1,2-Ethanediol, reaction products with branched 2-[(4-nonylphenoxy)methyl]oxirane

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study performed in accordance with GLP requirements (but not a GLP 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:

Study performed in accordance with GLP preactices, but was not a fully audited GLP study

Specific details on test material used for the study:

Test Material Name: EPON™ Resin CS-337
Lot/Reference/Batch Number: LL6I0011
Purity/Characterization (Method of Analysis and Reference): The non-GLP certificate of analysis lists the test material as containing 0.18% ethylene glycol and having a viscosity of 33633 cPs at 25°C (Babineaux, 2016).
Stability: The record of custody lists the test material as having a 2 year shelf life (Brown, 2016).

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The EpiDerm System consists of organized basal, spinous, granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Details on animal used as source of test system:

The EpiDerm System (model EPI-200) consists of normal, human-derived epidermal kerotinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous, granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Justification for test system used:

The EpiDerm three-dimensional human skin model has been extensively characterized (EC-ECVAM, 2009) and is approved (OECD TG 431, 2016) for identification and classification of non-corrosive and corrosive substances and mixtures in accordance with United Nations Globally Harmonized System (UN GHS). This procedure also allows a partial sub-categorization of corrosives into sub-category 1A and a combination of sub-categories 1B-and-1C. The EpiDerm model uses human-derived, non-transformed keratinocytes as cell source, which are cultured to form a multilayered, highly differentiated model that closely mimics human epidermis at biochemical and physiological levels.

Vehicle:

unchanged (no vehicle)

Details on test system:

BACKGROUND:
The potential for chemically-induced corrosion is an important consideration in establishing procedures for the safe handling, packing, and transport of chemicals (UN, 2007). The EpiDerm three-dimensional human skin model has been extensively characterized (EC-ECVAM, 2009) and is approved (OECD TG 431, 2016) for identification and classification of non-corrosive and corrosive substances and mixtures in accordance with United Nations Globally Harmonized System (UN GHS). This procedure also allows a partial sub-categorization of corrosives into sub-category 1A and a combination of sub-categories 1B-and-1C. The EpiDerm model uses human-derived, non-transformed keratinocytes as cell source, which are cultured to form a multilayered, highly differentiated model that closely mimics human epidermis at biochemical and physiological levels.
The test is based on the principle that chemicals with corrosive potential can cause cytotoxic response to the stratum corneum and the rate of cytotoxicity is proportional to corrosion potency. The test is designed to predict and classify the skin corrosion potential of a test substance by assessment of its effect upon a three dimensional human epidermis model following short term exposure.
This test consisted of topical application of the test material to the EpiDerm tissue for two time points (3 or 60 min) followed by thorough washing with Dulbecco’s phosphate buffered saline (DPBS). The EpiDerm tissues were then evaluated for viability using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Berridge et al., 1996). Relative cell viability was calculated for each tissue as a percent of the mean of the negative control-treated tissues. Skin corrosion potential was classified based on cell viability following exposure at 3 min and 60 min.

Principle of the Test System:
The EpiDerm System (model EPI-200) consists of normal, human-derived epidermal kerotinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous, granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues are cultured on polycarbonate membranes of cell culture inserts (MILLICELLs, 10 mm diameter, 0.6 cm² surface) and shipped as kits, containing 24 tissues mounted on agarose.

Supplier and Location:
MatTek Corporation; Ashland Massachusetts

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

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Route of Administration:
The test material and control substances were administered by topical application to the tissue disc. In this case, 50 μL of undiluted EPON™ Resin CS-337 was directly dispensed atop the tissue.

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 of 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 60 ± 5 min to acclimate the tissue prior to
treatment.
At the end of the first incubation period, 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 of test material), in conjunction with negative (sterile water) and positive controls (8N potassium hydroxide solution (KOH)) for two exposure periods: 3 or 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 the test substance. Following rinsing, tissue inserts were evaluated for cell viability using the MTT assay. Each tissue insert was transferred to a well containing 300 μL MTT (1 mg/mL) solution in a 24-well plate and were incubated for 3 ± 0.1 hr at standard cell culture conditions. After incubation the tissues were washed once 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 is measured spectrophotometrically at 570 nm (OD570) with a Microplate Reader.

