5-(3-Phenylpropioloyl)-1,3-isobenzofurandione

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 October 2014 to 21 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

OECD Test Guideline (439) for the In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, Organization for Economic Cooperation and Development (Adopted 26 July 2013).

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

5-(3-phenylprop-2-ynoyl)-1,3-dihydro-2-benzofuran-1,3-dione

EC Number:

935-606-2

Cas Number:

1329658-14-1

Molecular formula:

C17H8O4

IUPAC Name:

5-(3-phenylprop-2-ynoyl)-1,3-dihydro-2-benzofuran-1,3-dione

Test material form:

solid: particulate/powder

Details on test material:

Short Name: PETA
Long Name: NNEXAMITE™ A56 (PETA)
Chemical Name: Phenylacetylene modified trimellitic anhydride
Lot Number: NEX-X61-A02
CAS Number: 1329658-14-1
Description: Yellow powder
Purity: >99 %
Molecular weight: 276.24
Manufacture date: 04 September 2013
Expiry date: 01 May 2014
Storage conditions: Room temperature (15-25 °C), protected from humidity
Safety Precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety

Specific details on test material used for the study:

No further details specified in the study report.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

not specified

Details on animal used as source of test system:

Upon receipt of the EpiDerm™ Skin Kit (MatTek Corporation), the solutions were stored as indicated by the manufacturer. The EpiDerm™ tissues were stored at 2-8ºC until use. On the day prior to testing, EpiDerm™ Maintenance Medium was set to room temperature prior to use. Nine-tenths (0.9) mL of Maintenance Medium were aliquotted into the appropriate wells of 6-well plates. Each 6-well plate was labeled with the test article, positive control, or negative control. Each EpiDerm™ tissue was inspected for air bubbles between the agarose gel and cell culture insert prior to opening the sealed package. Tissue inserts with air bubbles covering greater than 50% of the cell culture insert area were not used. The 24-well shipping containers were removed from the plastic bag and their surfaces were disinfected with 70% ethanol. The EpiDerm™ tissues were transferred aseptically into the 6-well plates. The EpiDerm™ tissues were then incubated at 37 ± 1 ºC in a humidified atmosphere of 5 ±1% CO2 in air (standard culture conditions) for 60±5 minutes. After 60±5 minutes, the EpiDerm™ tissues were transferred to appropriate wells containing 0.9 mL of fresh warmed (to 37ºC) Maintenance Medium. The plates were returned to the incubator for 18 ± 3 hours to acclimate the tissues.

Justification for test system used:

The purpose of this study was to evaluate the skin irritation potential of the test article, NEXAMITE™ A56 (PETA) (CAS# 1329658-14-1), supplied by ADAS UK Ltd, in the context of identification and classification of skin irritation hazard according to the UN GHS and EU classification system (Category 2/R38 or No label). The skin irritation potential was evaluated based upon measuring the relative conversion of MTT (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide) in the test article-treated tissues after exposure to the test article for a 60-minute exposure period, followed by a 42-hour post-exposure expression period. Skin irritation potential of the test article was predicted if the relative viability was less than or equal to 50%. The protocol was based upon the EpiDerm™ SOP, Version 7.0 (Revised March 2009), Protocol for: In vitro EpiDerm™ skin irritation test (EPI-200-SIT), for use with MatTek Corporation's reconstructed human epidermal model EpiDerm (EPI-200). The protocol met the requirements of the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439).

Vehicle:

unchanged (no vehicle)

Details on test system:

Test Article Preparation
As instructed by the Sponsor, the test article was administered to the test system without dilution.

Assessment of Test Article/Nylon Mesh Compatibility
Since the test article was a powder, a mesh compatibility test was not performed. Therefore, a mesh was not applied after dosing of the test article onto the EpiDerm™ tissues.

Assessment of Direct Test Article Reduction of MTT
The test article was added to a 1.0 mg/mL MTT (Sigma) solution in warm Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2 mM L-glutamine (MTT Addition Medium) to assess its ability to directly reduce MTT. Approximately 25 mg of the test article were added to 1 mL of the MTT solution and the mixture was incubated in the dark at standard culture conditions for at least one hour. A negative control, 30 μL of sterile, Calcium and Magnesium Free Dulbecco’s Phosphate Buffered Saline (CMF-DPBS), was tested concurrently. If the MTT solution color turned blue/purple, the test article was presumed to have reduced the MTT.
The test article was not observed to directly reduce MTT in the absence of viable cells.

pH Determination
Since the test article was a powder, the pH of the test article could not be determined.

