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

Endpoint summary

Description of key information

Skin irritation - In vitro

Based upon the results of the assay, the test article, was predicted to be non-irritating to skin.

Skin corrosion - In vitro

NEXAMITE™ A56 (PETA), the results indicate that the test item is not a skin corrosive.

Eye irritation - In vitro

NEXAMITE™ A56 (PETA) is not classified as a severe irritant and not classified as non-irritant. On the basis of worst case hazard assessment, the substance is classified as eye irritation Category 2.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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

Specific details on test material used for the study:

No further details specified in the study report.

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

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.

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.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 February 2014 to 20 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EU B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”,

Version / remarks:

Commission Regulation (EC) No 440/2008, Annex Part B, B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142, dated May 31st, 2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

OECD Guidelines for the Testing of Chemicals, Section 4, No. 431, “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method” adopted 26 July 2013.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

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:

Human Skin
EPISKIN-SM (Manufacturer: SkinEthic, France, Batch No.:14-EKIN-005, Expiry Date: 24 February 2014) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Quality Control
EPISKIN-SM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control checks were conducted at SkinEthic laboratories, the supplier of the EpiSkin-SM Test Kit used in the present study.

Justification for test system used:

The EPISKIN-SM model has been validated for corrosivity testing in an international trial (Fentem, 1998) and it is the subject of an OECD guideline for corrosivity testing (OECD 431), therefore it is considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

Kit Contents
Units: EPISKIN-SM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKIN-SM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium”. (Batch No.: 14-MAIN3-006; Exp. Date: 26 February 2014)
A flask of sterile “Assay Medium”. (Batch No.: 14-ESSC-006; Exp. Date: 26 February 2014)

Number of Replicate Wells
In this assay 2 replicates per test item and 2 negative controls + 2 positive controls were used. As the test item was coloured, in addition to the normal procedure, two additional test item-treated tissues were used for the non specific OD evaluation.

Kit Reception
The pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- the indicator changes from white to grey at 40°C
The kit was found to be in good order at reception.

Storage
The EPISKIN-SM kit was kept in their packaging at 37°C and the assay medium supplied with the kit was stored at 2-8°C until the initiation of the test.

INDICATOR FOR POTENTIAL FALSE VIABILITY
Chemical action by the test material on MTT may mimic that of cellular metabolism leading to a false estimate of viability. This may occur when the test substance is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material directly acts on MTT (MTT-reducer), additional controls should be used to detect and correct for test substance interference with the viability measurement.

Check-method for possible direct MTT reduction with test substance
Approximately 20 mg of test item was added to 2 mL MTT ready to use solution and mixed. The mixture was incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 3 hours and then any colour change was observed:
-Test substances which do not interact with MTT: yellow
-Test substances interacting with MTT: blue or purple
After three hours incubation yellow colour of the mixture was detected; the test substance did not interact with MTT and therefore using of additional controls was not necessary. A false estimation of viability due to MTT interaction can be precluded.

Check-method to detect the colouring potential of test-substances
Prior to treatment, chemicals were evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment).
As the test item has an intrinsic colour, further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSC %) was determined in order to evaluate the ability of test substance to stain the epidermis by using additional control tissues.

Additional control(s) for dyes and chemicals able to colour the tissue
In addition to the normal procedure, two additional test item-treated living tissues were used for the non specific OD evaluation. These tissues followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. This is to mimic the amount of colour from the test item that may be present in the test disks. OD readings were made following the same conditions as for the other tissues.

PERFORMANCE OF THE STUDY
Pre-incubation (day [-1])
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of wells in an assay plate were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere.

Application
The assay medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
- 20 mg test item was applied evenly to each of two test and to the two additional control skin units. A volume of 100 μL NaCl (Salsol solution, NaCl 0.9% w/v) was added to the test item to ensure good contact with the epidermis.
- 50 μL saline (Salsol solution, NaCl 0.9% w/v) was added to each of the two negative control skin units.
- 50 μL glacial acetic acid was added to each of the two positive control skin units.
The plates with the treated epidermis units were incubated for the exposure time of 4 hours at room temperature (23.2-24.1°C) covered with the plate lids.

Rinsing
After the incubation time (4 hours), all test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with approximately 25 mL PBS 1x solution (0.9 % NaCl) to remove all of the test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly. The rest of the PBS was removed from the epidermal surface using a Pasteur pipette linked to a vacuum source (without touching the epidermis) and the culture medium was removed from the assay plate wells.

MTT test
MTT solution (2 mL of 0.3 mg/mL MTT) was added to each well below the skin units (except the additional chemical-treated tissues). The lid was replaced and the plate incubated at 37°C in an incubator with 5 % CO2 for 3 hours, protected from light.