Duration of treatment / exposure:

EpiDerm tissues were exposed to the test material (50 μL of test material), in conjunction with negative (sterile water) and positive controls (8N potassium hydroxide solution (KOH)) for two exposure periods: 3 or 60 minutes.

Duration of post-treatment incubation (if applicable):

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

Number of replicates:

Three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 Minute Treatment: Mean Viability (%) for 3 replicates

Value:

99

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 Minute Treatment: Mean Viability (%) for 3 replicates

Value:

106.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Assessment of Direct Test Material Reduction of MTT:
Results from this experiment suggested no direct reduction of MTT dye by EPON™ Resin CS-337, as the test material did not turn the MTT solution to a blue/purple color.

Assessment of Test Material Color Interference:
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.

All parameters for the test to be considered acceptable were met.

Interpretation of results:

GHS criteria not met

Conclusions:

The mean tissue viabilities of EPON™ Resin CS-337-dosed tissues following the 3- minute exposure period was 99.0% and following the one hour exposure period was 106.9%. Since the mean tissue viability was > 50% at the 3 minute exposure and >15% at the 60 minute exposure, EPON™ Resin CS-337 was interpreted as a potential noncorrosive (NC) in the EpiDerm corrosion assay.

Executive summary:

EPON™ Resin CS-337 was evaluated for skin corrosion potential in an in vitro EpiDerm 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. In this assay, EPON™ Resin CS-337 was topically applied to the EpiDerm tissue for 3 and 60 minutes. 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. Sterile water and 8N potassium hydroxide served as the negative and positive controls, respectively.

The mean tissue viabilities of EPON™ Resin CS-337-dosed tissues following the three minute exposure period was 99.0% and following the one hour exposure period was 106.9%. Therefore, under these conditions, EPON™ Resin CS-337 was interpreted as non-corrosive in the EpiDerm corrosion assay.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study performed in accordance with GLP requirements (but not a GLP study).

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Remarks:

Study performed in accordance with GLP preactices, but was not a fully audited GLP study

Specific details on test material used for the study:

Test Material Name: EPON™ Resin CS-337
Lot/Reference/Batch Number: LL6I0011
Purity/Characterization (Method of Analysis and Reference): The record of custody lists the purity as 100% (Brown, 2016).
Test Material Stability Under Storage Conditions: The record of custody lists the test material as having a 2 year shelf life (Brown, 2016).

Test system:

human skin model

Source species:

human

Cell source:

other: human-derived epidermal keratinocytes (NHEK)

Justification for test system used:

The EpiDerm three-dimensional human skin model has been extensively characterized (EC-ECVAM, 2009) and is approved (OECD TG 439, 2013) for identification and classification of skin irritation hazard according to the UN GHS and EU classification system (Category 2/R38 or No label).

Vehicle:

unchanged (no vehicle)

Details on test system:

Test System:
The potential for chemically-induced skin irritation and corrosion are important considerations in establishing procedures for the safe handling, packing, and transport of chemicals (UN, 2007). The EpiDerm three-dimensional human skin model has been extensively characterized (EC-ECVAM, 2009) and is approved (OECD TG 439, 2013) for identification and classification of skin irritation hazard according to the UN GHS and EU classification system (Category 2/R38 or No label). The EpiDerm model uses human-derived epidermal keratinocytes (NHEK) as the cell source, which are cultured to form a multi-layered, highly differentiated model that closely mimics human epidermis at biochemical and physiological levels.
The test is based on the principle that chemicals with irritant potential can cause a cytotoxic response to the stratum cornea and the rate of cytotoxicity is proportional to irritation potency. In the assay, the EpiDerm tissue model was incubated with the test chemical for 60 minutes, followed by 42-hour incubation (recovery) under standard cell culture conditions. Following the post-treatment incubation period, cell viability was assessed using the MTT (3-[4,5- dimethylthiazol-2-yl] -2,5 – diphenyltetrazolium bromide) assay (Mosmann, 1983). Relative cell viability was calculated for each tissue as % of the mean of the negative control-treated tissues. A test chemical was interpreted as a potential skin irritant (UN GHS Cat 1 or 2) or non-irritant (UN GHS Cat NC), when the cell viability was ≤ or > 50%, respectively (OECD, 2013).

