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

Endpoint summary

Description of key information

In vitro Skin Irritation and Corrosion assays

In vitro Eye irritation assay

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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).

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).

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

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

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.

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.

Reference 2

Endpoint:

skin 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

no

Remarks:

This is a non-GLP study, based on OECD 439 test guideline (In Vitro Skin Irritation: Reconstructed Human Epidermis (RHE) Test Method).

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).

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.

Irritation Experimental Procedure:
On the day of treatment, all EpiDerm tissues were transferred to new 6-well plates containing 0.9 mL/well of fresh pre-warmed assay medium and 50 µL liquids of test material in conjunction with negative (DPBS) and positive controls (1% TRITON™ X-100) was dispensed atop the tissue. After dosing, the tissue plates were incubated for 35 ± 1 minutes at standard culture conditions. Following incubation, the plates were transferred to the laminar flow hood and incubated at room temperature for 25 minutes. Following the total of 60 ± 1 minutes treatment period, the tissues were rinsed with sterile DPBS to remove test substance (extra rinses were implemented as necessary). Following rinsing, the tissue inserts were transferred to new 6-well plates containing 0.9 mL/well of fresh pre-warmed assay medium and incubated at standard culture conditions for an initial post-treatment incubation of 24±2 hours. After the initial post-treatment incubation, the inserts were transferred into clean wells of 6 well plates pre-filled with 0.9 mL fresh pre-warmed assay medium and further incubated for additional 18±2 hours (total post-treatment incubation period (recovery): 42 ± 2 hours).
Following post-treatment incubation, tissue inserts were evaluated for cell viability using MTT assay. Each tissue insert was 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. After incubation the tissues were washed with DPBS and formazan was extracted in 2 mL extractant solution 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 439 testing guideline.

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

Duration of treatment / exposure:

On the day of treatment, all EpiDerm tissues were transferred to new 6-well plates containing 0.9 mL/well of fresh pre-warmed assay medium and 50 μL liquids of test material in conjunction with negative (DPBS) and positive controls (1% TRITON™ X-100) was dispensed atop the tissue. After dosing, the tissue plates were incubated for 35 ± 1 minutes at standard culture conditions. Following incubation, the plates were transferred to the laminar flow hood and incubated at room temperature for 25 minutes.

Duration of post-treatment incubation (if applicable):

Following the total of 60 ± 1 minutes treatment period, the tissues were rinsed with sterile DPBS to remove test substance (extra rinses were implemented as necessary). Following rinsing, the tissue inserts were transferred to new 6-well plates containing 0.9 mL/well of fresh pre-warmed assay medium and incubated at standard culture conditions for an initial post-treatment incubation of 24±2 hours. After the initial post-treatment incubation, the inserts were transferred into clean wells of 6-well plates pre-filled with 0.9 mL fresh pre-warmed assay medium and further incubated for additional 18±2 hours (total post-treatment incubation period (recovery): 42 ± 2 hours).

Number of replicates:

3 replicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

3.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

2.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

3.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

In the irritation assay, the positive control (1% TRITON™ X-100) reduced relative mean tissue viability to 6.1% following 60 minute exposure. 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.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The mean relative cell viability of methacryloxyisopropyl acid phthalate and positive control-treated tissues were 3.0% and 6.1% (i.e. ≤ 50%), respectively. Therefore, methacryloxyisopropyl acid phthalate was interpreted as an irritant (UN GHS Cat 2) in the EpiDerm irritation assay.

Executive summary:

Methacryloxyisopropyl acid phthalate was evaluated for skin corrosion and irritation potential in anin vitroEpiDerm skin corrosion assay (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. To assess irritation potential, methacryloxyisopropyl acid phthalate was topically applied to the EpiDerm tissue for 60 minutes, followed by a 42-hour post-exposure recovery. Following the irritation post-exposure recovery and corrosion exposure, the cell viability was then measured in treated and control tissues using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and the data reported as a percentage of the mean of negative control.

Skin corrosion potential of the test substance was classified according to tissue viability following the two exposure times. The test substance is considered corrosive if the mean viability is ≤ 50% at three minutes or ≥ 50% at three minutes but less than 15% at one hour. In this corrosion study, sterile water and 8N potassium hydroxide served as the negative and positive controls, respectively.A test chemical was considered to possess skin irritation potential (UN GHS Cat 1 or 2) if the relative cell viability was less than or equal to 50%. In this irritation study, Dulbecco’s Phosphate Buffered Saline (DPBS) and 1% TRITON™ X-100served as the negative and positive controls, respectively.

Corrosion: The mean tissue viabilities ofmethacryloxyisopropyl acid phthalate-dosed tissues following the three minute exposure period was 82.1% and following the one hour exposure period was 36.5%. Therefore, under these conditions, methacryloxyisopropyl acid phthalate was interpreted as non-corrosive in the EpiDerm corrosion assay.

Irritation: The mean relative cell viability of methacryloxyisopropyl acid phthalate and positive control-treated tissues were 3.0% and 6.1% (i.e.≤50%), respectively. Therefore, methacryloxyisopropyl acid phthalatewas interpreted as anirritant (UN GHS Cat 2) in the EpiDerm irritation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

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

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

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

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.

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

	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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The in vitro corrosion assay was negative, however the in vitro irritation assay was positive. Based on these two results, it is concluded that this substance meets the criteria for classification as a Skin irritant Category 2

Eye irritation

The in vitro eye irritation/corrosion assay was positive, with the ET-40 value of <2 minutes. This substance is therefore considered to be capable of causing irreversible damage to the eye.

Justification for classification or non-classification

Skin irritation: Criteria for classification as irritant category 2 are met.

Eye irritation: Criteria for cateogry 1 are met