Registration Dossier

Administrative data

Description of key information

Skin Corrosion in vitro: Negative

Skin Irritation in vitro: Negative

Eye Irritation in vitro: Negative

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:
02 August 2016 to 05 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
No correction was made for the purity/composition of the test material.
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: EpiDerm Skin Model (EPI-200, kit E and F)
- Tissue batch number(s): Lot no.: 24318
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- Source: MatTek Corporation, Ashland MA, USA

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

CELL CULTURE
- Tissues: On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL supplemented DMEM medium (Dulbecco’s Modified Eagle’s Medium, serum-free).
- MTT medium: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Environmental conditions: All incubations, with the exception of the test material incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 to 100 %, containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C.

NUMBER OF REPLICATE TISSUES: 2 per exposure time

TEST FOR THE INTERFERENCE OF THE TEST MATERIAL WITH THE MTT ENDPOINT
The test material was checked for possible colour interference before the study was started. To assess the colour interference, at least 25 mg of the test material or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.

TEST FOR REDUCTION OF MTT BY THE TEST MATERIAL
The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test material to reduce MTT, at least 25 mg was added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the exposure time it was checked if a blue / purple colour change or a blue / purple precipitate was observed.

APPLICATION/TREATMENT OF THE TEST MATERIAL
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for approximately 2.5 hours at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM medium just before the test material was applied. The test was performed on a total of 4 tissues per test material together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test material and two for a 1-hour exposure. The skin was moistened with 25 μL Milli-Q water to ensure close contact of the test material to the tissue and 25.7 to 29.5 mg of the solid test material was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μL Milli-Q water (negative control) and with 50 μL 8 N KOH (positive control), respectively. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test material. Rinsed tissues were kept in 24 well plates on 300 μL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

CELL VIABILITY MEASUREMENT
The DMEM medium was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37 °C in air containing 5 % CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol overnight at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test material was classified according to remaining cell viability following exposure of the test material with either of the two exposure times.

INTERPRETATION
- Acceptability of the assay
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 to 100 % viability, the Coefficient of Variation (CV) between tissue replicates should be ≤30 %.

- Data evaluation and statistical procedures
A test material is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50 %.
b) In addition, a test material considered non-corrosive (viability ≥ 50 %) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test material is decreased below 15 %.
A test material is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50 %.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25.7 to 29.5 mg of the solid test material was added into the 6-well plates on top of the skin tissues.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL Milli-Q water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL KOH
- Concentration (if solution): 8 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:
Experiment 1 - 3 minutes of exposure
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Experiment 2 - 1 hour of exposure
Value:
96
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test material was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test material to MTT medium. As the solutions did not turn blue / purple nor was a blue / purple precipitate observed, it was concluded that the test material did not interfere with the MTT endpoint.
The mean absorption at 570 nm measured after treatment with the test material and controls are presented in Table 1.
Table 2 shows the mean tissue viability obtained after 3-minute and 1-hour treatments compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 100 and 96 %, respectively. As the mean relative tissue viability for the test material was not below 50 % after 3 minutes of treatment and not below 15 % after 1 hour of treatment, the test material is considered to be not corrosive.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 9 %.
In the range of 20 to 100 % viability the Coefficient of Variation between tissue replicates was ≤8 %, indicating that the test system functioned properly.

Table 1: Mean absorption in the in vitro skin corrosion test

 

3-minute application

1-hour application

A (OD570)

B (OD570)

Mean (OD570) ± SD

A (OD570)

B (OD570)

Mean (OD570) ± SD

Negative control

1.462

1.459

1.461 ± 0.002

1.657

1.563

1.610 ± 0.066

Test material

1.478

1.434

1.456 ± 0.031

1.612

1.485

1.548 ± 0.090

Positive control

0.138

0.169

0.154 ± 0.021

0.180

0.104

0.142 ± 0.054

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

 

Table 2: Mean tissue viability

 

3-minute application viability (% of control)

1-hour application viability (% of control)

Negative control

100

100

Test material

100

96

Positive control

11

9

 

Table 3: Coefficient of Variation between tissue replicates

 

3 minute exposure

1 hour exposure

Negative control

0.2

5.6

Test material

3.0

7.9

Positive control

18

42

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.

The ability of the test material to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)) was investigated. The possible corrosive potential was tested through topical application for 3 minutes and 1 hour.

Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of the test material was applied directly on top of the skin tissue. Milli-Q water and 8 N KOH served as the negative and positive control, respectively.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments compared to the negative control tissues was 100 and 96 %, respectively. As the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment, the test material is considered to be not corrosive.

