Registration Dossier

Administrative data

Endpoint:
skin 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
This is a screening non-GLP study. The current protocol is a streamlined version of OECD TG 439 test guideline.
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.

In vitro test system

Test system:
human skin model
Source species:
human
Cell source:
other: human-derived epidermal keratinocytes (NHEK)
Justification for test system used:
The EpiDerm three-dimensional human skin model has been extensively characterized (EC-ECVAM, 2009) and is approved (OECD TG 439, 2013) for identification and classification of skin irritation hazard according to the UN GHS and EU classification system (Category 2/R38 or No label).
Vehicle:
other: Hydrocal 45 solvent was used for formulation.
Details on test system:
Test System:
The EpiDerm model uses human-derived epidermal keratinocytes (NHEK) as the cell source, which are cultured to form a multi-layered, highly differentiated model that closely mimics human epidermis at biochemical and physiological levels.
The test is based on the principle that chemicals with irritant potential can cause a cytotoxic response to the stratum cornea and the rate of cytotoxicity is proportional to irritation potency. In the assay, the EpiDerm tissue model was incubated with the test chemical for 60 minutes, followed by 42-hour incubation (recovery) under standard cell culture conditions. Following the post-treatment incubation period, cell viability was assessed using the MTT (3-[4,5- dimethylthiazol-2-yl] -2,5 – diphenyltetrazolium bromide) assay (Mosmann, 1983). Relative cell viability was calculated for each tissue as % of the mean of the negative control-treated tissues. A test chemical was interpreted as a potential skin irritant (UN GHS Cat 1/2) or non-irritant (UN GHS Cat NC), when the cell viability was ≤ or > 50%, respectively (OECD TG 439, 2013).

Study Design:
The EpiDerm System (EPI-200) consists of normal, human-derived epidermal kerotinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered 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 kit, containing 24 tissues mounted on agarose.

Supplier and Location:
MatTek Corporation; Ashland, MA
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Preparation of the Treatment Solution:
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)

Route of Administration:
The test chemical was administered by topical application to the surface of the EpiDerm tissues.

Experimental Procedure:
EpiDerm tissue cultures were used within 24 to 48 hours of receipt from the supplier. Upon receipt, the EpiDerm tissues were stored at 2-8ºC until used. On the day prior to testing, EpiDerm assay medium was warmed to room temperature and an aliquot of 0.9 mL was dispensed into each well of a 6-well plate. The EpiDerm tissues were transferred from a 24-well shipping plate to a 6-well plate containing the fresh, pre-warmed assay medium. Prior to transferring, each EpiDerm tissue was inspected for air bubbles between the agarose gel and MILLICELL insert. Tissues with air bubbles greater than 50% of the MILLICELL area were discarded. Any unused tissues were stored in an air tight bag (filled with 5% CO2/95% air) and stored at 2-8ºC for subsequent use. The EpiDerm 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 and were incubated overnight (18 ± 3 hrs) to acclimate the tissue.
On the day of treatment, the EpiDerm tissues were exposed to the test chemical as described above (30 μL of test material or controls). After dosing, the tissue plates were transferred to a cell culture incubator and were incubated for 35 ± 1 minutes at standard culture conditions. Following incubation, the plates were transferred to the laminar flow hood and incubated at room temperature for 25 minutes. Following the total of 60 ± 1 minutes treatment period, the tissues were rinsed with sterile DPBS to remove test substance. The tissues were then transferred to new 6-well plates containing 0.9 mL/well fresh pre-warmed assay medium and incubated at standard culture conditions for an initial post-treatment incubation of 24±2 hours. After the initial post-treatment incubation, the inserts were transferred into new 6-well plates pre-filled with 0.9 mL fresh pre-warmed assay medium and incubated for additional 18±2 hours (total post-treatment incubation period of 42 ± 2 hours).
Following post-treatment incubation, tissue inserts were evaluated for cell viability using the MTT assay. Each tissue insert was 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 formazan was extracted in 2 mL extractant solution with 2 hours shaking at room temperature. The amounts of extracted formazan from each tissue was measured spectrophotometrically at 570 nm (OD570) using a Microplate Reader (Molecular Devices).
Duration of treatment / exposure:
60 ± 1 minutes treatment period
Duration of post-treatment incubation (if applicable):
Following the total of 60 ± 1 minutes treatment period, the tissues were rinsed with sterile DPBS to remove test substance. The tissues were then transferred to new 6-well plates containing 0.9 mL/well fresh pre-warmed assay medium and incubated at standard culture conditions for an initial post-treatment incubation of 24 ± 2 hours. After the initial post-treatment incubation, the inserts were transferred into new 6-well plates pre-filled with 0.9 mL fresh pre-warmed assay medium and incubated for additional 18 ± 2 hours (total post-treatment incubation period of 42 ± 2 hours).
Number of replicates:
Four

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: Mean Viability %
Run / experiment:
Mean Viability % for 4 replicates
Value:
86.4
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Assessment of Direct Test Material Reduction of MTT:
As some test chemicals may interfere with MTT assay readout by directly reducing MTT dye (turn MTT solution to blue/purple formazan in the absence of viable cells), 1-phenyl was evaluated for this property. In this experiment, 1-phenyl (30 μL) was incubated with 1 mL of the MTT reagent in the dark at standard cell culture conditions (37°C) 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 1-phenyl, as the test material did not turn MTT solution to blue/purple colour.

ACCEPTANCE CRITERIA:
The results for negative and positive controls met 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 3.077 (i.e. ≥ 1.00; criteria set by the tissue manufacturer).
2) The relative mean viability of positive control (5% SDS) was 6.8% (i.e. < 50% compared to negative control).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The mean relative tissue viability for EpiDerm tissues treated with 1-phenyl and positive control (5% SDS) were 86.4% and 6.8%, respectively. According to the EpiDerm skin irritancy prediction model, a test substance is considered irritant to skin if the mean tissue viability is ≤ 50%. Therefore, based on the present EpiDerm study results, 1-phenyl was interpreted to be a non- irritant (UN GHS Cat NC) to skin.
Executive summary:

1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone (with solvent) was evaluated for skin irritation potential in an in vitro EpiDerm skin irritation assay (MatTek Corporation; Ashland, MA). The EpiDerm tissue consists of normal, human-derived epidermal keratinocytes (NHEK) that are cultured to form a multilayered, differentiated model of human epidermis. In this assay, 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone (with solvent) was topically applied to the EpiDerm tissue for 60-minutes, followed by a 42- hour 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. A test chemical was considered to possess skin irritation potential (UN GHS Cat 1/2) if the relative cell viability was less than or equal to 50%. In this study, Dulbecco Phosphate Buffered Saline (DPBS) and 5% SDS served as the negative and positive controls, respectively. The mean relative cell viability of 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone (with solvent) and positive control-treated tissues were 86.4% and 6.8% (i.e. ≤ 50%), respectively. Therefore, 1-Phenyl-3-methyl-4-(p-dodecylphenylazo)-5-pyrazolone (with solvent) was interpreted as a non-irritant (UN GHS Cat NC) to skin in the EpiDerm irritation assay.