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Description of key information

In Vitro Eye irritation and In vitro Skin irritation studies

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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).
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
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 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
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).
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
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).
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
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.
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

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).
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.
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).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation:

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.

Eye Irritation:

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 non-irritant (UN GHS No Category).

Justification for classification or non-classification

Criteria for classification for irritation (skin and Eye) are not met