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

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

no

Remarks:

This is a non-GLP study, based on OECD test guideline 431 (In vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method).

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

Test material form:

liquid

Specific details on test material used for the study:

Test Material Name: Methacryloxyisopropyl acid phthalate
Lot/Reference/Batch Number: YY00GCV000
Purity/Characterization (Method of Analysis and Reference): The non-GLP purity of the test material was determined to be 62.8% (Sathiosatham, 2017).
Test Material Stability Under Storage Conditions: The record of custody lists the test material as having a 1 year shelf life (Sathiosatham, 2017).

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Cultured human derived epidermal keratinocytes, MatTek Corporation

Vehicle:

unchanged (no vehicle)

Details on test system:

Experiment Procedure:
Upon receipt, the EpiDerm tissue transwell discs were stored at 2-8 ºC and used within 48 hours of receipt from the supplier. On the day prior to testing, each EpiDerm tissue transwell disc was inspected for air bubbles between the agarose gel and Millicell® insert prior to opening the sealed package. Tissue discs with air bubbles greater than 50% of the Millicell® area were not used for testing. An appropriate number of EpiDerm tissues were aseptically removed from the 24-well shipping plate and transferred to a 6-well plate containing pre-warmed assay medium. The EpiDerm tissues were then incubated at approximately 37ºC in a humidified atmosphere of approximately 5% CO2 for 18 ± 3 hours to acclimate the tissue prior to treatment.

Corrosion Experimental Procedure:
On the day of treatment, the EpiDerm tissues were transferred to new 6-well plates containing 0.9 mL/well of fresh pre-warmed assay medium and exposed to the test material (50 µL), in conjunction with negative (water) and positive controls (8N Potassium hydroxide solution (KOH)) for two exposure periods: 3 and 60 minutes. The 3 minute exposure groups were held at room temperature during the treatment incubation, while the 60 minute exposure groups were placed in the incubator at standard culture conditions (approximately 37ºC with 5% CO2/95% air) during treatment. Following the exposure period, the tissues were rinsed with sterile DPBS (approximately 20 times) to remove test substance (extra rinses were implemented as necessary). Following rinsing, tissue inserts were evaluated for cell viability using MTT assay. Each tissue insert was transferred to a well containing 300 µL MTT (1mg/mL) solution in a 24-well plate and tissues were incubated for 3 ± 0.1 hr at standard cell culture conditions. After incubation the tissues were washed with DPBS and formazan was extracted in 2 mL extractant solution (isopropanol) overnight at room temperature. The extract solution was mixed and 2 x 200 µL aliquots were transferred to the appropriate wells of a 96-well plate. The amount of extracted formazan was measured spectrophotometrically at 570 nm (OD570) with a Microplate Reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Preparation of the Test Material:
Test material(s) will be tested as neat (100%) or as provided, following the OECD 431 guideline.

Route of Administration:
The test material and control substances were administered by topical application to the tissue disc. In the corrosion assay, 50 μL of undiluted methacryloxyisopropyl acid phthalate was directly dispensed atop the tissue.

Duration of treatment / exposure:

Corrosion Experimental Procedure:
On the day of treatment, the EpiDerm tissues were transferred to new 6-well plates containing 0.9 mL/well of fresh pre-warmed assay medium and exposed to the test material (50 μL), in conjunction with negative (water) and positive controls (8N Potassium hydroxide solution (KOH)) for two exposure periods: 3 and 60 minutes. The 3 minute exposure groups were held at room temperature during the treatment incubation, while the 60 minute exposure groups were placed in the incubator at standard culture conditions (approximately 37ºC with 5% CO2/95% air) during treatment.

Duration of post-treatment incubation (if applicable):

Corrosion Experimental Procedure:
Following the exposure period, the tissues were rinsed with sterile DPBS (approximately 20 times) to remove test substance (extra rinses were implemented as necessary). Following rinsing, tissue inserts were evaluated for cell viability using MTT assay. Each tissue insert was transferred to a well containing 300 μL MTT (1mg/mL) solution in a 24-well plate and tissues were incubated for 3 ± 0.1 hr at standard cell culture conditions.

Number of replicates:

3 replicates

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates (3 minute)

Value:

82.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates (60 minutes)

Value:

36.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

In the corrosion assay, the positive control (KOH) reduced the relative mean tissue viability to 19.8% and 11.2%, following 3 and 60 minute exposures, respectively. The positive control results demonstrated appropriate study conduct and tissue responsiveness.

Assessment of Direct Test Material Reduction of MTT:
One limitation of this assay method is a possible interference of the test material with the MTT assay. A colored test substance or one that directly reduces MTT (and thereby mimics dehydrogenase activity of the cellular mitochondria) may interfere with the MTT end point. To assess potential direct test material reduction, 50 μL of methacryloxyisopropyl acid phthalate was incubated with 1 mg/mL of the MTT reagent at standard cell culture conditions for 60 min. Untreated (no test material) MTT medium was used as the negative control.
Results from this experiment suggested no direct reduction of MTT dye by methacryloxyisopropyl acid phthalate, as the test material did not turn the MTT solution to a blue/purple color.

Assessment of Test Material Color Interference:
Potential color interference can arise from colored test chemicals or test chemicals that become colored when in contact with water (environment during exposure) and/or isopropanol (extracting solution). To assess the potential of color interference, 50 μL of methacryloxyisopropyl acid phthalate was incubated with 300 μL of H2O at standard cell culture conditions for 60 min. Untreated (no test material) H2O was used as the negative control. As the solution was not colored at the end of the 60 min incubation, it can be concluded that the test material does not possess the potential to induce color interference.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean tissue viabilities of methacryloxyisopropyl acid phthalate-dosed tissues following the 3 minute exposure period was 82.1% and following the one hour exposure period was 36.5%. Since the mean tissue viability was > 50% at the 3 minute exposure and >15% at the 60 minute exposure, methacryloxyisopropyl acid phthalate was interpreted as a non-corrosive (NC) in the EpiDerm corrosion assay.

Executive summary:

Methacryloxyisopropyl acid phthalate was evaluated for skin corrosion and irritation potential in in vitro EpiDerm skin corrosion and irritation assays (MatTek Corporation; Ashland, MA). The EpiDerm tissue model consists of normal, human-derived epidermal keratinocytes that are cultured to form a stratified, squamous epithelium similar to that found in human epidermis tissue. To assess corrosion potential, methacryloxyisopropyl acid phthalate was topically applied to the EpiDerm tissue for 3 and 60 minutes. In this study, sterile water and 8N potassium hydroxide (KOH) served as the negative and positive controls, respectively. At the end of exposure, cell viability in treated and control tissues were measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The test substance is considered corrosive (UN GHS Corrosive) if the relative mean viability is ≤ 50% at three minutes or ≥ 50% at three minutes but less than 15% at one hour.

In the corrosion assay, the mean tissue viability of the positive control (KOH) following the 3 minute and 60 minute exposures were 19.8% and 11.2%, respectively, thereby demonstrating appropriate assay responsiveness. The mean tissue viabilities of methacryloxyisopropyl acid phthalate-dosed tissues following the 3 minute and 60 minute exposures were 82.1% and 36.5%, respectively. Therefore, under these conditions, methacryloxyisopropyl acid phthalate was interpreted as negative for corrosion potential in the EpiDerm corrosion assay (UN GHS Non-Corrosive).

Therefore, under the conditions of the EpiDerm corrosion study, methacryloxyisopropyl acid phthalate is predicted to be negative for dermal corrosion potential.