Study Design:
The EpiDerm System (model number EPI-200 EpiDerm cultures) consists of normal, human-derived epidermal kerotinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis (Figure 1). It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues are cultured on polycarbonate membranes of cell culture inserts (MILLICELLs, 10 mm diameter, 0.6 cm² surface) and shipped as kit, containing 24 tissues mounted on agarose.

Supplier and Location:
MatTek Corporation; Ashland, MA.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Preparation of the Treatment Solution:
The chemical was tested at a concentration of 100% (neat/as provided), following supplier’s protocol (EpiDerm SOP) and OECD 439 test guideline. Thirty microliters of undiluted EPON™ Resin CS-337 was directly dispensed atop the tissue.

Route of Administration:
The test chemical was administered by topical application to the surface of the EpiDerm tissues.

Experimental Procedure:
EpiDerm tissue cultures were used within 24 to 48 hours of receipt from the supplier. Upon receipt, the EpiDerm tissues were stored at 2-8ºC until used. On the day prior to testing, EpiDerm assay medium was warmed to room temperature and an aliquot of 0.9 mL was dispensed into each well of a 6-well plate. The EpiDerm tissues were transferred from a 24-well shipping plate to a 6-well plate containing the fresh, pre-warmed assay medium. Prior to transferring, each EpiDerm tissue was inspected for air bubbles between the agarose gel and MILLICELL insert. Tissues with air bubbles greater than 50% of the MILLICELL area were discarded. The EpiDerm tissues were incubated at approximately 37ºC in a humidified atmosphere of approximately 5% CO2 for 60 ± 5 min. At the end of the first preincubation period, the inserts were transferred into wells containing fresh warm assay medium and were incubated overnight (18 ± 3 hrs) to acclimate the tissue.
On the day of treatment, the EpiDerm tissues were exposed to the test chemical as described above (30 μL of test material or controls). After dosing, the tissue plates were transferred to a cell culture incubator and were incubated for 35 ± 1 minute 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 minute treatment period, the tissues were rinsed with sterile DPBS to remove the test substance. The tissues were then transferred to new 6-well plates containing 0.9 mL/well 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 new 6-well plates pre-filled with 0.9 mL fresh pre-warmed assay medium and incubated for additional 18±2 hours (total post-treatment incubation period of 42 ± 2 hours).
Following post-treatment incubation, tissue inserts were evaluated for cell viability using the 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 with 2 hours shaking at room temperature. The amounts of extracted formazan from each tissue was measured spectrophotometrically at 570 nm (OD570) using a Microplate Reader (Molecular Devices).

Duration of treatment / exposure:

60 ± 1 minute treatment period

Duration of post-treatment incubation (if applicable):

Following the total of 60 ± 1 minute treatment period, the tissues were rinsed with sterile DPBS to remove the test substance. The tissues were then transferred to new 6-well plates containing 0.9 mL/well 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 new 6-well plates pre-filled with 0.9 mL fresh pre-warmed assay medium and incubated for additional 18±2 hours (total post-treatment incubation period of 42 ± 2 hours).

Number of replicates:

Three

Irritation / corrosion parameter:

other: Mean Viability %

Run / experiment:

Mean Viability % for 3 replicates

Value:

2.6

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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, 30 μL of EPON™ Resin CS-337 was incubated with 1 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 EPON™ Resin CS-337, as the test material did not turn the MTT solution to a blue/purple color.

ACCEPTANCE CRITERIA:
The results for negative and positive controls met assay acceptance criteria, suggesting appropriate conduct of the study.
1) The corrected mean OD570 value of the negative control tissues (exposed for 60 minutes) was 2.604 (i.e. ≥ 1.00; criteria set by the tissue manufacturer).
2) The relative mean viability of positive control (1% TRITON™ X-100) was 3.2% (i.e. <50% compared to negative control).

Interpretation of results:

other: EPON™ Resin CS-337 was interpreted as a potential irritant (UN GHS Cat 1 or 2) in the EpiDerm irritation assay.