Controls
The definitive assay included a negative control and a positive control. The negative control was 30 μL of sterile, CMF-DPBS and the positive control was 30 μL of 5% Sodium Dodecyl Sulfate (SDS). Both the positive and negative controls were tested in triplicate, and at the same exposure time as the test article (60±1 minutes).

Skin Irritation Test (SIT) Definitive Assay
The test article, NEXAMITE™ A56 (PETA), was tested using the EpiDerm™ Skin Model for the Skin Irritation Test (SIT) in two definitive trials.
After the overnight incubation for 18±3 hours, the 6-well plates containing the EpiDerm™ tissues were removed from the incubator and placed at room temperature for at least 5 minutes prior to dosing.
The EpiDerm™ tissues were treated in triplicate with the test article, NEXAMITE™ A56 (PETA), for 60±1 minutes. Since the test article was a powder, immediately before application of the solid test article, each tissue surface was moistened with 25 μL of sterile CMF-DPBS to improve contact of the tissue surface with the test chemical. After adding the CMF-DPBS, 25 mg of the test article was added to each of three tissues at 1 minute intervals per tissue using a 25 mg sharp spoon (Aesculap #FK 623R). The sharp spoon was filled with the test article and then the spoon was leveled. After the three tissues were dosed with the test article, the test article was gently mixed and spread over the tissue surface using a sterile bulb-headed rod. The EpiDerm™ tissues were tested in triplicate with the positive or negative control for 60±1 minutes. Thirty microliters of each control were applied to each of three tissues at 1 minute intervals per tissue. Immediately after control administration onto the tissue, a nylon mesh was placed gently over the dose to spread the negative and positive controls. The plates with dosed tissues were kept in the laminar flow hood until the last tissue was dosed. After the last tissue was dosed, all of plates were transferred to the incubator for 35±1 minutes at standard culture conditions. After 35±1 minutes, all of the plates were removed from the incubator, placed into the laminar flow hood and kept at room temperature until the exposure period was completed for the first dosed tissue.
After 60±1 minutes of test or control article exposure, the tissues were rinsed with sterile, CMF-DPBS by filling and emptying the tissue insert 15 times. A stream of CMF-DPBS was directed onto the tissue surface. For the control articles where a mesh was used, the mesh was carefully removed with forceps (if necessary) after the 5th rinse. After the removal of the mesh, the rinsing procedure of the tissue continued for 10 times. After the 15th rinse, each of the 3 inserts per treatment group (test article, positive and negative control) was completely submerged, gently swirled, and rinse media dumped in a beaker containing approximately 150 mL of CMF-DPBS and specifically assigned for each treatment group; this procedure was repeated three times for each insert of each treatment group. Finally, the tissues were rinsed once more on the inside and outside of the tissue insert with sterile CMF-DPBS from the wash bottle, and the excess CMF-DPBS was decanted. The bottoms of the tissue inserts were blotted on sterile paper towels and the inserts were transferred to new 6-well plates containing 0.9 mL of fresh warmed (to 37ºC) Maintenance Medium. The tissue surface was carefully blotted with sterile cotton-tipped applicators to remove any excess moisture, and the tissue surface was visually observed for residual test article using a dissecting scope. In cases where residual test article was observed, sterile cotton-tipped applicators pre-moistened with CMF-DPBS were used to attempt to remove any residual test article from the tissue surface. The tissues were then incubated at standard culture conditions for a post-treatment expression incubation of 42±2 hours. After an initial 24±1 hours of incubation, the 6-well plates were removed from the incubator and the tissues were transferred into new 6-well plates pre-filled with 0.9 mL fresh Maintenance Medium warmed to approximately 37ºC. The tissues were placed back into the incubator at standard culture conditions for an additional 18±1 hours for the remainder of the 42±2 hour post-treatment expression incubation.