The positive control had a mean relative tissue viability of 9 % after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of 20 to 100 % viability the Coefficient of Variation between tissue replicates was ≤8 %, indicating that the test system functioned properly. The test was concluded to be valid.

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

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 September 2016 to 12 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other 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:
2012
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
No correction was made for the purity/composition of the test material.
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 Small Model™ (EPISKIN-SM™, 0.38 cm²)
- Tissue batch number(s): Lot no.: 16-EKIN-036
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Source: SkinEthic Laboratories, Lyon, France

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

NUMBER OF REPLICATE TISSUES: 3

CELL CULTURE
- Tissues: On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 22 hours at 37 °C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories.
- MTT medium: MTT concentrate (3 mg/mL in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL).
- Environmental conditions: All incubations, with the exception of the test material incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 to 100 % containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C.

TEST FOR THE INTERFERENCE OF THE TEST MATERIAL WITH THE MTT ENDPOINT
The test material was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model; as solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test material did not interfere with the MTT endpoint.

APPLICATION/TREATMENT OF THE TEST MATERIAL
The test was performed on a total of 3 tissues per test material together with negative and positive controls. The skin was moistened with 5 μL Milli-Q water to ensure close contact of the test material to the tissue and the solid test material (10.0 to 13.0 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (phosphate buffered saline, negative control) and 3 tissues with 25 μL 5 % SDS (sodium dodecyl sulphate, positive control), respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test material. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37 °C.

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-well plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37 °C. After incubation the tissues were placed on blotting paper to dry. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labelled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for 71 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

CALCULATION OF CELL VIABILITY
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed. The corrected OD for each sample or control were calculated by subtracting the value of ODblank mean from each reading (ODraw).
ODcorrected = ODraw - ODblank mean
The OD value representing 100 % cell viability is the average OD of the negative controls. For the EpiSkin® Irritation assay this is the average OD of the negative controls.
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODcorrected / mean ODnegative controls) x 100

INTERPRETATION
- Acceptability of the assay
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤50 % relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

- Data evaluation and statistical procedures
A test material is considered irritant in the skin irritation test if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is ≤ 50 % of the mean viability of the negative controls.
A test material is considered non-irritant in the in vitro skin irritation test if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is > 50 % of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10.0 to 13.0 mg was added into 12-well plates on top of the skin tissues

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL phosphate buffered saline (PBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL sodium dodecyl sulphate (SDS)
- Concentration (if solution): 5 %
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
incubated for 3 hours with MTT
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
105
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The mean absorption at 570 nm measured after treatment with the test material and controls are presented in Table 1.
Table 2 shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 105 %. Since the mean relative tissue viability for the test material was above 50 %, the test material is considered to be non-irritant.
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 5.3 %. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was 12 % or less, indicating that the test system functioned properly.

Table 1: Mean absorption in the in vitro skin irritation test

 

A (OD570)

B (OD570)

C (OD570)

Mean (OD570) ± SD

Negative control

1.216

1.031

1.006

1.084 ± 0.115

Test material

1.114

1.024

1.274

1.137 ± 0.127

Positive control

0.068

0.045

0.060

0.058 ± 0.012

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

 

Table 2: Mean tissue viability in the in vitro skin irritation test

 

Mean tissue viability (percentage of control)

Standard deviation (percentage)

Negative control

100

11

Test material

105

12

Positive control

5.3

1.1

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:

An in vitro skin irritation test using a human skin model was carried out in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

This study tests the ability of the test material to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SM™)). The possible skin irritation potential was tested through topical application for 15 minutes.

Skin tissue was moistened with 5 μL of Milli-Q water and at least 10 mg of the test material was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 105 %. Since the mean relative tissue viability for the test material was above 50 % after 15 ± 0.5 minutes treatment the test material is considered to be non-irritant.

The positive control had a mean cell viability of 5.3 % after 15 minutes of exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was 12 % or less, indicating that the test system functioned properly. Under the conditions of this study, the test material is 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:
25 July 2016 to 26 July 2016
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:
no
GLP compliance:
yes
Specific details on test material used for the study:
No correction was made for the purity/composition of the test material.
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Bovine eyes were used as soon as possible after slaughter.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularisation by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: None reported
Vehicle:
physiological saline
Remarks:
20 % w/v suspension
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): 20 % (w/v) suspension

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): 20 % (w/v) solution
Duration of treatment / exposure:
4 hours (240 ± 10 minutes)
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects; those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1 % (v/v) L-glutamine and 1 % (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
Yes, physiological saline

