Registration Dossier

Administrative data

Description of key information

In vitro skin corrosion test

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

In vitro skin irritation test

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

In vitro eye irritation test

Under the conditions of the study, the test material had an IVIS of 3.5 therefore no prediction of eye irritation can be made.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 July 2017 to 06 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue lot number: 25828
- Delivery date: 04 July 2017
- Date of initiation of testing: 04 July 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): Room temperature for MTT extraction

TEST FOR DIRECT MTT REDUCTION
- A test material may interfere with the MTT endpoint if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test material was checked for the ability to directly reduce MTT: 25 mg of the test material was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37°C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test material turns blue/purple relative to the control, the test material was presumed to have reduced the MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- 25 mg of test material was added to 300 µL of sterile water. The solution was incubated in the dark at 37°C, 5% CO2 in air for 60 minutes. A visual assessment of the colour was then made. The test material was found to produce a coloured solution which may interfere with the MTT endpoint. Therefore, colour correction tissues were incorporated into the test to correct for this possibility. These tissues were treated identically to the tissues of the main test with the exception of being placed into assay medium for three hours post-exposure instead of MTT. Two tissues were dosed with the test material and two tissues were dosed with the negative control for each exposure period.

Main test
- The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labelled 6-well plates for both the 3 minute and 60 minute exposure periods. EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37°C, 5% CO2) for approximately 1 hour before dosing.
- Before pre-incubation was complete, a 24 well plate was prepared for use as a “holding plate” for both the 3 minute and 60 minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24 well plate was prepared for the MTT loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
- After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 minute exposure period was returned to the incubator, while the other was being dosed for the 60 minute exposure. For the 60 minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 25 mg of the test material and 50 µL of 8.0 N Potassium hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 µL of sterile water was added for wetting of the test material to increase tissue surface contact. The plate was returned to the incubator (37°C, 5% CO2) for the 60 minute exposure period. When dosing for the 60 minute exposure period was complete, the same procedure was repeated for the 3 minute exposure period.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After rinsing plates were then blotted and transferred to the 24 well plate prepared for MTT loading. The plate was incubated (37 °C, 5 % CO2) for 3 hours. Once the 60 Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated. After the 3 hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

NUMBER OF REPLICATE TISSUES: 2

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS
- After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre labelled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader.

QUANTITATIVE MTT ASSESSMENT (% TISSUE VIABILITY)
- The corrosivity potential of the test material was predicted from the relative mean tissue viabilities obtained after the 3 and 60 minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. Relative mean viability (%) = (mean OD570 of the test material/ mean OD570 of negative control) x 100
- Classification of corrosivity potential is based on relative % viabilities for both exposure times according to the following relative mean tissue viability (% of negative control):
3 min: < 50%: H314 Category 1A
3 min: ≥ 50%, 1 hour: < 15%: H314 Category 1B or 1C
3 min: ≥ 50%, 1 hour: ≥ 15: Not classified for corrosivity.

QUALITY CRITERIA
- Negative Control: The mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
- Positive Control: An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15%.
- Coefficient of Variation: In the range 20 and 100 % viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8.0 N
Duration of treatment / exposure:
3 minutes of exposure and 1 hour of exposure
Duration of post-treatment incubation (if applicable):
Incubated for 3 hours with MTT
Number of replicates:
2 per exposure time
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
111.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
96.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- Direct MTT Reduction: The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.
- Assessment of Colour Interference with the MTT endpoint: The solution containing the test material was a black to dark grey colour, therefore additional colour correction tissues were incorporated into the testing procedure. However, the results obtained showed that no colour interference occurred. It was therefore considered unnecessary to use the results of the colour correction tissues for quantitative correction of results or for reporting purposes.
- Test Material, Positive Control Material and Negative Control Material: Results are summarised in Table 1.

