methyl acrylate; methyl propenoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

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:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methyl acrylate

EC Number:

202-500-6

EC Name:

Methyl acrylate

Cas Number:

96-33-3

Molecular formula:

C4H6O2

IUPAC Name:

methyl prop-2-enoate

Specific details on test material used for the study:

Identity : Methyl acrylate
Provided by : Basic Acrylic Monomer Manufacturers, Inc. (BAMM)
Date Received : 18 Oct 2017
Storage : Room temperature and humidity
Description : Clear colorless liquid
Sample Preparation : The test article was used as received.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System:
Bovine eyes (at least six months old) were obtained from an abattoir and transported to the laboratory in a refrigerated container containing Hanks’ Balanced Salt Solution (HBSS) with penicillin-streptomycin. The bovine eyes were transported to MB Research on 02 Nov 2017, within 24 hours of harvest.

Pretest Procedures:
Fresh assay solutions were prepared prior to use. MEM solution was prepared by stirring together one jar (9.3 g) of MEM powder (sufficient to make one liter of solution), 2.2 g of sodium bicarbonate, 0.292 g L-glutamine, 10 ml of fetal bovine serum (FBS) and brought to a final volume of 1000 ml with distilled water. The MEM solution was kept in a 32 (±1)°C incubator for the duration of testing. HBSS was prepared by stirring together HBSS powder (sufficient to make one liter) and 0.35 g of sodium bicarbonate and brought to a final volume of 1000 ml with distilled water. HBSS was maintained at room temperature. In addition, MEM solution with phenol red was prepared by stirring together 9.3 g of MEM with phenol red (sufficient to make one liter), 2.2 g of sodium bicarbonate, 0.292 g L-glutamine, 10 ml of fetal bovine serum (FBS) and brought to a final volume of 1000 ml with distilled water. The MEM solution with phenol red was kept in a 32 (±1)°C incubator for the duration of testing.
The eyes will be examined after receipt from the abattoir. Any eye with a cornea exhibiting evidence of neovascularization, pigmentation, opacity or scratches will be discarded.
Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS.
The dissected corneas were mounted in specially designed holders (MC2, formerly Electro-Design – the manufacturer of the Op-KIT opacitometer) that were separated into anterior and posterior chambers and
filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each cornea was visually inspected again to ensure there were no defects.
Prior to collection of the pretest opacity scores, the OP-KIT opacitometer (Electro-Design Corporation, RIOM, France) was calibrated. The calibrator (an empty corneal holder designed to hold the calibrators)
was placed into the calibration chamber of the opacitometer. The opacitometer was first calibrated to zero opacity units by measuring the opacity without a calibrator (i.e., a polyester filter insert provided with
the OP-KIT). Then the opacitometer is calibrated to 75 opacity units using calibrator filter 1. During the test, if the opacity scores exceed 75, the opacitometer is recalibrated in the same manner, using the other
calibrators provided with the OP-KIT equipment (i.e., calibrator filter 2 or 3 that represent opacity of 150 and 225, respectively). A pre-exposure determination of opacity was made for each cornea by measuring
each against the blank supplied by the opacitometer. Fresh MEM was added before taking these baseline opacity readings. Any cornea with a value greater than 7 units was discarded.
The entire holder was incubated at 32 (±1)°C and allowed to equilibrate for at least one hour, but not longer than two hours.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

A volume of 0.75 ml of test article, ethanol, or MEM solution was applied to the epithelium of each of the three test article corneas, three positive control corneas and three negative control corneas in a manner which ensured that the entire cornea was covered. All corneas were dosed via the closed-chamber method.

Duration of treatment / exposure:

10-minute exposure

Duration of post- treatment incubation (in vitro):

All holders and corneas were placed in a horizontal position (anterior chamber facing upward) in the 32 (±1)°C water-jacketed incubator (VWR, model: 1815 TC). After 10 (±1) minutes), the test article, ethanol, or MEM was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red.
The anterior and posterior chambers of the holders were then refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill.
All corneas were incubated at 32 (±1)°C for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. A measurement of opacity was taken with each treated cornea compared to the blank supplied with the OP-KIT. This reading was used in the final IVIS calculations.
Immediately following the two-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.4% sodium fluorescein solution in Dulbecco's phosphatebuffered saline (PBS). Each holder was then returned to the 32 (±1)°C incubator in a horizontal position (anterior chamber facing upward) ensuring contact of the fluorescein with the cornea.

Details on study design:

Study Procedure:
Following the pretest observations, the MEM solution was removed from the anterior chamber. A volume of 0.75 ml of test article, ethanol, or MEM solution was applied to the epithelium of each of the three test
article corneas, three positive control corneas and three negative control corneas in a manner which ensured that the entire cornea was covered. All corneas were dosed via the closed-chamber method.

All holders and corneas were placed in a horizontal position (anterior chamber facing upward) in the 32 (±1)°C water-jacketed incubator (VWR, model: 1815 TC). After 10 (±1) minutes), the test article, ethanol, or MEM was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill.

All corneas were incubated at 32 (±1)°C for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. A
measurement of opacity was taken with each treated cornea compared to the blank supplied with the OP-KIT. This reading was used in the final IVIS calculations.

Immediately following the two-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.4% sodium fluorescein solution in Dulbecco's phosphatebuffered
saline (PBS). Each holder was then returned to the 32 (±1)°C incubator in a horizontal position (anterior chamber facing upward) ensuring contact of the fluorescein with the cornea.

