Registration Dossier

Diss Factsheets

Administrative data

Description of key information

The test substance was neither corrosive nor irritating to skin (LPT, 2016).

Based on the available data no prediction concerning eye irritant or corrosive potential of the test item can be made (LPT, 2016).

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:
2016-03-17 to 2016-04-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
Reconstructed Human Epidermis (RHE) Test Method, adopted July 28, 2015.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item was applied topically as solid to the model skin surface, which was moistened with sterile deionised water.
Test system:
human skin model
Cell type:
other: three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum
Details on animal used as source of test system:
Human skin model
Justification for test system used:
Skin corrosion refers to the production of irreversible tissue damage in the skin following the application of a test item [as defined by the Globally Harmonised System for the Classification and Labelling of Chemical Substances and Mixtures (GHS)]. The OECD Guideline 431 does not require the use of live animals or animal tissue for the assessment of skin corrosivity.
Vehicle:
unchanged (no vehicle)
Details on test system:
The following Reconstructed Human Epidermis Model was used:
EpiDerm™ (EPI-200-SCT, Lot no. 23328) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.


Cell viability measurements
- MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1) reduction, which had been shown
to give accurate and reproducible results, was used to measure cell viability
- Each skin sample was placed in an MTT assay solution of 1 mg/mL (37°C incubation temperature, 5% CO2, 95% humidity) for 3 hours
- The precipitated blue formazan product was extracted using the solvent propanol-2, and the concentration of the formazan was measured by
determining the optical density (OD) at a wavelength of 540 nm in a spectrophotometer
- Cell viability measurements were carried out at the end of the exposure period (1st period: 3 min; 2nd period: 60 min). The measurements were
made for each of the two tissues in triplicate.
- Previous checks for interference of the test item with the MTT assay, the nylon mesh or the tissues were performed. No discoloration or test item
interference with the vital dye was noted.

ADMINISTRATION
- EpiDerm tissues were conditioned by pre-incubation (1 hour) in Maintenance Medium1 for release of transport stress related compounds and
debris in the incubator (37°C, 5% CO2, 95% humidity).
- After pre-incubation tissues were transferred to fresh Maintenance Medium and topically exposed with the test chemicals for 3 min and 1 h,
respectively.
- Two tissues were used per treatment, negative and positive control and exposition time (12 tissues in total).
- 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface which was moistened with
sterile deionised water to ensure adequate contact with the skin. A minimum of 30 mg or 70 μL substance applied per cm2 is required by the
guidelines.
- Positive control item was 8 N KOH4, 50 µl
- Negative control was sterile deionised water, 50µl
- After exposure tissues were rinsed, blotted and assay medium was replaced by MTT assay medium2 (final concentration: 1 mg
MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue)/mL medium).
- After 3-h incubation, tissues were washed with Dulbecco's phosphate buffered saline (D-PBS), blotted and the blue formazan salt was extracted with propanol-2.
- The optical density of the formazan extract was determined spectrophotometrically at 540 nm and cell viability was calculated for each tissue as
% of the mean of the negative control tissues.
- Skin corrosion potential of the test materials was classified according to the remaining cell viability obtained after 3 minutes or 1 hour exposure
with the test chemical.


Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Test item: 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface which was
moistened with sterile deionised water to ensure adequate contact with the skin.
- Positive control item was 8 N KOH4, 50 µl
- Negative control was sterile deionised water, 50µl
- At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS).
Duration of treatment / exposure:
3 minutes and 1 hour
Number of replicates:
Two tissues were used for each treatment and concurrent control groups.
Irritation / corrosion parameter:
other: cut-off percentage cell viability value
Run / experiment:
The EpiDerm™ model was employed.
Value:
105.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Under the present test conditions test item tested at two exposure periods of 3 minutes (cell viability value = 105.5 %) or 1 hour (cell viability value = 108,6 %) was non-corrosive to skin.
Irritation / corrosion parameter:
other: cut-off percentage cell viability value
Run / experiment:
The EpiDerm™ model was employed.
Value:
108.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Under the present test conditions test item tested at two exposure periods of 3 minutes (cell viability value = 105.5 %) or 1 hour (cell viability value = 108,6 %) was non-corrosive to skin.
Other effects / acceptance of results:
no other effects

Assay acceptability criteria

Assay acceptance criterion 1: Negative control

The absolute OD of the negative control (NC) tissues (treated with sterile deionised water) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The tissues treated with the negative control should not be below historically established boundaries.