- Quality Criteria:
The mean OD570 for the negative control treated tissues was 1.596 for the 3 minute exposure period and 1.812 for the 60 minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control following the 60 minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Table 1: Summary of results

Exposure period

Percentage viability

Negative control

Positive control

Test material

3 minutes

100*

4.7

111.7

60 minutes

100*

3.3

96.2

*The mean viability of the negative control tissues is set at 100%

Interpretation of results:
other: Not corrosive in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.
Executive summary:

An in vitro skin corrosion test was carried out with the test material using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 BIS under GLP conditions.

During the study, duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test material was found to have the potential to cause colour interference with the MTT end-point therefore additional tissues were incorporated into the testing to correct for this. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT loading. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96 well plate. The optical density (OD) was measured at 570 nm (OD570).

Under the conditions of the study the percentatge viability of the tissues was 111.7 at 3 minutes and 96.2 after 60 minutes. The quality criteria required for acceptance of results in the test were satisfied.

The test material was therefore not considered to be corrosive to the skin, in vitro.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 August 2017 to 04 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended in international guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis
- Supplier: SkinEthic Laboratories, Lyon, France
- Tissue lot number(s): 17-EKIN-035
- Delivery date: 29 August 2017

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

TEST FOR DIRECT MTT REDUCTION
- To identify possible interference, the test material is checked for the ability to directly reduce MTT according to the following procedure: 10 mg of the test material was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
- If the MTT solution containing the test material turns blue/purple, the test material is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- A test material may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed. 10 mg of test material was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the colour was made.
- The test material was found to produce a coloured solution which may interfere with the MTT endpoint. Therefore, colour correction tissues were incorporated into the test to correct for this possibility. These tissues were treated identically to the tissues of the main test with the exception of being placed into assay medium for 3 hours post-exposure instead of MTT. Three tissues were dosed with the test material and three remained untreated to act as negative controls.

PRE-INCUBATION (DAY 0- TISSUE ARRIVAL)
- Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert, whether the tissues were satisfactory, if the temperature indicator colour was satisfactory and if the agar medium colour was satisfactory.
- 2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test material and each control material. The tissues were incubated at 37°C, 5% CO2 in air overnight.

MAIN TEST

APPLICATION
- 2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the second column of 3 wells of the 12-well plate.
- Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was applied topically to the corresponding tissues ensuring uniform covering. 5 μL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test material and the epidermis. Approximately 10 mg (26.3 mg/cm²) of the test material was then applied to the epidermal surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control material the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test material). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.

REMOVAL OF TEST MATERIAL AND CONTROLS
- At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test material. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 hours.

MTT LOADING/ FORMAZAN EXTRACTION (DAY 3)
- Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30°C for possible inflammatory mediator determination.
- 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS
- At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
- For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

NUMBER OF REPLICATE TISSUES: 3

QUANTITATIVE MTT ASSESSMENT (% TISSUE VIABILITY)
- For the test material the relative mean tissue viabilities obtained after the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way: relative mean viability (%)= (mean OD570 of test material/ mean OD570 of negative control) x 100
- Classification of irritation potential is based upon relative mean tissue viability following the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period according to the following:
- Relative mean tissue viability is ≤ 50% = irritant, H315 Category 2
- Relative mean tissue viability is > 50% = Not classified for irritation

QUALITY CRITERIA
- The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
- Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤ 40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤ 18%.
- Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is ≥ 0.6 and ≤ 1.5, and the SD value of the percentage viability is ≤ 18%.
- Test Material: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤ 18%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 mg (26.3 mg/cm²)

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration: 5% w/v aqueous solution.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- Direct MTT Reduction:
The MTT solution containing the test material did not turn blue or purple which indicated that the test material did not directly reduce MTT.

- Assessment of Colour Interference with the MTT endpoint:
The solution containing the test material was a grey colour, therefore additional colour correction tissues were incorporated into the testing procedure. However, the results obtained showed that negligible colour interference occurred. It was therefore considered unnecessary to use the results of the colour correction tissues for quantitative correction of results or for reporting purposes.

