Registration Dossier

Administrative data

Description of key information

Based on a battery of in vitro assays, Luperox® 520M50 is considered as irritating for the skin and not irritating for the eyes.

In vitro skin irritation

OECD 439 study

The skin irritation potential of Luperox® 520M50 was evaluated using the EpiskinTM reconstructed human epidermis model (Gerbeix, 2017a). The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46). The study was conducted in compliance with CiToxLAB France standard operating procedures and the principles of Good Laboratory Practice. Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +37°C, 5% CO2in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability). In addition, the concentration of the inflammatory mediator IL-1a was evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential. All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid. Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 78% with a standard deviation of 7%. As the mean viability was > 50% after the MTT reduction, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The mean IL-1a concentration for test item-treated tissues was 104 pg/mL. Due tothis value being above the limit of 60 pg/mL, the results met the criteria for anin vitroclassification as irritant to skin.

Under the experimental conditions of this study, Luperox® 520M50 is considered to be irritant to skin; however, it is not possible to discriminate the classification between Category 1 (H314) and Category 2 (H315) (Regulation (EC) No. 1272/2008).

OECD 431 study

An in vitro skin corrosivity test of Luperox 520M50 was performed in a reconstructed human epidermis model (Orovecz, 2017). EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline. Disks of EPISKINTM(SM) (two units) were treated with Luperox 520M50 test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). For each treated tissue viability was expressed as a % relative to the negative control. If

the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with Luperox 520M50, the mean cell viability was 129.3% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test with Luperox 520M50, the results indicate that the test item is non-corrosive to the skin.

In vitro eye irritation

OECD 437 study

The potential irritant and corrosive properties of the LUPEROX® 520M50 to the eye was evaluated with the Bovine Corneal Opacity and Permeability (BCOP) test method (Gerbeix, 2017b). The BCOP assay can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. The design of this study was based on the guideline OECD Guideline 437 and the study was performedin compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C. A single experiment was performed using three corneas for test item and negative control and two corneas for positive control. Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item, applied undiluted, and the negative and positive controls were evaluated in a single experiment using a treatment time of 10 minutes and the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

No notable opaque spots or irregularities were observed on test item-treated corneas. All acceptance criteria were fulfilled. The study was therefore considered as valid. The meanIn VitroIrritancy Score (IVIS) of the test item-treated corneas was: 0. As the mean IVIS was < 3, the test item was considered asa testchemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Under the experimental conditions of this study, LUPEROX® 520M50 was identified as not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 March 2017 - 31 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
July 2009
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
erature
Expiry date: 20 September 2017
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Episkin Laboratories, Lyon, France.
Details on animal used as source of test system:
The EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis.

Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin Small Model (0.38cm²)
- Tissue batch number: 17-EKIN-012
- Date of initiation of testing: 22 March 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: +37°C
- Temperature of post-treatment incubation: +37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 12 times with 2 mL D-PBS 1X
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Histology, Quality controls and Biological Safety verified by the test system supplier.
All information available in the test system certificate of analysis.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): EpiSkin tissues are placed in an incubator (set at +37°C, 5% CO2) for 48 hours (± 1 hour). At the end of the incubation period, the tissues will are dried before being placed in a freezer (-20°C) for at least one night.
- N. of replicates : 3
- Method of calculation used: the percentage of OD due to this non-specific MTT reduction is calculated and subtracted before calculation of the “true” viability percentages.
Non-Specific MTT reduction (NSMTT) calculation = [(mean cODTIK – mean cODUK) / mean cODNC] x 100
where cOD Untreated Killed (UK) tissues = ODUK – mean ODblank and cOD Test Item treated Killed (TIK) tissues = ODTIK – mean ODblank
If the NSMTT is > 30% relative to the negative control OD, additional steps may be undertaken (at additional cost; will be detailed in a study plan amendment) or the chemical will be considered as incompatible with the test.
If the NSMTT is 0% = NSMTT = 30% relative to the negative control OD, the corrected OD value of the test item-treated water-killed tissues may be subtracted from the mean cOD of the test item-treated living tissues to obtain the true amount of MTT reduction (i.e. the MTT reduction produced by metabolic conversion only).