Formazan extraction
At the end of incubation with MTT a formazan extraction was undertaken:
A disk of epidermis was cut from each skin unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 μL acidified isopropanol (One tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements
Following the formazan extraction, 2×200 μL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer using wavelength filter of 540 nm and acidified isopropanol solution as blank (6×200 μL).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

20 mg test item was applied

Duration of treatment / exposure:

exposure time of 4 hours

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2 replicates per test group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item - mean value

Value:

91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

VALIDITY OF THE TEST
The mean OD value of the two negative control tissues was 0.659. The positive control result showed 2.2 % viability.

INDICATOR FOR POTENTIAL FALSE VIABILITY
Possible direct MTT reduction with Test Substance
No colour change was observed after three hours of incubation of the test item in MTT solution. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Colouring potential of test substances
As the test item is coloured, two additional test item-treated tissues were used for the non specific OD evaluation. The mean optical density (measured at 540 nm) of these tissue was determined as 0.101, Non Specific Colour % was calculated as 15%. Therefore additional data calculation was necessary.

CELL VIABILITY
The OD value for the test item treated skin (after adjustment for colour) showed a viability of 91%.
The variability in the viability of the two test item-treated tissue samples in the MTT assay was 30.7%. Although this value was slightly higher than the criteria (30%), but the given limit value refers to the 20-100% viability range. The observed higher than usual variability value was resulted by the fact that this test one of the replicates resulted 105% relative viability. Therefore, the observed value was considered to be acceptable. Furthermore, both replicate samples showed a clear negative result in the test, so this fact did not affect the conclusion of the study.

Historical control data

	Negative control Saline	Positive control Glacial acetic acid
Minimum optical density (OD)	0.611	0.005
Maximum optical density (OD)	0.972	0.027
Mean optical density (OD)	0.761	0.012
Standard Deviation (SD)	0.120	0.006
Number of Sample	14	14

 

Optical Density (OD) and the calculated Non Specific Colour % (NSC%) of the Additional Control Tissues

Additional controls	Mean Optical Density (OD)	NSC%
Treated with NEXAMITE™ A56 (PETA)	0.101	15

Note: Mean blank value was 0.045.

 

Optical Density (OD) and the calculated % viability of test item treated samples

Substance	Optical Density (OD)		OD after adjustment*	Viability (% RV)
Test Item: NEXAMITE™ A56 (PETA)	1 2	0.608 0.795	0.507 0.697	77 105
	Mean		0.601	91

Notes:

1. Mean blank value was 0.045

2. The test item had a residual colour which was expected to cause an OD of 0.101 in the final solutions. This value was subtracted from the measured OD values.

 

Optical Density (OD) and the calculated % viability of the negative and positive controls

Substance	Optical Density (OD)		Viability (%RV)
Negative Control: Saline (Salsol solution, NaCl 0.9% w/v)	1 2	0.627 0.690	95 105
	Mean	0.659	100
Positive Control: Glacial acetic acid	1 2	0.016 0.013	2.4 2.0
	Mean	0.015	2.2

Note: Mena blank value was 0.045

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with NEXAMITETM A56 (PETA) the mean cell (after adjustment for colour) was 91% compared to the negative control and therefore non-corrosive. All validity criteria were within acceptable limits and therefore the study can be considered as valid.
In this in vitro EPISKIN model test with NEXAMITE TM A56 (PETA), the results indicate that the test item is not a skin corrosive.

Executive summary:

The reconstructed human epidermis model EPISKIN-SM is designed to predict and classify the corrosive potential of chemicals, by measuring its cytotoxic effect, as reflected in the MTT assay, on the EPISKIN reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of corrosives in the in vivo Rabbit skin assay (OECD 404) and within OECD 431 is specifically approved as a replacement for the in vivo skin corrosivity test.

 

Disks of EPISKIN (two units / chemical) were treated with test item NEXAMITE™ A56 (PETA) and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution (0.9 % NaCl). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Saline (Salsol solution, NaCl 0.9% w/v) and glacial acetic acid treated epidermis were used as negative and positive controls. Two additional disks were used to provide an estimate of colour contribution from the test item. For each treated tissue viability was expressed as a % relative to negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test substance is considered to be corrosive to skin.

 

Following exposure with NEXAMITE™ A56 (PETA), the mean cell viability (after adjustment for colour) was 91% compared to the negative control and therefore non-corrosive. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

 

In this in vitro EPISKIN model test with NEXAMITE™ A56 (PETA), the results indicate that the test item is not a skin corrosive.

