Reaction mass of 2-(((1-(methacryloyloxy)propan-2-yl)oxy)carbonyl)benzoic acid and 2-((2-(methacryloyloxy)propoxy)carbonyl)benzoic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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

Deviations:

no

GLP compliance:

no

Remarks:

Study performed in accordance with principles of GLP

Test material

Test material information

Constituent 1

Reference substance name:

1-methyl-2-[(2-methyl-1-oxoallyl)oxy]ethyl hydrogen phthalate

EC Number:

265-951-8

EC Name:

1-methyl-2-[(2-methyl-1-oxoallyl)oxy]ethyl hydrogen phthalate

Cas Number:

65859-45-2

Molecular formula:

C15H16O6

IUPAC Name:

2-{[2-(methacryloyloxy)-1-methylethoxy]carbonyl}benzoic acid

Details on test material:

Lot/Reference/Batch Number: YY00GCV000

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcular model (model number OCL-200) uses Normal Human Epidermal Keratinocytes (NHEK) from a single donor as the cell source. The cells are cultured on polycarbonate membranes of cell culture inserts (MILLICELLs, 10 mm diameter,
0.6 cm² surface), in serum-free medium to form a multilayered (5-8 cell layers), highly differentiated stratified, squamous epithelia that closely mimics human eye (corneal) epithelium at biochemical and physiological levels

Supplier: MatTek Corporation; Ashland, Massachusetts

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

Duration of post- treatment incubation (in vitro):

The test consisted of topical application of the test material to the EpiOcular tissue for three time points (2, 15, or 30 min) followed by thorough washing with DPBS and incubating with cell culture medium for an initial 12 minutes and then an additional 120 minutes.

Details on study design:

Upon receipt, the EpiOcular tissue transwell discs were stored at 2-8ºC and used within
48 hours of receipt from the supplier. On the day of testing, an aliquot of 0.9 mL of EpiOcular assay medium (MatTek Corporation) was dispensed into the wells of 6-well plates. Each EpiOcular tissue disc was inspected for air bubbles between the agarose gel and Millicell insert prior to opening the sealed package. Cultures with air bubbles greater than 50% of the Millicell (transwell disc) area were not used for testing.
The EpiOcular tissues were incubated at approximately 37ºC in a humidified atmosphere of approximately 5% CO2 for 60 ± 5 min. At the end of the first pre-incubation period, the inserts were transferred into wells containing fresh, warm assay medium. The testing included treating the inserts with 50 µL of DPBS (negative control; 30±2 minutes exposure time), 0.3% TRITON™ X-100 (positive control; 30±2 minutes exposure time), and the test material(s) (three exposure times; 2, 15, or 30 min). Following the exposure periods, the EpiOcular tissues were carefully washed with DPBS (at least 5 times) to remove residual test substance. Following washes, the Millicell inserts were submerged in fresh assay media and incubated at 37ºC and 5% CO2 for approximately 12±2 minutes to remove any test chemical absorbed into the tissue. Subsequently, the Millicell inserts were further incubated in fresh medium for 120±15 minutes at standard culture conditions (post-exposure incubation). After incubation, the tissue inserts were transferred to a well containing 300 µL MTT solution in a 24-well plate and tissues were incubated for 3 ± 0.1 hr at standard cell culture conditions. Following incubation, the tissues were washed with DPBS and the MTT dye (formazan crystals) was solubilized and extracted from the inserts by incubating each insert in 2 mL of extract reagent (MatTek Corporation) overnight at room temperature. The extract solution was mixed and 2 x 200 µL aliquots of the extract solution was transferred to a 96-well plate and the optical density of the extracted formazan was quantified at 570 nm (OD570) using a Microplate Reader.

Results and discussion

In vitro

Results

Irritation parameter:

other: ET-40

Remarks:

minutes

Run / experiment:

mean

Value:

< 2

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

	Treatment plus 120±15 Min Recovery
Chemical Name	Replicate 1	Replicate 2	Replicate 3	Mean Viability (%)
Methacryloxyisopropyl Acid Phthalate 2 minute	15.4	11.7	14.6	13.9
Methacryloxyisopropyl Acid Phthalate 15 minute	5.7	7.0	8.6	7.1
Methacryloxyisopropyl Acid Phthalate 30 minute	3.8	3.0	5.4	4.1
Negative Control^*	98.6	99.9	101.5	100.0
Positive Control^*	18.3	28.8	21.6	22.9
^*Negative Control: DPBS; Positive Control: 0.3% TRITON™ X-100

Chemical Name	ET-40 (min)	EpiOcular Classification (Neat)
Methacryloxyisopropyl acid phthalate	< 2	UN GHS Cat 1
Positive Control^*	< 30	UN GHS Cat 1 or 2
^*Positive Control: 0.3% TRITON™ X-100

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The ET-40 value of methacryloxyisopropyl acid phthalate was < 2 minutes. Therefore, under these conditions, methacryloxyisopropyl acid phthalate was interpreted as a potential severe ocular irritant (UN GHS Cat 1) in the EpiOcular assay.