The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 0.8 and ≤ 2.8.

Assay acceptance criterion 2: Positive control

A8N KOHwas used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay.

Tissues treated with the PC, should reflect the ability of the tissues to respond to a corrosive chemical under the conditions of the test method (viability after 1hour exposure: < 15%).

Assay acceptance criterion 3:variability between tissue replicates

Associated and appropriate measures of variability between tissue replicatesshould not exceed 30% (in the range of 20 – 100% viability).

Interpretation of results

The OD values obtained for each test sample was used to calculate a percentage viability relative to the negative control, which is arbitrarily set at 100%. The cut-off percentage cell viability value distinguishing corrosive from non-corrosive test items (or discriminating between different corrosive classes, e.g. subcategories 1A, 1B and 1C, or the statistical procedure(s) used to evaluate the results and identify corrosive materials, is clearly defined and documented, and shown to be appropriate. The criteria of corrosivity associated with the EpiDermTM model are as follows:

- the test item is considered to be corrosive to skin and classified as category 1 (or optional category 1A8), if the viability after 3

minutes exposure is less than 50%;

- the test item is considered to be corrosive to skin and classified as sub-category 1B-and-1C, if the viability after 3 minutes

exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%;

- the test item is considered to be non-corrosive to skin, if the viability after 3 minutes exposure is greater than or equal to 50% and

the viability after 1 hour exposure is greater than or equal to 15%.

Conclusions:
Under the present test conditions test item tested at two exposure periods of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.
Executive summary:

The purpose of this study was to assess the corrosive properties of test item to human skin,in anexperiment with an artificial three-dimensional model of human skin. TheEpiDerm™model was employed.

Two tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assayand expressed as relative percentage of viability of the negative control-treated tissues.

Test item was applied topically as solid test item to the model skin surface, which was moistened with sterile deionised water. Sterile deionised water was used as the negative control. 8N KOHwas used as the positive reference item.The test item and the reference items were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 105.5% after a 3-minute exposure period and 108.6% after an 1‑hour exposure.The 3-minute and the1-hour exposure values were above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of ≥ 50% and ≥ 15%, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean optical density (OD) of the negative control of 2 tissues was 1.960 (3‑minute exposure) or 2.243 (1-hour exposure) and was

well within the acceptable range of ≥ 0.8 to ≤ 2.8. The viability of cells treated with the positive reference item 8N KOH were

8.4% (3-minute exposure) and 7.1% (1-hour exposure) of the negative control and, hence well below the 15% cut-off value at the

1-hour exposure. The difference of viability between the two tissue replicates(at 20 - 100% viability) was below the limit of acceptance of 30%. Hence, all acceptance criteria were fulfilled.

Conclusion

Under the present test conditions test item tested at two exposure periods of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-02 to 2016-05-27
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:
adopted July 28, 2015
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted July 06, 2012
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be
soluble or insoluble in water.


Vehicle:
other: Dulbecco’s phosphate buffered saline.
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:The following Reconstructed Human Epidermis Model was used: EpiDermTM (EPI-200, Lot no. 23338) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- The test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 6.4
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination.
- MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm lot.
- The ET50 must fall within a range established based on a historical database of results.

NUMBER OF REPLICATE TISSUES: Three replicate tissues were employed

TEST FOR INTERFERENCE OF TEST ITEM WITH MTT REDUCTION ASSAY
- Prior to the testing, the test item was evaluated of colour changes under aqueous conditions. Therefore, 25 mg test item was diluted in
300 µL deionized water and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. At the end of exposure time, the mixture was evaluated of the presence and intensity of the staining. No discolorations were noted.
- Furthermore the test item was evaluated for its potential to interfere with the MTT assay reagent (e.g. reduction). Therefore, 25 mg test item was
dissolved in 1 mL MTT solution and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. Untreated MTT solution was used as
control. No change of colour was noted.
Hence, no possible interacting with the MTT measurement had to be considered and no additional test had to be performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:



ADMINISTRATION
- 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 moistened with Dulbecco’s phosphate buffered saline to uniformly cover the skin surface.
- The test item is a fine powder. For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline.
- Three replicate tissues were employed
- At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)
- The whole exposure period for the used EpiDermTM skin model was 60 minutes
- The incubation conditions were 37°C, 5% CO2 and 95% humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood.
- Concurrent negative and positive controls were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue
sensitivity (PC) of the tissues are within a defined historical acceptance range
- Positive control item was 5% aqueous sodium dodecyl sulphate (SDS)
- Negative control was D-PBS5
- 30 μL of negative and positive controls were used

