Registration Dossier

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:
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 predictive, non-GLP study that is based on Organization for Economic Cooperation
and Development (OECD) Testing Guideline 492: Reconstructed human
Cornea-lie Epithelium (RhCE) test method for identifying chemicals not requiring
classification and labeling for eye irritation or serious eye damage, July 2015.
Deviations:
no
GLP compliance:
no
Remarks:
performed in accordance with GLP but not reported as GLP due to lab not offering a GLP report

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Specific details on test material used for the study:
The test material is a formulation containing 2,4-Dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one 4-((4-C7-17- branched alkylphenyl)azo) derivatives and Hydrocal 45 (1:1)

Purity/Characterization (Method of Analysis and Reference):
The non-GLP purity of the active is listed as >99% on the record of custody (Asif, 2016).

Test Material Stability Under Storage Conditions:
The test material 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone (with solvent), lot CAC05262016, 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 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 (Klausner et al., 2000).

Supplier and Location:
MatTek Corporation; Ashland Massachusetts

Test system

Vehicle:
other: Hydrocal 45 solvent was used for formulation.
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.

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) and of DPBS (negative control; 30±2 exposure time), 0.3% Triton X-100 (positive control; 30±2 exposure time) and 1-phenyl (30±2 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) and of DPBS (negative control; 30±2 exposure time), 0.3% Triton X-100 (positive control; 30±2 exposure time) and 1-phenyl (30±2 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: % Viability
Value:
105.8
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 60 minutes) was 2.043 (i.e., ≥ 1.00; criteria set by the tissue manufacturer).
2) The mean tissue viability of the positive control was 20.0% (<50% relative to the negative control).

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, 1-Phenyl was evaluated for potential interaction with the MTT dye.
Approximately 100 mg of 1-Phenyl 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 1-Phenyl 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:
GHS criteria not met
Conclusions:
The percent viability of 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone (with solvent) and positive control were 105.8% and 20.0% respectively. As the percent cell viability of 1-phenyl was 105.8%, 1-phenyl was classified as a UN GHS No Category.
Executive summary:

1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone (with solvent) 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. In this assay, 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5- pyrazolone (with solvent) was topically applied to the EpiOcular tissue for 30 ± 2 minutes, followed by approximately 120 minutes post-exposure recovery. Following recovery, the cell viability 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. Criteria for classification as an irritant (UN GHS Cat 1/2) or Non-Classified (UN GHS NC) was based on a cell viability of ≤60% or >60%, respectively. 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 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone (with solvent) was 105.8%. Therefore, 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone (with solvent) was interpreted as a potential non-irritant (UN GHS No Category).