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Toxicological information

Eye irritation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[bis(2-ethylhexyl)amino]ethanol
EC Number:
202-912-6
EC Name:
2-[bis(2-ethylhexyl)amino]ethanol
Cas Number:
101-07-5
Molecular formula:
C18H39NO
IUPAC Name:
2-[bis(2-ethylhexyl)amino]ethan-1-ol
Details on test material:
Test Material Name: ETHOX 4256
Chemical Name: 2-(Bis(2-ethylhexyl)amino)ethanol
Lot #: 26D647
Specific details on test material used for the study:
Test Material Name: ETHOX 4256
Chemical Name: 2-(Bis(2-ethylhexyl)amino)ethanol
Lot/Reference/Batch Number: 26D647
Purity/Characterization (Method of Analysis and Reference): The non-GLP certificate of analysis lists the purity as 99.80% POE (1) DI 2 EH AMINE by gas chromatography (Tucker, 2016).
Stability: The test material ETHOX 4256, Lot 26D647, was not tested for neat test material stability.

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 (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

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 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 2-8ºC 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 (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.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: Mean Viability %
Run / experiment:
2 minute
Value:
109.9
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Mean Viability %
Run / experiment:
15 minute
Value:
103.6
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Mean Viability %
Run / experiment:
30 minute
Value:
90.5
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 ETHOX 4256 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 ETHOX 4256, 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).

Any other information on results incl. tables

Text Table 1. Culture Viability of ETHOX 4256 in the EpiOcular Eye Irritation Model

Chemical Name

Treatment plus 120±15 Min Recovery

Mean

Viability (%)

Replicate 1

Replicate 2

Replicate 3

ETHOX 4256 2 minute

114.8

114.3

100.6

109.9

ETHOX 4256 15 minute

100.2

109.5

101.1

103.6

ETHOX 4256 30 minute

89.1

89.9

92.4

90.5

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 ETHOX 4256 in EpiOcular Eye Irritation Model

Chemical Name

ET-40 (min)

EpiOcular Classification

(Neat)

ETHOX 4256

> 30.0

UN GHS Cat NC

Positive Control*

< 30.0

UN GHS Cat 1 or 2

*Positive Control: 0.3% TRITON™ X-100

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The mean relative cell viability of the positive control-treated tissue was 13.7% (i.e. ≤ 40%) and the ET-40 value of ETHOX 4256 was > 30 minutes (Text Tables 1 and 2). Therefore, under these conditions, ETHOX 4256 was interpreted as a potential ocular non-irritant (UN GHS Cat NC) in the EpiOcular assay.
Executive summary:

ETHOX 4256 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, ETHOX 4256 was topically applied to the EpiOcular tissue for 2, 15, or 30 minutes followed by a 120±15 minute 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. An ET-40 value was calculated for ETHOX 4256, 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 ETHOX 4256 was > 30 minutes. Therefore, under these conditions, ETHOX 4256 was interpreted as a potential ocular non-irritant (UN GHS Cat NC) in the EpiOcular assay.