Conclusions:

The mean relative tissue viability for EpiDerm tissues treated with EPON™ Resin CS-337 and positive control (1% TRITON™ X-100) were 2.6% and 3.2%, respectively. According to the EpiDerm skin irritancy prediction model, a test substance is considered irritant to skin if the mean tissue viability is ≤ 50%, therefore, based on the present EpiDerm study results, EPON™ Resin CS-337 was interpreted to be a potential irritant (UN GHS Cat 1 or 2) to skin.

Executive summary:

EPON™ Resin CS-337 was evaluated for skin irritation potential in an in vitro EpiDerm skin irritation assay (MatTek Corporation; Ashland, MA). The EpiDerm tissue model consists of normal, human-derived epidermal keratinocytes that are cultured to form a multilayered, differentiated model of human epidermis. In this assay, EPON™ Resin CS-337 was topically applied to the EpiDerm tissue for 60 minutes, followed by a 42-hour postexposure recovery. Following recovery, the cell viability was 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. 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 study, Dulbecco’s Phosphate Buffered Saline (DPBS) and 1% TRITON™ X-100 served as the negative and positive controls, respectively.

The mean relative cell viability of EPON™ Resin CS-337 and positive control-treated tissues were 2.6% and 3.2% (i.e. ≤ 50%), respectively, therefore, EPON™ Resin CS-337 was interpreted as a potential irritant (UN GHS Cat 1 or 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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This is a non-GLP study and is based on OECD TG 492.

Qualifier:

equivalent or similar 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:

This is a non-GLP study and is based on OECD TG 492.

Deviations:

no

GLP compliance:

no

Remarks:

Study performed in accordance with GLP preactices, but was not a fully audited GLP study

Specific details on test material used for the study:

Test Material Name: EPON™ Resin CS-337
Lot/Reference/Batch Number: LL6I0011
Purity/Characterization (Method of Analysis and Reference): The non-GLP certificate of analysis lists the test material as containing 0.18% ethylene glycol and having a viscosity of 33633 cPs at 25°C (Babineaux, 2016). The record of custody lists the test material as 100% of the desired substance (Brown, 2016).
Test Material Stability Under Storage Conditions; The record of custody lists the test material as having a 2 year shelf life (Brown, 2016).

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcular three-dimensional model has been extensively characterized and currently has an OECD test guideline (OECD 492) for identifying chemicals not requiring classification and labeling for eye irritation or serious eye damage. The EpiOcular model estimates the potential ocular irritation of a test substance by measuring cytotoxicity following topical exposure (Freeman et al., 2010) (MatTek Corporation, Ashland, MA). This assay assumes that in vitro cytotoxicity is directly
proportional to in vivo damage that a test substance would inflict upon exposure to the eye (cornea) (Jackson et al., 2006). This assumption is based in part on the Maurer et al. (2002) proposed hypothesis, which suggests that the level of ocular irritation is related to the extent of initial injury, regardless of the processes leading to tissue damage.

Principle of the Test System:
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. The EpiOcular tissue is mitotically and metabolically active and releases many of the pro-inflammatory agents (cytokines) that are important in ocular irritation and inflammation (Klausner et al., 2000).

Supplier and Location:
MatTek Corporation; Ashland, Massachusetts

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Preparation of the Test Material:
Test material was tested at a concentration of 100% (neat/as provided), following supplier’s protocol (MatTek Corporation).

Route of Administration:
The test material was administered by topical application to the ocular tissue.

Experiment Procedure:
Upon receipt, the EpiOcular tissue transwell discs were stored at 2-8 ºC 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 (liquids) or 50 mg (solids), DPBS (negative control; 30±2 minutes exposure time), 0.3% TRITON™ X-100 (positive control; 30±2 minutes exposure time), or 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.

Duration of treatment / exposure:

The testing included treating the inserts with 50 μL (liquids) or 50 mg (solids), DPBS (negative control; 30±2 minutes exposure time), 0.3% TRITON™ X-100 (positive control; 30±2 minutes exposure time), or test material(s) (three exposure times; 2, 15, or 30 min).

Duration of post- treatment incubation (in vitro):

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).

Number of animals or in vitro replicates:

3 replicates per time-point and for positve and negative control.

