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Description of key information

Skin Irritation

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

BCOP Test

Under the conditions of this study, the test material induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made.

RhCE

Under the conditions of this study, the test material is potentially irritant or corrosive when treated neat but non-irritant for solutions of 50 % and downwards in the EpiOcular™ test.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 August 2017 to 14 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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.: 17-EKIN-032
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 23 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 % (actual range 74 - 90%), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.6 - 37.5 °C).

TEST FOR COLOUR INTERFERENCE BY THE TEST MATERIAL
The test material was checked for possible color interference before the study was started. Some non-colored test materials may change into coloured items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the colour interference, at least 10 mg of the test material was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.

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 10 mg of the test material was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37 °C. A negative control, 25 μL sterile Milli-Q water was tested concurrently. At the end of the incubation period a colour check was performed.

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 (17.0 to 23.7 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 69.5 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate.

CALCULATION OF CELL VIABILITY
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed. The corrected OD (ODc) for each sample or control were calculated by subtracting the value of blank mean (ODbl) from each reading (ODraw).
ODc = ODraw - ODbl
The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc / mean ODlt_u+MTT) 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.

≤ 50 % of the mean viability of the negative controls = Category 1 or Category 2 (additional information on corrosion needed)
> 50% of the mean viability of the negative controls = No category
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 17.0 to 23.7 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):
42 hours and then incubated for 3 hours with MTT
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
94
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
INTERFERENCE WITH MTT
- The test material was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test material to MTT medium. Because no colour changes were observed it was concluded that the test material did not interact with the MTT endpoint.

TISSUE VIABILITY
- Results can be seen in Tables 1 and 2.
- The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 94 %. Since the mean relative tissue viability for the test material was above 50 % it is considered to be non-irritant.
- The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 4.8 %. 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 <3 %, 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

1.222

1.227

1.252

1.234 ± 0.016

Test material

1.172

1.170

1.135

1.159 ± 0.021

Positive control

0.032

0.087

0.059

0.059 ± 0.028

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 (0.042). 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

1.3

Test material

94

1.7

Positive control

4.8

2.3

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 94 %. 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 4.8 % after 15 ± 0.5 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 < 3 %, 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

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 June 2017 to 30 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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)
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.
- A solubility test in physiological saline was performed based on visual assessment.
- A 20% (w/v) solution of the test material was prepared in physiological saline.
- Any residual volumes were discarded.
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
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration (if solution): 20 % (w/v)

VEHICLE
- Amount(s) applied: 750 µL
Duration of treatment / exposure:
240 ± 10 minutes
Number of animals or in vitro replicates:
3
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 (Life Technologies, Bleiswijk, The Netherlands) 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) of BASF (Ludwigshafen, Germany) 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 (BASF-OP3.0, BASF, Ludwigshafen, Germany). 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.

VEHICLE CONTROL USED: physiological saline

POSITIVE CONTROL USED: 20% (w/v) Imidazole solution prepared in physiological saline.

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 240 ± 10 minutes at 32 ± 1 °C.

TREATMENT METHOD
- The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or 20 % (w/v) solution of the test material 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
- 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. 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:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- 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:
17
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
TEST MATERIAL
- The corneas treated with the test material showed opacity values ranging from 6.2 to 24 and permeability values ranging from 0.016 to 0.071. The corneas were translucent after the 240 minutes of treatment with the test material.
- A pH effect of the test material was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 6.4 to 25 after 240 minutes of treatment with the test material.
- The test material induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 17 after 240 minutes of treatment.

CONTROL GROUPS
- The individual in vitro irritancy scores for the negative controls ranged from -0.1 to 2.3.
- The individual positive control in vitro irritancy scores ranged from 124 to 150. The corneas treated with the positive control were turbid after the 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 (20% (w/v) Imidazole) was 134 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.