MTT Preparation
A 10X stock of MTT prepared in PBS (filtered at time of batch preparation) was thawed and diluted in warm MTT Addition Medium to produce a 1.0 mg/mL solution no more than two hours before use. Three hundred microliters of the MTT solution were added to each designated well of a pre-labeled 24-well plate.
After the total 42 ± 2 hours post-exposure expression incubation, the 6-well plates were removed from the incubator. Each tissue was blotted on a sterile paper towel and transferred to an appropriate well containing 0.3 mL of MTT solution. The 24-well MTT plates were incubated at standard culture conditions for 3±0.1 hours.
After the 3±0.1 hours incubation, the EpiDerm™ tissues were submerged, gently swirled, and rinse media decanted in a beaker containing approximately 150 mL of CMF-DPBS three times. The tissue was then blotted on absorbent paper, cleared of excess liquid, and transferred to a prelabelled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plate was covered with parafilm and shaken for at least 2 hours at room temperature to extract the MTT. At the end of the extraction period, the insert was gently agitated up and down in its extractant well. The tissues were pierced with forceps to allow the extract to flow back into the well from which the insert was removed, and the cell culture inserts were discarded. The extract solution was mixed (homogenized by pipetting up and down three times) and two x 200 μL aliquots were transferred to the appropriate wells of a 96-well plate. Two hundred μL of isopropanol were added to the wells designated as blanks. The absorbance at 570 nm (OD570) of each well was measured with a Molecular Devices Vmax plate reader with the AUTOMIX function selected.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg of the test article was added to each of three tissues.

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 replicates per test group

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean Viability

Value:

105.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test article, NEXAMITE™ A56 (PETA), was tested using the EpiDerm™ Skin Model for the Skin Irritation Test (SIT) in two definitive trials.
The mean OD570 of the negative control, CMF-DPBS, was 1.748. The mean viability of the positive control, 5% SDS, was 3.20%. The standard deviation calculated from individual percent tissue viabilities of the 3 identically treated replicates was < 18% for the positive control and negative control. Since the acceptance criteria were met, the assay was considered valid.
Cotton-tipped applicators pre-wetted in DPBS were used to attempt to remove residual test article. A dissecting scope was used to check for residual test article before and after use of the pre-wetted cotton swabs. The residual test article could not be completely removed from the EpiDerm™ tissues. The residual test article prolonged the exposure to the tissues, which may have influenced any toxic effect.
The test article was not observed to directly reduce MTT in the absence of viable cells.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based upon the results of the assay, the test article, was predicted to be non-irritating to skin, and thus would not require classification.

Executive summary:

The purpose of this study was to evaluate the skin irritation potential of the test article, NEXAMITE™ A56 (PETA) (CAS# 1329658-14-1), supplied by ADAS UK Ltd, in the context of identification and classification of skin irritation hazard according to the UN GHS and EU classification system (Category 2/R38 or No label). The skin irritation potential was evaluated based upon measuring the relative conversion of MTT (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide) in the test article-treated tissues after exposure to the test article for a 60-minute exposure period, followed by a 42-hour post-exposure expression period. Skin irritation potential of the test article was predicted if the relative viability was less than or equal to 50%. The protocol was based upon the EpiDerm™ SOP, Version 7.0 (Revised March 2009), Protocol for: In vitro EpiDerm™ skin irritation test (EPI-200-SIT), for use with MatTek Corporation's reconstructed human epidermal model EpiDerm (EPI-200). The protocol met the requirements of the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439).

 

The test article was tested in two definitive assays to determine the identification and classification of skin irritation hazard according to the UN GHS and EU classification system (Category 2/R38 or No label). The first definitive trial, was considered invalid due to the use of an expired batch of the assay positive control (5% SDS). The second definitive trial (using a newly prepared batch of the assay positive control) was considered valid and met the acceptance criteria; thus, only the data obtained in the second trial are included in this report.

 

The mean OD570 of the negative control, CMF-DPBS, was 1.748. The mean viability of the positive control, 5% SDS, was 3.20%. The standard deviation calculated from individual percent tissue viabilities of the 3 identically treated replicates was < 18% for the positive control and negative control. Since the acceptance criteria were met, the assay was considered valid.

Cotton-tipped applicators pre-wetted in DPBS were used to attempt to remove residual test article. A dissecting scope was used to check for residual test article before and after use of the pre-wetted cotton swabs. The residual test article could not be completely removed from the EpiDerm™ tissues. The residual test article prolonged the exposure to the tissues, which may have influenced any toxic effect.

The test article was not observed to directly reduce MTT in the absence of viable cells.

 

Based upon the results of this assay, the test article, was predicted to be non-irritating to skin, and thus would not require classification.