POSITIVE CONTROL USED
Yes, 20 % w/v imidazole solution prepared in physiological saline

APPLICATION DOSE AND EXPOSURE TIME
750 µL of a 20 % w/v suspension for 240 ± 10 minutes

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 µL of either the test material, negative or positive control was introduced onto the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test material were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies).
- Post-exposure incubation: Yes, with sodium fluorescein

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
The opacity of a cornea was measured by the diminution of light passing through the cornea.
The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity = [(I0 - I) - 0.9894] / 0.0251
With I0 being the empirically determined illuminance through a cornea holder but with windows and medium and I being the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test material or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test material or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1 °C.
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.
- Others: Possible pH effects of the test material on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test material induced irritation through only one of the two endpoints.

DECISION CRITERIA:
The IVIS cut-off values for identifying the test materials as inducing serious eye damage (UN GHS Category 1) and test materials not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are:
In vitro score range: ≤ 3 = UN GHS No Category; > 3 but ≤ 55 = No prediction can be made; and >55 = UN GHS Category 1

- Acceptability of the assay
The assay is considered acceptable if:
a) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
b) The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
0.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Table 1 summarises the opacity, permeability and in vitro irritancy scores of the test material and the controls.
The individual in vitro irritancy scores for the negative controls ranged from -0.6 to 0.9. The individual positive control in vitro irritancy scores ranged from 109 to 136. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with the test material showed opacity values ranging from -0.2 to 1.3 and permeability values ranging from 0.002 to 0.033. The corneas were clear but yellow after the 240 minutes of treatment with the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.0 to 1.3 after 240 minutes of treatment.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control was 121 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.6 after 240 minutes of treatment.

Table 1: Summary of opacity, permeability and in vitro scores

Treatment

Mean Opacity

Mean Permeability

Mean In vitro Irritation Score*

Negative control

0.1

0.004

0.2

Positive control

85.9

2.323

120.7

Test material

0.4

0.015

0.6

*Calculated using the negative control mean opacity and mean permeability values for the positive control and test material. In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, as the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

The hazard potential of the test material to the eye was evaluated in vitro in accordance with the standardised guideline OECD 437 under GLP conditions using the Bovine Corneal Opacity and Permeability test (BCOP test).

The eye damage of the test material was tested in isolated bovine corneas through topical application for approximately 240 minutes. The test material was applied as a 20 % w/v suspension in physiological saline (750 µL). Concurrent negative and positive controls were run using physiological saline and 20 % imidazole solution in physiological saline, respectively.

The test material did not induce ocular irritation through both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score of 0.6 after 240 minutes of treatment.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control was 121 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Under the conditions of this study, as the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro skin corrosion

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).

The ability of the test material to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)) was investigated. The possible corrosive potential was tested through topical application for 3 minutes and 1 hour.

Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of the test material was applied directly on top of the skin tissue. Milli-Q water and 8 N KOH served as the negative and positive control, respectively.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments compared to the negative control tissues was 100 and 96 %, respectively. As the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment, the test material is considered to be not corrosive.

The positive control had a mean relative tissue viability of 9 % after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of 20 to 100 % viability the Coefficient of Variation between tissue replicates was ≤8 %, indicating that the test system functioned properly. The test was concluded to be valid.

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

In vitro skin irritation

An in vitro skin irritation test using a human skin model was carried out 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).

This study tests the ability of the test material to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SM™)). The possible skin irritation potential was tested through topical application for 15 minutes.

Skin tissue was moistened with 5 μL of Milli-Q water and at least 10 mg of the test material was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 105 %. Since the mean relative tissue viability for the test material was above 50 % after 15 ± 0.5 minutes treatment the test material is considered to be non-irritant.

The positive control had a mean cell viability of 5.3 % after 15 minutes of exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was 12 % or less, indicating that the test system functioned properly. Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

In vitro eye irriation

The hazard potential of the test material to the eye was evaluated in vitro in accordance with the standardised guideline OECD 437 under GLP conditions using the Bovine Corneal Opacity and Permeability test (BCOP test). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The eye damage of the test material was tested in isolated bovine corneas through topical application for approximately 240 minutes. The test material was applied as a 20 % w/v suspension in physiological saline (750 µL). Concurrent negative and positive controls were run using physiological saline and 20 % imidazole solution in physiological saline, respectively.

The test material did not induce ocular irritation through both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score of 0.6 after 240 minutes of treatment.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control was 121 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Under the conditions of this study, as the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

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 and eye corrosion or irritation.