Table 1: Opacity, Permeability and In Vitro Scores

Treatment

Final Opacity

Final OD490

IVIS

Negative control

1

2.1

0.008

2.3

2

-0.1

0.015

0.1

3

-0.1

0.001

-0.1

Mean

0.6

0.008

0.7

Positive control

1

91

2.167

124

2

102

1.773

129

3

102

3.183

150

Mean

98

2.375

134

Test material

1

6.2

0.017

6.4

2

24

0.071

25

3

18

0.016

18

Mean

16

0.034

17

Interpretation of results:
other: No prediction on the classification can be made
Conclusions:
Under the conditions of this study, the test material induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made.
Executive summary:

The potential of the test material to cause irritation to the eye was investigated in accordance with the standardised guideline OECD 437, under GLP conditions.

The eye hazard potential of the test material was measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The eye damage of the test material was tested through topical application for approximately 240 minutes. The test material was applied as a 20% (w/v) solution (750 μL) directly on top of the corneas.

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 (20% (w/v) Imidazole) was 134 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.

Thiamine hydrochloride induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 17 after 4 hours of treatment.

Under the conditions of this study, the test material induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 November 2017 to 17 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes
Species:
human
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27406 Kit Q)
- The EpiOcular tissue construct is a non-keratinised epithelium (0.6 cm^2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialised medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.
- Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).
- Source: MatTek Corporation, Ashland MA, U.S.A.
- On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37 °C in 1.0 mL fresh pre-warmed Assay Medium, which was refreshed after approximately 1 hour. Assay Medium was supplied by MatTek Corporation, Ashland, USA.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 µL or 59.1 to 66.0 mg
- Concentration (if solution): 25 or 50 % (w/v) or neat
Duration of treatment / exposure:
- 30 ± 2 minutes for the 25 and 50 % (w/v) solutions
- 6 hours ± 15 minutes for the neat solution
Duration of post- treatment incubation (in vitro):
- 12 ± 2 minutes immersion incubation followed by 120 ± 15 minutes incubation for the 25 and 50 % (w/v) solutions
- 25 ± 2 minutes immersion incubation followed by 18 hours ± 15 minutes incubation for the neat solution
- The tissues were then incubated for 180 ± 10 minutes with MTT.
Number of animals or in vitro replicates:
The test was performed on a total of 2 tissues per test material together with a negative control and positive control.
Details on study design:
COLOUR INTERFERENCE BY THE TEST MATERIAL
- The test material was checked for possible colour interference before the study was started. Some non-coloured test materials may change into coloured materials in aqueous conditions and thus stain the tissues during the exposure. To assess the colour interference, approximately 50 mg of the test material or 50 μL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0 °C in the dark. Furthermore, approximately 50 mg of the test material or 50 μL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g.
- At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Centrifugation was not considered necessary. If after subtraction of the negative control, the OD for the test material solution is >0.08, the test material is considered as possibly interacting with the MTT measurement.

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, approximately 50 mg of the test material was added to 1 ml MTT solution (1 mg/ml MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0 °C in the dark. A negative control, 50 μL sterile Milli-Q water was tested concurrently. If the MTT solution colour turned blue / purple or if a blue / purple precipitate was observed the test material interacts with MTT. Only test materials which bind to the tissue after rinsing can interact with MTT in the main assay.

APPLICATION/ TREATMENT
- The test was performed on a total of 2 tissues per test material together with a negative control and positive control.
- Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free- DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Two tissues were treated with 50 μl Milli-Q water (negative control) and 2 tissues with 50 μl Methyl Acetate (positive control) respectively.
- 25 and 50 % (w/v) solutions: 50 μL of the undiluted test material was added into the 6-well plates on top of the tissues. After the exposure period with the test material (30 ± 2 minutes at 37.0 ± 1.0 °C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test material. After rinsing, the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 120 ± 15 minutes at 37 °C.
- Neat: At least 50 mg solid was added into the 6-well plates on top of the tissues. After the exposure period with the test material (6 hours ± 15 minutes at 37.0 ± 1.0 °C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test material. After rinsing, the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37 °C.

CELL VIABILITY MEASUREMENT
- After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37 °C.
- 25 and 50 % (w/v) solutions: After incubation with MTT-medium, the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol is flowing into the insert on the tissue surface. Formazan was extracted with 2 mL isopropanol for 18 ± 2 hours refrigerated in the dark. At the end of the extraction period, the liquid within each insert was decanted/pipetted into the well from which it was taken.
- Neat: After incubation with MTT-medium, the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking.
- The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test material was classified according to remaining cell viability following exposure of the test material.