NUMBER OF INDEPENDENT EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL:
- The test substance is considered to be irritant to skin if the mean viability obtained for the test substance is less than or equal to 50%, or if the mean viability os greater than 50% but the mean IL1a release exceeds 60 pg/mL
- The test substance is considered to be non-irritant if the mean viability value obtained for the test substance is less than 50% together with a mean IL1a below or equal to 60 pg/mL.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 µL (± 2 µL) of neat test item
Duration of treatment / exposure:
15 minutes (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Type of coverage:
other: not applicable (in vitro)
Preparation of test site:
other: not applicable (in vitro)
Vehicle:
unchanged (no vehicle)
Details on study design:
Preliminary tests
Test for direct MTT reduction with the test item
As a test substance may directly reduce MTT, thus mimicking mitochondrial succinate dehydrogenase activity, it is necessary to test the ability of the test item to directly reduce MTT before performing the main test.
To identify any test item interference with the MTT endpoint, the following preliminary test was performed:
- 10 µL of the test item were added to 2 mL of a 0.3 mg/mL freshly prepared MTT solution,
- a negative control was tested concurrently by adding 10 µL of water for injections to 2 mL of a 0.3 mg/mL freshly prepared MTT solution,
- both mixtures were incubated in darkness at +37°C for 3 hours (± 5 minutes).
Then the colour of the solutions obtained was evaluated.

Test for the detection of the colouring potential of the test item
The intrinsic colour or the ability of the test item to become coloured in contact with water (simulating a tissue humid environment) was evaluated by adding 10 µL of test item to 90 µL of water for injections in a transparent recipient. After 1 hour (± 3 minutes) of mixing, the presence and intensity of the colouration was evaluated.

Main test
One 12-well plate was used for each test and control items: one plate for the test item, one plate for the negative control and one plate for the positive control. Each item was applied onto triplicate tissues.

Pre-incubation of the tissues on their day of arrival (Day 0)
A volume of 2 mL of pre-warmed (at +37°C) maintenance medium was added to 3 wells on a 12 well plate (one plate per item).
Each EpiskinTM tissue was then transferred into the maintenance medium pre-filled wells (three tissues per plate). The plates were incubated at +37°C, 5% CO2 in a humidified incubator for at least 24 hours.
Treatment of tissues (Day 1)
A volume of 2 mL of pre-warmed maintenance medium was added into 3 wells on the 12 well plate for each item, respectively.
Each test or control item was then applied on three tissues for an exposure period of 15 minutes (± 1 minute) at room temperature and according to quantities defined in § Test item, positive and negative controls administrations. The items were applied topically to the corresponding tissues and gently spread over the epidermal surfaces to ensure uniform covering of the tissues. The tissues were processed (treatment and rinsing) in the same order and at regular time-intervals to ensure each tissue received an equal exposure period.

For the positive control only, the SDS solution was re-spread after 7 minutes (± 1 minute) contact time with a curved spatula to improve the distribution of the SDS for the remainder of the contact period.

Rinsing of tissues and incubation for 42 hours (Day 1)
At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Rinsing was achieved by gently filling and emptying several times each tissue with D PBS to gently remove any residual test or control items.
The rinsed tissues were transferred to the second column of three wells containing 2 mL of maintenance medium in each well and the plates were incubated at +37°C, 5% CO2 in a humidified incubator for 42 hours.

MTT viability assay (Day 3)
Following the 42 hours incubation period, each 12-well plate was placed for 15 minutes ± 2 minutes on a plate shaker to homogenize the released inflammatory mediators in the maintenance medium. Then, 1.6 mL of incubation medium from beneath each tissue were retained in pre-labeled micro tubes and stored at -20°C until possible quantification for inflammatory mediators.

Two milliliters of a freshly prepared MTT solution (0.3 mg/mL) were added into 3 wells on each 12-well plate. The tissues were then transferred to the MTT filled wells and incubated for 3 hours at +37°C, 5% CO2 in a humidified incubator.

At the end of the MTT incubation period, a total biopsy of the epidermis was made by using the Episkin™ biopsy punch. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated. The epidermis was separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into micro tubes and incubated with 500 µL of acidic isopropanol. Then, to extract the formazan (reduced MTT) from the MTT-loaded tissues, each tube was stored after vortexing at +5°C and protected from light until Day 6 of the experiment.