- Test Material, Positive Control Material and Negative Control Material:
The initial test was repeated due to a failure to meet the assay acceptance criteria. The individual and mean OD570 values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.
The relative mean viability of the test material treated tissues was 99.0% after a 15-Minute exposure period and 42-Hour post-exposure incubation period. It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

- Quality Criteria:
The relative mean tissue viability for the positive control treated tissues was 5.7% relative to the negative control treated tissues and the standard deviation value of the viability was 1.5%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.795 and the standard deviation value of the viability was 8.6%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test material treated tissues was 5.8%. The test material acceptance criterion was therefore satisfied.

Table 1: Mean OD570 Values and Viabilities for the Negative Control, Positive Control and Test Material

Material

OD570 of tissues

Mean OD570 of triplicate tissues

±SD of OD570

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control

0.717

0.795

0.069

90.2

100*

8.6

0.823

103.5

0.846

106.4

Positive Control

0.049

0.045

0.012

6.2

5.7

1.5

0.032

4.0

0.055

6.9

Test Material

0.817

0.787

0.046

102.8

99.0

5.8

0.734

92.3

0.810

101.9

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100 %

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.
Executive summary:

The skin irritation potential of the test material was determined in accordance with the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions.

During the study, the skin irritation potential of the test material was evaluated using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the coluorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. 

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test material was found to have the potential to cause colour interference with the MTT endpoint therefore additional tissues were incorporated into the testing to correct for this. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre labelled 96 well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test material treated tissues was 99.0% after the 15 Minute exposure period and 42 Hours post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material was considered to be a non-irritant in the in vitro skin irritation test.

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:
14 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2013
Deviations:
yes
Remarks:
(see "Principles of method if other than guideline")
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2008
Deviations:
yes
Remarks:
(see "Principles of method of other than guideline")
Principles of method if other than guideline:
The positive control group had an overall IVIS of 121.2. This was marginally higher than the criteria range set for an acceptable test. However, as the score was only marginally exceeded, it was decided that this result was acceptable as the positive control group was still providing its intended function which is to show the sensitivity of the test system to a known ocular irritant.
This deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the purpose of this study the test material was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution.
The test material was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Characteristics of donor animals: 12 to 60 months old
- Storage, temperature and transport conditions of ocular tissue: The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
Vehicle:
other: sodium chloride 0.9% w/v
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration: 20% w/v

VEHICLE
- Amount(s) applied: 0.75 mL
- Concentration: 0.9% w/v
- Batch no.: 3012488
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 minutes with sodium fluorescein
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (MEM) without phenol red and plugged. The holders were incubated at 32 ± 1°C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
The medium from both chambers of each holder was replaced with fresh complete MEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test material and three corneas to the positive control material.

NUMBER OF REPLICATES: 3

VEHICLE CONTROL USED: Sodium chloride 0.9% w/v

POSITIVE CONTROL USED: Imidazole 20% w/v in sodium chloride 0.9% w/v solution.

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL for 240 minutes

TREATMENT METHOD
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test material preparation or control materials were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1°C for 240 minutes.

REMOVAL OF TEST MATERIAL
At the end of the exposure period the test material and control materials were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A post-treatment opacity reading was taken and each cornea was visually observed.
- Corneal permeability: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1°C for 90 minutes. 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labelled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
- Others: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
- Opacity Measurement:
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Permeability Measurement:
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
- In Vitro Irritancy Score:
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
- Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.

DECISION CRITERIA:
The test material was classified according to the following prediction model:
- IVIS ≤ 3: No category. Not requiring classification to UN GHS or EU CLP
- IVIS > 3; ≤55: No prediction of eye irritation can be made
- IVIS > 55: Category 1. UN GHS or EU CLP Causes serious eye damage.

ACCEPTABILITY CRITERIA
- The test was acceptable if the positive control (20% w/v Imidazole ) produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 50.8 to 100.4.
- The test was acceptable if the negative control (0.9% w/v sodium chloride solution) produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2015 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤ 5.4 and for permeability ≤ 0.070.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
3.5
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not valid
Other effects / acceptance of results:
CORNEAL EPITHELIUM CONDITION
The corneas treated with the test material or negative control were clear post treatment. The corneas treated with the positive control were cloudy post treatment.

