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

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

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

Test material

Test material information

Constituent 1

Reference substance name:

Oxirane, [(4-nonylphenoxy)methyl]-, reaction product with ethylene glycol

EC Number:

807-586-4

Cas Number:

634602-80-5

Molecular formula:

UVCB - so Molecular formula i snot applicable

IUPAC Name:

Oxirane, [(4-nonylphenoxy)methyl]-, reaction product with ethylene glycol

Test material form:

liquid: viscous

Remarks:

Reddish-Brown

Details on test material:

Lot #: LL6I0011

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).

In vitro test system

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

Results and discussion

In vitro

Results

Results 1

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

Results 2

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.

Applicant's summary and conclusion

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.