid: 0.86%
Appearance: Colourless to yellowish, liquid
Expiry Date: 01 February 2018
Storage Conditions: At room temperature

Test animals / tissue source

Species:

other: EpiOcular Kit and MTT-100

Details on test animals or tissues and environmental conditions:

EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists
of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar
to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface
of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²)
were cultured on specially prepared cell culture inserts (MILLICELL(R), 10 mm ).
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate.
On day of receipt (19 September 2017) of the EpiOcular™ tissues, the equilibration step
(15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

120 min

Number of animals or in vitro replicates:

2

Details on study design:

After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 30 minutes.
Test item exposure: After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 μL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.
At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilized a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 mL of warm Assay Medium. The tissues were incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

MTT Assay
After post-treatment incubation of 120 minutes, the MTT assay was performed.
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate
wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

Results and discussion

In vitro

Results

Irritation parameter:

other: Mean tissue viability

Remarks:

Mean relative absorbance [%]

Run / experiment:

Mean of duplicate test

Value:

45.3

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

36.3 %

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Prediction Model
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant (no Category according to UN GHS).
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant (Category 2 or Category 1 according to UN GHS; no differentiation between the categories possible).

Acceptability of the Assay
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.

Any other information on results incl. tables

Results after treatment for 30 minutes with Glycerol dimethacrylate and the controls

Dose Group	Ab-sorbance Well 1 (Tissue 1/2)	Ab-sorbance Well 2 (Tissue 1/2)	Mean Absor-bance (Tissue 1/2)	Mean Absorbance* Tissue 1 and 2	Mean Absorbance of 2 Tissues*	Rel. Absorbance [%] Tissue 1 and 2**	Absolute Value of the Difference of the Rel.Absorbances [%] Tissue 1 and 2	Mean Rel. Absorbance [%]***
Blank	0.036	0.036	0.036
Negative Control	1.628	1.644	1.636	1.601	1.590	100.6	1.3	100.0
	1.617	1.614	1.616	1.580		99.4
Positive Control	0.585	0.635	0.610	0.574	0.577	36.1	0.3	36.3
	0.618	0.613	0.615	0.580		36.4
Test Item	0.694	0.744	0.719	0.683	0.721	43.0	4.7	45.3
	0.787	0.800	0.793	0.758		47.6

 

* Mean of two replicate wells after blank correction

** Relative absorbance [rounded values]

 

*** Mean relative absorbance [rounded values]

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not lead to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue/purple colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 45.3% (threshold for irritancy: ≤ 60%), consequently the test item was irritant to eye.

Concerning acceptance criteria:

• The negative control OD is > 0.8 and < 2.5 (1.614 and 1.644).

• The mean relative viability of the positive control is below 50% of the negative control viability (36.3%).

• The difference of viability between the two relating tissues of a single item is < 20% (values between 0.3% and 4.7%) in the same run (for positive and negative control tissues and tissues of single test items).

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

It can be stated that in this in vitro GLP study and under the experimental conditions reported, Glycerol dimethacrylate possesses an eye irritating potential.

Executive summary:

An in vitro study was performed to assess the eye irritation potential of Glycerol dimethacrylate by means of the Human Cornea Model Test according to OECD 492 and current GLP requirements.

Additional tests with viable or freeze-killed tissues were not performed, since the test item was not coloured intensively, did not dye water or isopropanol, and did not prove to be a MTT reducer.

Tissues of the human cornea model EpiOcular™ were treated with the test item, the positive and the negative control for 30 minutes each in duplicate.

50 µL of the test item and of the controls, respectively, were applied to each tissue, spread to match the tissue size.

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 36.3%, thus the validity of the test system is ensured.

Relevant irritating effects were observed following 30 minutes incubation with Glycerol dimethacrylate. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 45.3% compared with the value of the negative control (threshold for irritancy: ≤ 60%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, Glycerol dimethacrylate possesses an eye irritating potential.

A limitation of the Test Guideline OECD 492 is that it does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B), as defined by UN GHS.