Irritation parameter:

other: Mean Viability %

Run / experiment:

2 minute

Value:

70.4

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Mean Viability %

Run / experiment:

15 minute

Value:

42.3

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Mean viability %

Run / experiment:

30 minute

Value:

30.6

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Assessment of Direct Test Chemical 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, 30 μL of EPON™ Resin CS- 337 was incubated with 1 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 EPON™ Resin CS-337, as the test material did not turn the MTT solution to a blue/purple color.

Criteria for Determination of a Valid Test:
The results for negative and positive controls met the following assay acceptance criteria, suggesting appropriate conduct of the study:
1) The corrected mean OD570 value of the negative control tissues (exposed for 60 minutes) was 2.009 (i.e., ≥ 1.00 criteria set by the tissue manufacturer).
2) The relative mean viability of the positive control (0.3% TRITON™ X-100) was 13.7% (i.e. < 50% compared to the negative control).

Text Table 1. Culture Viability of EPON™ Resin CS-337 in the EpiOcular Eye Irritation Model

Chemical Name	Treatment plus 120±15 Min Recovery			Mean Viability (%)
	Replicate 1	Replicate 2	Replicate 3
EPON™ Resin CS-337 2 minute	79.9	53.6	77.7	70.4
EPON™ Resin CS-337 15 minute	49.8	37.4	39.8	42.3
EPON™ Resin CS-337 30 minute	33.4	24.8	33.5	30.6
Negative Control*	99.3	102.6	98.1	100.0
Positive Control*	15.9	11.2	14.0	13.7

*Negative Control: DPBS; Positive Control: 0.3% TRITON™ X-100

Text Table 2. ET-40 of EPON™ Resin CS-337 in EpiOcular Eye Irritation Model

Chemical Name	ET-40 (min)	EpiOcular Classification (Neat)
EPON™ Resin CS-337	9.5	UN GHS Cat 2
Positive Control	< 30.0	UN GHS Cat 1 or 2

*Positive Control: 0.3% TRITON™ X-100

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The mean relative cell viability of the positive control-treated tissue was 13.7% (i.e. ≤ 40%) and the ET-40 value of EPON™ Resin CS-337 was 9.5 minutes (Text Tables 1 and 2). Therefore, under these conditions, EPON™ Resin CS-337 was interpreted as a potential ocular irritant (UN GHS Category 2) in the EpiOcular assay.

Executive summary:

EPON™ Resin CS-337 was evaluated for eye irritation potential in an in vitro EpiOcular eye irritation assay (MatTek Corporation; Ashland, MA). The EpiOcular tissue model consists of normal, human-derived epidermal keratinocytes that are cultured to form a stratified, squamous epithelium similar to that found in the cornea. In this assay, EPON™ Resin CS-337 was topically applied to the EpiOcular tissue for 2, 15, or 30 minutes followed by a 120±15 minute post-exposure recovery. Following recovery, the cell viability was 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. An ET-40 value was calculated for EPON™ Resin CS-337, which is the time of exposure that resulted in reduction in cell viability to 40% of negative control level.

The test substance was considered a severe irritant (UN GHS Cat 1) or an irritant (UN GHS Cat 2) in the EpiOcular assay if the ET-40 was less than 3 or 30 minutes, respectively. The test substance was considered a non-irritant (UN GHS Cat NC) if the ET-40 was > 30 minutes. In this study, Dulbecco’s Phosphate Buffered Saline (DPBS) and 0.3% TRITON™ X-100 served as the negative and positive controls, respectively. The mean relative cell viability of the positive control-treated tissue was 13.7% (i.e. ≤ 40%) and the ET-40 value of EPON™ Resin CS-337 was 9.5 minutes. Therefore, under these conditions, EPON™ Resin CS-337 was interpreted as a potential ocular irritant (UN GHS Category 2) in the EpiOcular assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation - in the In vitro skin irritation assay, the test material was identified as a potential irritant, however the assay used was not able to discriminate between irritants and corrosive substances. Therefore an in vitro corrosion assay was performed and the results confirmed the substance was not corrosive, and therefore based on the weight of evidence, it is concluded the substance is a category 2 skin irritant.

In the in vitro eye irritation assay, the test substance met the criteria for a category 2 eye irritant.