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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
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:
GLP requirements are not applicable for in vitro methods.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals / tissue source

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. 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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
120 ± 15 min post-exposure recovery
Number of animals or in vitro replicates:
3
Details on study design:
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.

Supplier and Location
MatTek Corporation; Ashland Massachusetts

Preparation of the Test Material
The chemical was tested as provided by sponsor (neat), following supplier’s protocol (MatTek Corporation) and OECD 492 test guideline.

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

Experimental 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 exposure time), 0.3% TRITON™ X-100 (positive control; 30±2 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 hours 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 were transferred to a 96-well plate and the optical density of the extracted formazan was quantified at 570 nm (OD570) using a microplate reader.

Results and discussion

In vitro

Results
Irritation parameter:
other: mean relative cell viability (%)
Remarks:
reduction in cell viability compared to negative control level
Value:
30.8
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of irritation

Any other information on results incl. tables

The mean relative cell viability of the positive control-treated tissue was 13.7%. Exposure of the tissue model to the test material vinylbenzyl chloride for 30 min reduced the mean relative cell viability to 30.8 %. Therefore, under these conditions, vinylbenzyl chloride was interpreted as a potential ocular irritant (UN GHS Category 1 or 2) in the EpiOcular assay.

Applicant's summary and conclusion

Interpretation of results:
other:
Remarks:
Potential ocular irritant. The assay does not allow discrimination between UN GHS Cat. 1 or 2.
Conclusions:
Vinylbenzyl chloride was interpreted as a potential ocular irritant (UN GHS Category 1 or 2) in the EpiOcular assay.
Executive summary:

Vinylbenzyl chloride was evaluated for eye irritation potential in an in vitro EpiOcular eye irritation assay according to OECD Testing Guideline 492 (Supplier: 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, vinylbenzyl chloride was topically applied to the EpiOcular tissue for 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. The test substance is considered a (severe) irritant (UN GHS Cat 1 or 2) if the mean relative cell viability is <= 60%. The test substance was considered a nonirritant (UN GHS Cat NC) if the relative mean cell viabiliy is >60%. 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%. Exposure of the tissue model to the test material vinylbenzyl chloride for 30 min reduced the mean relative cell viability to 30.8 %. Therefore, under these conditions, vinylbenzyl chloride was interpreted as a potential ocular irritant (UN GHS Category 1 or 2) in the EpiOcular assay.