- Viability measurements were not performed immediately after the exposure to the test item, but after a post-treatment incubation period of the
rinsed tissues in fresh medium of 42 hours.
- This period allows both for recovery from weakly irritant effects and for appearance of clear cytotoxic effects
- Each skin sample was placed in an MTT solution of 1 mg/mL (37°C incubation temperature, 5% CO2, 95% humidity) for 3 hours
- The precipitated blue formazan product was extracted using the extraction solution (isopropanol)
- Concentration of the formazan was measured by determining the optical density (OD) at a wavelength of 540 nm in a spectrophotometer
(Tecan Sunrise Magellan Version 6.4)
- Measurements were made for each of the three tissues in duplicate
Control samples:
yes, concurrent negative control
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 moistened with Dulbecco’s phosphate buffered saline to uniformly cover the skin surface.
- The test item is a fine powder.
- For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline.
- Three replicate tissues were employed.
- At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)

VEHICLE
- Dulbecco's phosphate buffered saline (D-PBS)

NEGATIVE CONTROL
- 30 µL Dulbecco's phosphate buffered saline (D-PBS)

POSITIVE CONTROL
- 30 µL 5% aqueous sodium dodecyl sulphate (SDS)
Duration of treatment / exposure:
An exposure time of 60 minutes was employed.
Duration of post-treatment incubation (if applicable):
Post-treatment incubation period of the rinsed tissues in fresh assay medium of 42 hours.
Number of replicates:
Three replicate tissues were employed.
Vehicle:
unchanged (no vehicle)
Irritation / corrosion parameter:
other: cut-off percentage cell viability
Run / experiment:
The test method is based on reconstructed human epidermis models
Value:
68.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
Test for interference of chemicals with MTT
Optical properties of the test item or its chemical action on the MTT may interfere with the assay leading to a false estimate of viability, as the test item may prevent or reverse the colour generation as well as cause it. This may occur when a specific test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.

Prior to the testing, the test item was evaluated of colour changes under aqueous conditions. Therefore, 25 mg test item was mixed with 300 µL deionized water and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. At the end of exposure time, the mixture was evaluated of the presence and intensity of the staining. No discolorations were noted.

Furthermore, the test item was evaluated for its potential to interfere with the MTT assay reagent (e.g. reduction). Therefore, 25 mg test item were mixed with 1 mL MTT solution and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. Untreated MTT solution was used as control. No change of colour was noted.

Hence, no possible interacting with the MTT measurement had to be considered and no additional test had to be performed.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Quality controls (QC) of the model
The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm lot. The ET50 must fall within a range established based on a historical database of results.


ACCEPTANCE OF RESULTS:
Assay acceptability criteria
Assay acceptance criterion 1: Negative control
The absolute OD of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use.
The assay meets the acceptance criterion if the mean ODof the NC tissues is ≥ 1.0 and ≤ 2.5.

Assay acceptance criterion 2: Positive control
A 5% SDS (in H2O) solution was used as a positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is required per testing day.
The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%.

Assay acceptance criterion 3: Standard deviation
Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low.
The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18%.
Other effects:
no other effects

 Interpretation of results

The OD values obtained for each test sample were used to calculate mean percentage viability relative to the negative control, which is set at 100%. The cut-off mean percentage cell viability value that distinguishes irritant from non-classified test substances is given below:

According to the EU and GHS classification (R38/ Category 1/2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or equal to 50% of the mean viability of the negative controls.

mean tissue viability ≤50% Irritant (I), (R38 or GHS category 1 or 2)

mean tissue viability > 50% non-irritant (NI).



Conclusions:
Under the present test conditions, test item tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin. Hence, the test item did not show irritant properties and is therefore not classified as irritant (UN GHS no category).
Executive summary:

The purpose of this study was to determine cytotoxic properties of test item to skin cells, which might lead to irritation ofhuman skin, by using an artificial three-dimensional model of human skin. TheEpiDermTMmodel was employed.

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assayand expressed as relative percentage of viability of the negative control-treated tissues.