Optical Density measurements (Day 6)
At the end of the formazan extraction period, each tube was vortexed until the solution colour became homogenous.
Each tube was used to fill two wells of a 96-well plate with 200 µL of extract per well. One 96-well plate was used for the negative and positive controls (placed at opposite ends of the plate), and a separate 96-well plate was used for the test item.

For each 96-well plate, the average Optical Density value (OD) of six wells containing 200 µL of acidified isopropanol only was used as the blank.

The OD was measured at 570 nm using a plate reader.

Quantification of IL-1a by ELISA.
Irritation / corrosion parameter:
% tissue viability
Remarks:
15 min
Run / experiment:
Mean
Value:
78
Vehicle controls valid:
not examined
Negative controls valid:
yes
Remarks:
100%
Positive controls valid:
yes
Remarks:
5%
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: IL1a release
Remarks:
(pg/mL)
Run / experiment:
Main
Value:
104
Vehicle controls valid:
not examined
Negative controls valid:
yes
Remarks:
11.7
Positive controls valid:
not applicable
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
Evaluation of the colouration of tissues at the end of the MTT incubation period
The qualitative evaluation of the MTT staining was performed with the naked eye.
All test item-treated tissues appeared blue which was considered indicative for viable tissues.

Evaluation of the MTT results
All of the acceptance criteria for the negative and positive controls were fulfilled, therefore the study was considered to be valid.

Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 78% with a standard deviation of 7% as assessed by the MTT assay (Table 2).
As the mean viability was > 50% after the MTT reduction, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA.
The mean IL-1a concentration for test item-treated tissues was 104 pg/mL (Appendix 2). Due to this value being above 60 pg/mL, the results met the criteria for an in vitro classification as irritant to skin.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean cOD of the three negative controls should be = 0.6 and the Standard Deviation (SD) value of the % viability should be = 18%,
- Acceptance criteria met for positive control: the relative mean viability of the positive control should be = 40% of the relative mean viability of the negative control and the SD value of the % viability should be = 18%.

 

 

 

Interpretation of results:
study cannot be used for classification
Remarks:
Category 1 (H314) or Category 2 (H315)
Conclusions:
The test item is considered to be irritant to skin; however, it is not possible to discriminate the classification between Category 1 (H314) and Category 2 (H315) (Regulation (EC) No. 1272/2008).
Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item using the EpiskinTM reconstructed human epidermis model. The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46). The study was conducted in compliance with CiToxLAB France standard operating procedures and the principles of Good Laboratory Practice. Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability). In addition, the concentration of the inflammatory mediator IL-1a was evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential. All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid. Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 78% with a standard deviation of 7%. As the mean viability was > 50% after the MTT reduction, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The mean IL-1a concentration for test item-treated tissues was 104 pg/mL. Due tothis value being above the limit of 60 pg/mL, the results met the criteria for an in vitro classification as irritant to skin.

Under the experimental conditions of this study, the test item is considered to be irritant to skin; however, it is not possible to discriminate the classification between Category 1 (H314) and Category 2 (H315) (Regulation (EC) No. 1272/2008).

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 July 2017 to 14 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: EU Method B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test” (31 May 2008)
Version / remarks:
Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142 (31 May 2008)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
OECD Guidelines for the Testing of Chemicals, No. 431, (29 July 2016) “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
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
Details on animal used as source of test system:
EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-028, Expiry Date: 17 July 2017) is a three-dimensional human epidermis model. Adult humanderived 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.
Justification for test system used:
The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
Quality Control
EPISKINTM(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).

Storage
The EPISKINTM(SM) kits were kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.

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 was 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 (Day 0)
The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was 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.
- 50 µL of test item was applied evenly to the epidermal surface of each of two test units.
- 50 µL of physiological saline was added to each of the two negative control skin units.
- 50 µL of glacial acetic acid was added to each of the two positive control skin units.
The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (22.4-24.8°C) covered with the plate lids.

Rinsing (Day 0)
After the incubation time (4 hours), all test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining 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 pipette (without touching the epidermis).