ACCEPTABILITY CRITERIA
The in vitro eye irritation test is considered acceptable if it meets the following criteria:
- The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
- The mean relative tissue viability of the positive control should be <50% relative to the negative control.
- The difference between the % tissue viabilities of the two identically treated replicates should be <20.

INTERPRETATION
- The test material is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60 %. In this case no further testing in other test methods is required.
- The test material is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60 %.

ANALYSIS
- Calculation of Cell Viability:
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) x 100
Irritation parameter:
other: Mean Tissue Viability
Run / experiment:
Test material 25 %
Value:
93
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Difference between two tissues (percentage): 14
Irritation parameter:
other: Mean Tissue Viability
Run / experiment:
Test material 50 %
Value:
85
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Difference between two tissues (percentage): 3.59
Irritation parameter:
other: Mean Tissue Viability
Run / experiment:
Neat test material
Value:
1.97
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Difference between two tissues (percentage): 0.19
Other effects / acceptance of results:
INTERFERENCE OF THE TEST MATERIAL WITH THE MTT ENDPOINT
- The test material was checked for possible direct MTT reduction by adding the test material to MTT medium. Because no colour changes were observed it was concluded that the test material did not interact with the MTT endpoint.
- The test material was checked for colour interference in aqueous conditions. Addition of the test material to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.0421 and 0.0439, respectively. Therefore it was concluded that the test material did not induce colour interference.
- In addition, because no colour change was observed in the presence of MTT it was concluded that the test material did not interact with the MTT endpoint.

MAIN ASSAY
- Results can be seen in Tables 1, 2, 3 and 4.
- Eye hazard potential is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 30 ± 2 minutes (25 and 50 % solutions) and 6 hours ± 15 minutes treatment (neat) with the test material compared to the negative control tissues was 93 %, 85 % and 1.97 %, respectively. Since the mean relative tissue viability for the test material was below 60 % when treated neat, it is considered to be potentially irritant or corrosive to the eye. 50 % solutions and downwards is considered to be non-irritant.
- 25 and 50 % (w/v) solutions: The positive control had a mean cell viability of 15 % after 30 ± 2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically with the positive control was less than 25 %. However, both individual tissues treated with the positive control were both below 50 % relative to the negative control. The difference between the percentage of viability of two tissues treated identically with the negative control and test material was less than 15 %, indicating that the test system functioned properly.
- Neat: The positive control had a mean cell viability of 31 % after 6 hours ± 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 11 %, indicating that the test system functioned properly.

Table 1: Mean Absorption in the EpiOcular™ Test with 25 and 50 % (w/v) Test Material Solutions

Group

A (OD570)

B (OD570)

Mean (OD570) ± SD

Negative control

1.785

1.693

1.739 ± 0.065

Test material 25 %

1.732

1.488

1.610 ± 0.172

Test material 50 %

1.513

1.451

1.482 ± 0.044

Positive control

0.047

0.462

0.254 ± 0.293

 

Table 2: Mean Absorption in the EpiOcular™ Test with Neat Test Material Solution

Group

A (OD570)

B (OD570)

Mean (OD570) ± SD

Negative control

1.537

1.421

1.479 ± 0.082

Test material

0.028

0.031

0.029 ± 0.002

Positive control

0.537

0.387

0.462 ± 0.106

 

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In these tables the values are corrected for background absorption (0.041 and 0.042, respectively). Isopropanol was used to measure the background absorption.

 

Table 3: Mean Tissue Viability in the EpiOcular™ Test with 25 and 50 % (w/v) Test Material Solutions

Group

Mean tissue viability (percentage

of control)

Difference between two tissues

(percentage)

Negative control

100

5.28

Test material 25 %

93

14

Test material 50 %

85

3.59

Positive control

15

24

 

Table 4: Mean Tissue Viability in the EpiOcular™ Test with Neat Test Material Solution

Group

Mean tissue viability (percentage

of control)

Difference between two tissues

(percentage)

Negative control

100

7.86

Test material

1.97

0.19

Positive control

31

10

Interpretation of results:
other: EU Criteria: Category 2, Causes serious eye irritation
Conclusions:
Under the conditions of this study, the test material is potentially irritant or corrosive when treated neat but non-irritant for solutions of 50 % and downwards in the EpiOcular™ test.
Executive summary:

The eye hazard potential of the test material was investigated in accordance with the standardised guideline OECD 492, under GLP conditions. For this purpose the test material was topically applied on the Reconstructed Human EpiOcular™ Model.

