N-butyl phenyl ether

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

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:

2016

Report date:

2016

Materials and methods

Test guideline

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 study is not designed to fulfill any testing guidelines. The current protocol is a
streamlined version of OECD TG 492 test guideline.

Deviations:

no

GLP compliance:

no

Remarks:

THe study was conducted in accordance with the principles of GLP (reporting, data auditing, data retention, GLP qualification fo all equipment and study personel) but was not officially conducted as a GLP study.

Test material

Test material information

Constituent 1

Reference substance name:

N-butyl phenyl ether

EC Number:

214-426-1

EC Name:

N-butyl phenyl ether

Cas Number:

1126-79-0

Molecular formula:

C10H14O

IUPAC Name:

butoxybenzene

Details on test material:

Test Material Name: Butyl phenyl ether
Chemical Name: Butoxy benzene
Synonyms: None
Lot/Reference/Batch Number: 06027KH
Purity/Characterization (Method of Analysis and Reference): The non-GLP certificate of analysis lists the purity as 99.5% by gas liquid chromatography with structure confirmation by infrared spectroscopy (Rajzer, 2007).

Specific details on test material used for the study:

Test Material Name: Butyl phenyl ether
Chemical Name: Butoxybenzene
Lot/Reference/Batch Number: 06027KH
Purity/Characterization (Method of Analysis and Reference): The non-GLP certificate of analysis lists the purity as 99.5% by gas liquid chromatography with structure confirmation by infrared spectroscopy (Rajzer, 2007).
Stability: Butyl phenyl ether, lot 22466, was determined to be stable for 2 weeks at 54°C which is equivalent to 24 months under ambient storage conditions as tested under USEPA OPPTS Guideline 830.6317. (Ferruzzi, 2016).
The test material butyl phenyl ether, lot 06027KH was not tested for neat test material stability. However, neat test material stability was conducted for butyl phenyl ether, Lot 22466, under GLP conditions determining the neat test material to be stable for 24 months under ambient storage conditions.

Test animals / tissue source

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 TG 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 (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 multi-layered (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.

Supplier and Location:
MatTek Corporation; Ashland Massachusetts

Test system

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 MatTek Corporation’s recommended procedure (Ocular Irritation Protocol: Neat Method-MTT).

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

Experiment Procedure:
Upon receipt, the EpiOcular tissue kits were stored at 2-8ºC and used within 24 to 48 hours of receipt from the supplier. On the day of testing, an aliquot of 0.9 mL of Dulbecco’s Modified Eagle’s medium (MatTek Corporation) was dispensed into the wells of 6-well plates. Each EpiOcular tissue was inspected for air bubbles between the agarose gel and Millicell insert prior to opening the sealed package. Tissue samples with air bubbles greater than 50% of the Millicell 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 exposure time), 0.3% Triton X-100 (positive control; 30±2 exposure time) and the test material (three exposure times; 2, 15, or 30 min). Following the exposure periods, the EpiOcular tissues were carefully washed with Dulbecco phosphate buffered saline (DPBS; GIBCO, Grand Island, NY) (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) at room temperature. 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. After incubation the tissues were washed with DPBS and the MTT dye (formazan crystals) is solubilized and extracted from the inserts by incubating each insert in 2 mL of extract reagent (MatTek Corporation) for at least 2 hours with gentle shaking or overnight at room temperature. The extract solution was mixed and two 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 exposure time), 0.3% Triton X-100 (positive control; 30±2 exposure time) and the test material (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) at room temperature. Subsequently, the Millicell inserts were further incubated in fresh medium for 120±15 minutes at standard culture conditions (post-exposure incubation).

Results and discussion

In vitro

Results

Irritation parameter:

other: ET-40

Remarks:

Value is in minutes

Value:

30

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Criteria for Determination of a Valid Test:
The results for negative and positive controls met the assay acceptance criteria, suggesting appropriate conduct of the study.
1) The corrected mean OD570 value of the negative control tissues (exposed for 30 minutes) was 2.454 (i.e., ≥ 1.00; criteria set by the tissue manufacturer).
2) The ET40 value of the positive control (26.9 minutes) was within two standard deviations of the historical mean of manufacture data (12.2 – 37.5 minutes; Kubilus et al., 2005)

Calculation of ET-40 value:
The ET-40 value represents the test material exposure time that results in reduction in cell viability to 40% of negative control level. The ET-40 is determined by linear interpolation between time-points above and below 40% cytotoxicity. To determine ET-40, the dosing time is represented in log scale and % viability in linear scale and a supplier provided Microsoft Excel 5.0 macro is used. 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 >30minutes.

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. This issue arises for test materials that remain on the tissue after several postexposure washes. In the present study, Butyl phenyl ether was evaluated for potential interaction with the MTT dye.
Approximately 100 mg of Butyl phenyl ether was added to 1 mL of MTT solution in a tube and the mixture was incubated in the dark in an incubator with shaking at approximately 37°C for one hour. A negative control (100 μL of DPBS) was tested concurrently. Results from this experiment suggested that Butyl phenyl ether not interfere with the MTT dye, as the test material did not turn MTT solution to blue/purple crystals.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The ET-40 values of Butyl phenyl ether and positive control were 30.0 minutes and 26.9 minutes respectively. As the ET-40 of Butyl phenyl ether was 30 minutes, Butyl phenyl ether was classified as a potential mild (UN GHS Cat 2) irritant to eyes.

Executive summary:

Butyl phenyl ether was evaluated for eye irritation potential in the in vitro EpiOcular eye irritation screening assay (MatTek Corporation; Ashland, MA). The EpiOcular tissue consists of normal, human-derived epidermal keratinocytes that are cultured to form a stratified, squamous epithelium similar to that found in the cornea. The test consisted of topical application of the test material to the EpiOcular tissue for 30 minutes followed by thorough washing with DPBS and incubating with cell culture medium for approximately 120 ± 5 minutes (post-exposure recovery). In this study, Butyl phenyl ether was tested at two additional exposure times (2 and 15 minutes) to better characterize its irritation potential. Following recovery, the cell viability in the EpiOcular tissue was measured in the treated and control tissues using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and the data was reported as a percentage of the mean of negative control. An ET-40 value was calculated for Butyl phenyl ether, 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 >30minutes. In this study, Dulbecco Phosphate Buffered Saline (DPBS) and 0.3% Triton X-100 served as the negative and positive controls, respectively.

The percent viability for Butyl phenyl ether-treated EpiOcular tissue was < 50% at 30 minutes, therefore, interpreted as a potential mild irritant (UN GHS Category 2) in the EpiOcular assay.