MTT test (Day 0)
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units. The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 in a >95% humidified atmosphere for 3 hours (±15 minutes), protected from light.

Formazan extraction (Day 0)
At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved 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 containing 500 µL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

Cell viability measurements (Day 1)
Following the formazan extraction, 2×200 µL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 µL/well) was used as blank.
The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 24072800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 µL of test item was applied evenly to the epidermal surface of each of two test units.
Duration of treatment / exposure:
4 hours
Number of replicates:
2 per test group
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean OD value of test item
Value:
129.3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
ADDITIONAL CONTROLS
As the test item was colourless and it had no colouring potential, no additional controls were used for the non specific OD evaluation.
As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

VIABILITY RESULTS
The mean OD value for the test item treated skin samples showed a 129.3% relative viability compared to the negative control.

VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the two negative control tissues was in the recommended range (0.889).
The two positive control treated tissues showed 0.9% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 5.1%.
The difference of viability between the two negative control tissue samples in the MTT assay was 13.0%.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.
High viability results (>>100%) do regularly occur in cases where the test item causes metabolic stimulation in the exposed cells, so the study result is not considered to be invalid.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Optical Density (OD) and the calculated relative viability % of the samples

Substance

Optical Density (OD)

Viability (% RV)

 

Measured

Blank corrected

Negative Control:

Physiological saline

(0.9% (w/v) NaCl)

1

0.994

0.947

106.5

2

0.879

0.831

93.5

Mean

--

0.889

100.0

Positive control:

Glacial acetic acid

1

0.061

0.013

1.5

2

0.051

0.004

0.4

Mean

--

0.008

0.9

Test Item:

Luperox 520M50

1

1.167

1.120

126.0

2

1.226

1.179

132.6

Mean

--

1.149

129.3

Notes:

1. Mean blank value was 0.047

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

 

HISTORICAL CONTROL DATA

(updated 13 May 2016)

 

Negative control

(physiological saline)

Positive control

(Glacial acetic acid)

Minimum optical density (OD)

0.611

0.005

Maximum optical density (OD)

1.516

0.051

Mean optical density (OD)

0.871

0.017

Standard Deviation (SD)

0.164

0.010

Number of cases

81

81

Note: All optical density (OD) values measured are background corrected values (measured at 570±30 nm).

Interpretation of results:
other: Not corrosive
Conclusions:
Following exposure with Luperox 520M50, the mean cell viability was 129.3% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKIN™(SM) model test with Luperox 520M50 (Batch number: 99569 569E09-P03), the results indicate that the test item is non-corrosive to the skin.
Executive summary:

An in vitro skin corrosivity test of Luperox 520M50 test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section). The corrosivity of the test item was evaluated according to the OECD No. 431 guideline. Disks of EPISKINTM(SM) (two units) were treated with Luperox 520M50 test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control).

For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin. 

Following exposure with Luperox 520M50, the mean cell viability was 129.3% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test with Luperox 520M50, the results indicate that the test item is non-corrosive to the skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
15 March 2017 - 04 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD guideline 437 (In vitro eye irritation)
Version / remarks:
26 July 2013
Deviations:
yes
Remarks:
The positive control was applied to only 2 corneas instead of 3. Resulting individual and mean IVIS fell within the acceptable range defined in CiToxLAB France historical database and the experiment was consequently considered valid.
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
cattle
Details on test animals or tissues and environmental conditions:
Species: bovine cattle (Bos taurus).
Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
Age: as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle were up to 12 months old (typically, 5 to 8 months old).
Reason for choice: bovine corneas are recommended by Regulatory Authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from Supplier to CiToxLAB France: the eyes were immerged in containers filled with cooled buffered Hanks medium and placed into a cooling-box with a sufficient amount of ice packs to ensure cooling until arrival at CiToxLAB France. Containers with smooth internal surfaces were used for the transport to avoid damage to the corneas. Hank’s medium contained an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 µL of the undiluted test item was applied on each cornea.
.
Duration of treatment / exposure:
10 minutes.
Observation period (in vivo):
Opacity measurement:
Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.
The test item, applied undiluted, and the negative and positive controls were evaluated in a single experiment using a treatment time of 10 minutes and the closed chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.
The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C.
At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

Permeability measurement:
- after 90-min incubation in water (and other procedures), following the 2nd opacity measurement
Duration of post- treatment incubation (in vitro):
2 hours (± 10 minutes)
Number of animals or in vitro replicates:
Triplicate corneas for each tested substance (test item, negative control, positive control).
Details on study design:
EYES SELECTION
Upon arrival at CiToxLAB France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swivelled in order to observe the fringe areas and any scratches directly under the light.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature. The corneas were used within a maximum of 24 hours.
Storage of the corneas: as the corneas were not used immediately, they were stored after washing. Each cornea was stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.