The Spectronic 20-D Colorimeter Spectrophotometer (Milton/Roy, model: 333175) was calibrated prior to use on the same day of dosing. Calibration entailed first adjusting the wavelength to 490nm and the transmittance to 0.0%. The transmittance for a sample of fresh distilled water (in a cuvette, inserted into the sample compartment) was adjusted to 100.0% on the spectrophotometer. The mode was then switched to absborance before collecting optical density study data. After 90 (±5) minutes, the fluid from the posterior chamber of each corneal holder was removed and the amount of dye that passed through
the cornea (permeability) was measured as the optical density at 490nm (i.e., the OD490nm) by a spectrophotometry.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

12.55

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The assay was accepted because the positive control had an IVIS that fell within two standard deviations of the historical mean.

Any other information on results incl. tables

RESULTS:

TEST ARTICLE: Methyl acrylate

Cornea ID	Pretest Opacity Score	10-Minute Opacity Score	2-Hour Opacity Score	Corrected Opacity Scores^1		OD490 nm (Permeability)	Corrected Optical Density^2
				10-Minute	2-Hour
7	5	9	7	4	2	0.880	0.864
8	5	6	13	1	8	0.164	0.148
9	5	7	12	2	7	0.845	0.829
Corrected Mean Optical Density^3=							0.614
2-Hour Corrected Mean Opacity Score^4=							3.34

¹Individual Corrected Opacity Score: 10-minute or 2-hour opacity score minus pretest opacity score

²Individual Corrected Optical Density: Individual test article OD minus the mean OD for the negative control group

³Corrected Mean Optical Density: Mean of the individual corrected optical density values for a given dose group

⁴2-Hour Corrected Mean Opacity Score: Mean of the individual 2-hour corrected opacity scores for a given dose group minus the mean opacity score for the negative control group

NEGATIVE CONTROL: MEM

Cornea ID	Pretest Opacity Score	10-Minute Opacity Score	2-Hour Opacity Score	Corrected Opacity Scores^1		OD490 nm (Permeability)
				10-Minute	2-Hour
1	4	2	5	-2	1	0.015
2	3	2	7	-1	4	0.016
3	3	2	5	-1	2	0.017
Mean of Individual Optical Density =							0.016
2-Hour Corrected Mean Opacity Score^4=							2.33

 

POSITIVE CONTROL: Ethanol

Cornea ID	Pretest Opacity Score	10-Minute Opacity Score	2-Hour Opacity Score	Corrected Opacity Scores^1		OD490 nm (Permeability)	Corrected Optical Density^2
				10-Minute	2-Hour
4	1	27	28	26	27	0.305	0.289
5	3	28	27	25	24	0.845	0.829
6	3	35	33	32	30	0.598	0.582
Corrected Mean Optical Density^3=							0.567
2-Hour Corrected Mean Opacity Score^4=							24.67

 

¹Individual Corrected Opacity Score:10 minute or 2-hour opacity score minus pretest opacity score

²Individual Corrected Optical Density:Individual positive control OD minus the mean OD for the negative control group

³Corrected Mean Optical Density:Mean of the individual corrected optical density values for a given dose group

⁴2-Hour Corrected Mean Opacity Score:Mean of the individual 2-hour corrected opacity scores for a given dose group minus the mean opacity score for the negative control group; no correction was made for the negative control

CALCULATED IN VITRO IRRITANCY SCORES

Test Article	Negative Control	Positive Control
Methyl acrylate	MEM	100% Ethanol
3.34 + (15 x 0.614)	2.33 + (15 x 0.016)	24.67 + (15 x 0.567)
3.34 + 9.21	2.33 + 0.24	24.67 + 8.505
IVIS = 12.55	IVIS = 2.57	IVIS = 33.18

 

Positive Control Historical Data
Mean IVIS	26.98
Standard Deviation	5.01
n	63

 

The ethanol positive control IVIS was 33.18, which fell within the acceptance range of 16.96 - 37.00

(± 2 standard deviations of the historical mean).

Applicant's summary and conclusion

Interpretation of results:

other: The results from this study support the weight of evidence that Methyl Acrylate is not corrosive.

Conclusions:

The objective of the study was to determine the potential for ocular irritation using an alternative to the Draize methodology. This protocol is based on the methodology described in the current OECD Guideline for the Testing of Chemicals No. 437. Three bovine corneas per group were dosed with 0.75 ml of Methyl acrylate, Minimal Essential Media (MEM, negative control), or 100% ethanol (EtOH)) (positive control). Following a 10-minute exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined. Results of the study indicate that the IVIS score for Methyl Acrylate is 12.55.

Executive summary:

The objective of the study was to determine the potential for ocular irritation using an alternative to the Draize methodology. This protocol is based on the methodology described in the current OECD Guideline for the Testing of Chemicals No. 437. Three bovine corneas per group were dosed with 0.75 ml of Methyl acrylate, Minimal Essential Media (MEM, negative control), or 100% ethanol (EtOH)) (positive control). Following a 10-minute exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined. Results of the study indicate that the IVIS score for Methyl Acrylate is 12.55.

Summary

Treatment	In VitroIrritancy Score (IVIS)	Corrected Mean Opacity Score	Corrected Mean Optical Density
Methyl acrylate	12.55	3.34	0.614
100% EtOH (Positive Control)	33.18	24.67	0.567

 

Treatment	In VitroIrritancy Score (IVIS)	Corrected Mean Opacity Score	Mean Optical Density
MEM (Negative Control)	2.57	2.33	0.016

 

Conclusions

Test Article ID	In VitroIrritancy Score (IVIS)	UN GHS Categorization	EURL ECVAM DB-ALM Protocol 127 Classification
Methyl acrylate	12.55	No Prediction Can Be Made	Mild Eye Irritant