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:

20 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

OECD Guidelines for Testing of Chemicals 438 (26th July 2013)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

EU Commission Regulation (EC) No 1152/2010 (8th December 2010) amending, Regulation (EC) No 440/2008: Method B 48.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

OPPTS 870.2400 (EPA 712-C-98-195) August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Species:

chicken

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

Strain of chicken: COBB 500
Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út. 129.
Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were transported to CiToxLAB Hungary Ltd. and received into the laboratory for use within 2 hours of collection.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was applied in an amount of 30 mg onto the entire surface of the cornea.

Duration of treatment / exposure:

An exposure period of 10 seconds from the end of the application the cornea surface.

Duration of post- treatment incubation (in vitro):

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

3 replicates per test group

Details on study design:

SELECTION AND PREPARATION OF EYES FOR THE TEST
Eyes selection
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes
The eye ball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Eyes examination and acclimatization time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline dripping from a stainless steel tube, at a rate of approximately 3-5 drops/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

Identification
The eyes were identified by chamber number, marked on the door of each chamber.

THE BASE LINE ASSESSMENTS
The base line assessments
At the end of the equilibration period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% or -7% between the -45 min and the zero time. No changes in thickness were observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

TEST PROCEDURE
Treatment
After the zero reference measurements, the eye (in its retainer) was removed from the chamber, and placed on a layer of tissue with the cornea facing upwards. The eyes were held in horizontal position, while the test item was applied onto the cornea. The test item was applied in an amount of 30 mg onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance, taking care not to damage or touch the cornea.
The positive control eyes were treated in a similar way with 30 mg powdered Imidazole. The negative control eye was treated with 30μL of 0.9% w/v physiological saline (Salsol solution).
One eye was treated with isotonic saline, three eyes with the test item and another three with Imidazole in accordance with the current OECD guideline.

Test item removal
The time of application was observed and recorded, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL isotonic saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item if possible.
The test item and the Imidazole were adhered to the corneal surface after the post-treatment rinse. Gentle rinsing with 20 mL saline was performed and the rate of saline-drops was increased at each observation time point.
The test item and the Imidazole treated cornea surfaces were not cleared 240 minutes after the post-treatment rinse.

OBSERVATION
Observation and assessment of corneal effects
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

Irritation parameter:

percent corneal swelling

Run / experiment:

Test Item

Value:

1.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Based on the in vitro eye irritation assay in the isolated chicken eyes test with NEXAMITETM A56 (PETA), the test item is not classified as a severe irritant and not classified as non-irritant. No conclusion of in vivo significance can be made from the adherence of the test item to the cornea, since in vivo eye lids will probably clear the surface, but abrasion may occur. It is concluded that further information is required for classification.

Validity of the test: The results from all eyes used met the quality control standards. The negative control and positive control results were in line with historic data. This experiment was considered to be valid.

Test Item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	1.8%	I
Mean maximum corneal swelling at up to 240 min	1.8%	I
Mean maximum corneal opacity	0.33	I
Mean fluorescein retention	1.67	III
Other observations	The test Item was adhered to all corneal surfaces after the post-treatment rinse. The cornea surfaces were not cleared 240 minutes after the post-treatment rinse.
Overall ICE Class	2xI 1xIII

 

Positive Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	3.5%	I
Mean maximum corneal swelling at up to 240 min	7.1%	II
Mean maximum corneal opacity	3.83	IV
Mean fluorescein retention	3.00	IV
Other observations	The Imidazole was adhered to all corneal surfaces after the post-treatment rinse. The cornea surfaces were not cleared 240 minutes after the post-treatment rinse.
Overall ICE Class	1xII 2xIV

 

Negative Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other observations	None
Overall ICE Class	3xI

 

Historical Control data (n=17, data from 2013):

Negative Control: Physiological saline (Salsol solution, NaCl 0.9% w/v)

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	0.0%	1.2%
Maximum corneal swelling at up to 240 min	0.0%	1.2%
Maximum corneal opacity change	0.00	0.00
Fluorescein retention	0.00	0.00

Positive Control: (Imidazole)

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	1.1%	5.9%
Maximum corneal swelling at up to 240 min	4.6%	10.6%
Maximum corneal opacity change	3.50	4.00
Fluorescein retention	2.00	3.00

 

Table of individual date (NEXAMITE™ A56 (PETA))

Chamber number ↓	Corneal thickness (instrument units)										Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	Change	30	75	Max change up to 75	120	180	240	Max change up to 240	0	30	75	120	180	240	Max ∆ Opac	0	30	∆ Flu ret
1	76	76	0.0%	78	78	2.6%	78	78	78	2.6%	0	0	0	0	1	1	1.0	0	2	2.0
2	76	76	0.0%	77	77	1.3%	77	77	77	1.3%	0	0	0	0	0	0	0.0	0	1	1.0
3	76	76	0.0%	77	77	1.3%	77	77	77	1.3%	0	0	0	0	0	0	0.0	0	2	2.0
Mean values:						1.8%				1.8%							0.33			1.67

Comment: The Test Item was adhered to all corneal surfaces after the post-treatment rinse. The cornea surfaces was not cleared 240 minutes after the post-treatment rinse.