CRITERIA FOR AN ACCEPTABLE TEST
- The positive control In Vitro Irritancy Score was not within the range of 50.8 to 100.4. The positive control acceptance criterion was therefore not satisfied, this was reported as a deviation.
- The negative control gave opacity of ≤ 5.4 and permeability ≤ 0.070. The negative control acceptance criteria were therefore satisfied.

A summary of the results is displayed in Table 1.

 Table 1: Summary of results

Treatment

Cornea Number

Opacity

Permeability (OD)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

3

4

1

 

0.014

 

 

2

3

3

0

 

0.016

 

 

3

5

5

0

 

0.018

 

 

 

 

 

0.3*

 

0.016**

 

0.6

Positive
Control

4

5

107

102

101.7

2.215+

2.199

 

5

4

91

87

86.7

1.845+

1.829

 

6

3

92

89

88.7

1.760+

1.744

 

 

 

 

 

92.3***

 

1.924***

121.2

Test Material

7

3

7

4

3.7

0.076

0.060

 

8

4

7

3

2.7

0.041

0.025

 

9

4

7

3

2.7

0.029

0.013

 

 

 

 

 

3.0***

 

0.033***

3.5

OD = Optical density      

* = Mean of the post-treatment - pre treatment values 

** = Mean permeability               

*** = Mean corrected value     

+ = 1 in 5 dilution performed

Interpretation of results:
other: EU Criteria: no prediction of eye irritation can be made
Conclusions:
Under the conditions of the study, the test material had an IVIS of 3.5 therefore no prediction of eye irritation can be made.
Executive summary:

The potential of the test material to cause eye irritation was investigated in accordance with the standardised guidelines OECD 437 and EU Method B.47 using the Bovine Corneal Opacity and Permeability (BCOP), test method under GLP conditions.

During the study the test material was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive controls were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The positive control In Vitro Irritancy Score was not within the range of 50.8 to 100.4. The positive control acceptance criterion was therefore not satisfied, this was reported as a deviation. The negative control gave opacity of ≤5.4 and permeability ≤0.070. The negative control acceptance criteria were therefore satisfied.

Under the conditions of the study, the test material had an IVIS of 3.5 therefore no prediction of eye irritation can be made.

Additional information

In vitro skin corrosion test

An in vitro skin corrosion test was carried out with the test material using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 BIS under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test material was found to have the potential to cause colour interference with the MTT end-point therefore additional tissues were incorporated into the testing to correct for this. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT loading. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96 well plate. The optical density (OD) was measured at 570 nm (OD570).

Under the conditions of the study the percentatge viability of the tissues was 111.7 at 3 minutes and 96.2 after 60 minutes. The quality criteria required for acceptance of results in the test were satisfied.

The test material was therefore not considered to be corrosive to the skin, in vitro.

In vitro skin irritation test

The skin irritation potential of the test material was determined in accordance with the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, the skin irritation potential of the test material was evaluated using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the coluorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. 

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test material was found to have the potential to cause colour interference with the MTT endpoint therefore additional tissues were incorporated into the testing to correct for this. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre labelled 96 well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test material treated tissues was 99.0% after the 15 Minute exposure period and 42 Hours post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material was considered to be a non-irritant in the in vitro skin irritation test.

In vitro eye irritation test

The potential of the test material to cause eye irritation was investigated in accordance with the standardised guidelines OECD 437 and EU Method B.47 using the Bovine Corneal Opacity and Permeability (BCOP), test method under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study the test material was applied at a concentration of 20 % w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive controls were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The positive control In Vitro Irritancy Score was not within the range of 50.8 to 100.4. The positive control acceptance criterion was therefore not satisfied, this was reported as a deviation. The negative control gave opacity of ≤5.4 and permeability ≤0.070. The negative control acceptance criteria were therefore satisfied.

Under the conditions of the study, the test material had an IVIS of 3.5 therefore no prediction of eye irritation can be made.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin corrosion/ irritation or eye irritation.