The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded. The corneas were then mounted in the corneal holders with the endothelial side against the O-ring of the posterior chamber. Each cornea was identified with the corresponding holder number. After pre-incubation, corneas that showed any macroscopic defect or an opacity value over 7 were discarded.

TREATMENT
The corneas from the same series were always processed in the same order at each step. The medium of the anterior chamber was removed and each item was applied onto the epithelium of the cornea for 10 minutes in a water bath at +32°C (± 1°C).

REMOVAL OF TEST SUBSTANCE
The purpose of rinsing was to eliminate as much items as possible, while taking care not to damage the cornea. On completion of the treatment period, the test item were removed from the front opening of the anterior chamber and the epithelium was rinsed as follows:
- the anterior chamber was emptied using a metal gavage tube attached to a vacuum pump,
- any test item adhering to the walls of the anterior chamber was removed with a cotton bud,
- the corneas were rinsed five times with pre-warmed cMEM containing phenol red (i.e. until the test item had been completely removed from the chamber or until the phenol red was not discoloured). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.

SCORING SYSTEM
-Opacity:
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. The average change in opacity for the corneas treated with the negative control was calculated and this value was subtracted from the change in opacity for each cornea treated with test item or positive control to obtain a corrected opacity value (cOPT).
The mean cOPT value of each series of three corneas was calculated from the individual corrected opacity values.

-Permeability:
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution (4mg/mL) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes). At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube.
The corrected OD490 nm (cOD490 nm) value (i.e. permeability) for each cornea treated by test item or positive control was calculated by subtracting the average negative control cornea OD490 nm value from the original OD490 nm value of each cornea.
The mean cOD490 nm value of each series of corneas was calculated from the individual cOD490 nm values.

- Scoring:
In vitro irritancy score (IVIS) = cOPT + (15 x cOD490 nm)

ACCEPTANCE OF RESULTS:
For the validation of an experiment, the following criteria had to be fulfilled:
- the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two Standard Deviations (SD) of the historical mean,
- the mean opacity and mean OD490 nm of the negative control-treated corneas should be less than the established upper limit of historical mean.
Irritation parameter:
in vitro irritation score
Run / experiment:
Main
Value:
0
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control.
- Acceptance criteria met for positive control.
Irritant / corrosive response data:
Not requiring classification for eye irritation or serious damage (UN GHS No Category).
Other effects:
OTHER EFFECTS (macroscopic examination):
Negative control and test item-treated corneas: None
Positive control-treated corneas: Opacity, fluorescein fixation and thickening of each cornea
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item was identified as not requiring classification for eye irritation or serious eye damage (UN GHS No Category).
Executive summary:

The potential irritant and corrosive properties of the LUPEROX® 520M50 to the eye was evaluated with the Bovine Corneal Opacity and Permeability (BCOP) test method. The BCOP assay can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. The design of this study was based on the guideline OECD Guideline 437 and the study was performedin compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C. A single experiment was performed using three corneas for test item and negative control and two corneas for positive control. Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item, applied undiluted, and the negative and positive controls were evaluated in a single experiment using a treatment time of 10 minutes and the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

No notable opaque spots or irregularities were observed on test item-treated corneas.  All acceptance criteria were fulfilled. The study was therefore considered as valid. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0. As the mean IVIS was < 3, the test item was considered asa testchemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Under the experimental conditions of this study, LUPEROX® 520M50 was identified as not requiring classification for eye irritation or serious eye damage (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

Justification for classification or non-classification

According to the available data and the CLP criteria, a classification as skin irritant cat. 2 is warranted.