The possible eye hazard potential of the test material was tested through topical application for 30 minutes (25 and 50 % solutions w/v in Milli-Q water) and 6 hours (neat). The test material was applied directly on top of the tissue for 30 ± 2 minutes for the tissues treated with 25 and 50 % solutions. In addition, the test material (59.1 to 66.0 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes for the tissues treated neat.

25 and 50 % (w/v) solutions: After exposure, the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control had a mean cell viability of 15 % after 30 ± 2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically with the positive control was less than 25 %. However, both individual tissues treated with the positive control were both below 50 % relative to the negative control. The difference between the percentage of viability of two tissues treated identically with the negative control and test item was less than 15 %, indicating that the test system functioned properly.

Neat: After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control had a mean cell viability of 31 % after 6 hours ± 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 11 %, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30 ± 2 minutes (25 and 50 % solutions) and 6 hours ± 15 minutes treatment (neat) with the test material compared to the negative control tissues was 93, 85 and 1.97 %, respectively. The test material was above 60 % after 30 ± 2 minutes treatment (25 and 50 % solutions). The test material was below 60 % after 6 hours ± 15 minutes treatment (neat).

Under the conditions of this study, the test material is potentially irritant or corrosive when treated neat but non-irritant for solutions of 50 % and downwards in the EpiOcular™ test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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 94 %. 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 4.8 % after 15 ± 0.5 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 < 3 %, 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.

Eye Irritation

BCOP Test

The potential of the test material to cause irritation to the eye was investigated in accordance with the standardised guideline OECD 437, 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 eye hazard potential of the test material was measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The eye damage of the test material was tested through topical application for approximately 240 minutes. The test material was applied as a 20% (w/v) solution (750 μL) directly on top of the corneas.

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 (20% (w/v) Imidazole) was 134 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.

Thiamine hydrochloride induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 17 after 4 hours of treatment.

Under the conditions of this study, the test material induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made.

RhCE

The eye hazard potential of the test material was investigated in accordance with the standardised guideline OECD 492, under GLP conditions. For this purpose the test material was topically applied on the Reconstructed Human EpiOcular™ Model. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The possible eye hazard potential of the test material was tested through topical application for 30 minutes (25 and 50 % solutions w/v in Milli-Q water) and 6 hours (neat). The test material was applied directly on top of the tissue for 30 ± 2 minutes for the tissues treated with 25 and 50 % solutions. In addition, the test material (59.1 to 66.0 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes for the tissues treated neat.

25 and 50 % (w/v) solutions: After exposure, the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control had a mean cell viability of 15 % after 30 ± 2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically with the positive control was less than 25 %. However, both individual tissues treated with the positive control were both below 50 % relative to the negative control. The difference between the percentage of viability of two tissues treated identically with the negative control and test item was less than 15 %, indicating that the test system functioned properly.

Neat: After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control had a mean cell viability of 31 % after 6 hours ± 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 11 %, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30 ± 2 minutes (25 and 50 % solutions) and 6 hours ± 15 minutes treatment (neat) with the test material compared to the negative control tissues was 93, 85 and 1.97 %, respectively. The test material was above 60 % after 30 ± 2 minutes treatment (25 and 50 % solutions). The test material was below 60 % after 6 hours ± 15 minutes treatment (neat).

Under the conditions of this study, the test material is potentially irritant or corrosive when treated neat but non-irritant for solutions of 50 % and downwards in the EpiOcular™ test.

Justification for classification or non-classification

It is taken into account that an OECD 439 study was negative, an OECD 437 study did not have an IVIS > 55, but no negative result could be obtained in an OECD 492 study. Thus, it is recommended to classify the test material as Category 2 for eye irritation as a precautionary principle: EU Criteria: Category 2, H319: Causes serious eye irritation