 

Table of individual date (Imidazole)

Chamber number ↓	Corneal thickness (instrument units)										Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	Change	30	75	Max change up to 75	120	180	240	Max change up to 240	0	30	75	120	180	240	Max ∆ Opac	0	30	∆ Flu ret
4	75	75	0.0%	76	78	4.0%	79	80	80	6.7%	0	4	4	4	4	4	4.0	0	3	3.0
5	76	76	0.0%	78	78	2.6%	80	81	82	7.9%	0.5	4	4	4	4	4	3.5	0	3	3.0
6	75	75	0.0%	76	78	4.0%	78	79	80	6.7%	0	4	4	4	4	4	4.0	0	3	3.0
Mean values:						3.5%				7.1%							3.83			3.00

Comment: The Imidazole adhered to all corneal surfaces after the post-treatment rinse. The cornea surfaces was not cleared 240 minutes after the post-treatment rinse.

 

Table of individual date (Saline (Salsol solution, NaCl 0.9% w/v))

Chamber number ↓	Corneal thickness (instrument units)										Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	Change	30	75	Max change up to 75	120	180	240	Max change up to 240	0	30	75	120	180	240	Max ∆ Opac	0	30	∆ Flu ret
7	75	75	0.0%	75	75	0.0%	75	75	75	0.0%	0	0	0	0	0	0	0.00	0	0	0.00

 

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the in vitro eye irritation in the isolated chicken eyes test with NEXAMITETM A56 (PETA), the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for definitive classification. On the basis of worst case hazard assessment, the substance is classified as eye irritation Category 2.

Executive summary:

An in vitro eye irritation study of the test item NEXAMITE™ A56 (PETA) was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No.: 438 (26th July 2013).

 

After the zero reference measurements, the eye was held in horizontal position and 30 mg of NEXAMITETM A56 (PETA) was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated with 30 mg Imidazole. The negative control eye was treated with 30 μL of Saline (Salsol solution, NaCl 0.9% w/v).

 

Based on this in vitro eye irritation in the isolated chicken eyes test with NEXAMITE™ A56 (PETA), the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for definitive classification. On the basis of worst case hazard assessment, the substance is classified as eye irritation Category 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation - In vitro

The purpose of this study was to evaluate the skin irritation potential of the test article, NEXAMITE™ A56 (PETA). 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 test article was tested in two definitive assays. 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 the report.

 

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 the assay, the test article, was predicted to be non-irritating to skin.

Skin corrosion - In vitro

Disks of EPISKIN (two units / chemical) were treated with test item NEXAMITE™ A56 (PETA) and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution (0.9 % NaCl). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Following exposure with NEXAMITE™ A56 (PETA), the mean cell viability (after adjustment for colour) was 91% compared to the negative control and therefore non-corrosive. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

In the in vitro EPISKIN model test with NEXAMITE™ A56 (PETA), the results indicate that the test item is not a skin corrosive.

Eye irritation - In vitro

An in vitro eye irritation study of the test item NEXAMITE™ A56 (PETA) was performed in isolated chicken’s eyes.

After the zero reference measurements, the eye was held in horizontal position and 30 mg of NEXAMITETM A56 (PETA) was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated with 30 mg Imidazole. The negative control eye was treated with 30 μL of Saline (Salsol solution, NaCl 0.9% w/v).

Based on the in vitro eye irritation in the isolated chicken eyes test with NEXAMITETM A56 (PETA), the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for definitive classification. On the basis of worst case hazard assessment, the substance is classified as eye irritation Category 2.

Justification for classification or non-classification

Skin irritation/corrosion

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

In the in vitro EPISKIN model test with NEXAMITE™ A56 (PETA), the results indicate that the test item is not a skin corrosive, therefore, no classification is required.

Eye irritation

Based on the in vitro eye irritation in the isolated chicken eyes test with NEXAMITETM A56 (PETA), the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for definitive classification. On the basis of worst case hazard assessment, the substance is classified as eye irritation Category 2.