The test item was applied as solid test item to the modelskin surface, which was moistened with Dulbecco’s phosphate buffered saline (D-PBS). D-PBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS)was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium.

The mean viability of cells exposed to test item was 68.7% of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. The test item was considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of the negative control of 3 tissues was 1.774 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.The viability of cells treated with the positive reference item, 5% SDS, was 6.0% of the negative control and fulfilled the acceptance criterion of ≤20%.

The standard deviation of all triplicates determined was below the limit of acceptance of 18%. Hence, all acceptance criteria were fulfilled.

 

Conclusion

Under the present test conditions, test item tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin. Hence, the test item did not show irritant properties and is therefore not classified as irritant (UN GHS no category).

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:
2016-03-17 to 2016-04-20
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 Ocular Corrosives and Severe Irritants)
Version / remarks:
adpted July 26, 2013
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
December 09, 2010
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
The test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% (w/v).

Species:
cattle
Details on test animals or tissues and environmental conditions:
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. To minimize deterioration and bacterial
contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution2 (HBSS) containing 1% Penicillin/Streptomycin3.
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation.
Only corneas from eyes free of defects were used.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
750 µl test item, 20% suspension in 0.9% sodium chloride solution (w/v
750 µl solvent control item, 0.9% sodium chloride solution
750 µl positive control item, 20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution
Duration of treatment / exposure:
Exposure period: 240 minutes
Duration of post- treatment incubation (in vitro):
After rinsing the corneas were incubated at 32°C ± 1°C for 90 ± 5 minutes. After this post incubation period, the corneas were examined.
Number of animals or in vitro replicates:
Three corneas were used for each treatment group (test item, solvent control and positive control).
Details on study design:
PREPARATION OF BOVINE EYES
- Corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium)
chambers
- The chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM)
- Corneal holder was equilibrated at 32 ± 1°C for at least one hour
- After equilibration period, fresh pre-warmed EMEM was added to both chambers
- Baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation,
neovascularisation) or an opacity >7 opacity units were discarded
- Mean opacity of all equilibrated corneas was calculated by use of an opacitometer
- A minimum of three corneas with opacity values close to the median value for all corneas were selected as solvent control corneas
- The remaining corneas were then distributed into treatment, solvent and positive control groups
ADMINISTRATION
- Three corneas were used for each treatment group
Negative control item: 0.9% sodium chloride solution
Positive control item: 20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution
Test item: 20% suspension in 0.9% sodium chloride solution (w/v)
- Exposure period: 240 minutes
- After exposure period of 240 minutes the test item, solvent control and positive control, were removed from each chamber and epithelium was
washed with EMEM at least three times
- Subsequently, the epithelium was washed with EMEM containing phenol red at least three times.
- Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible
- Corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber
- Chamber was then filled with EMEM without phenol red
EXAMINATION
- Corneal injury was assessed by evaluating the opacity and permeability of the cornea
- Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer
resulting in opacity values measured on a continuous scale
- To determine the corneal permeability 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior
chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM
- The holder was incubated in a horizontal position at 32 ± 1°C for 90 ± 5 minutes
- Amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader
(Tecan Sunrise Magellan Version 6.412).
- Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values
based upon a visible light spectrophotometer using a standard 1 cm path length






Irritation parameter:
in vitro irritation score
Run / experiment:
BOVINE CORNEAL OPACITY AND PERMEABILITY TEST
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: yes

Opacity Values

 

Cornea No.

Opacity [Opacity Units]

Corrected Opacity

 

Mean of group

Standard deviation

0.9% NaCl

1

-0.398

-0.544

-0.544

0.128

2

-0.597

3

-0.637

20% Imidazol

 

4

67.569

68.113

75.205

10.188

5

70.079

70.623

6

86.335

86.879

20% test item

 

7

4.581

5.125

4.435

0.718

8

3.148

3.692

9

3.944

4.488

Permeability OD Values (490 nm)

 

Cornea no.

Permeability
[OD]

 

Mean of Triplicates

Corrected Permeability [OD]

 

Per Cornea

Per Group

 

Mean

SD

Mean

SD

0.9% NaCl

1

-0.001

0.001

-

 

 

0.027

0.031

0.000

-

 

 

0.003

-

0.001

0.002

2

0.063

0.061

-

0.061

0.002

0.061

-

0.060

-

3

0.017

0.019

-

0.019

0.002

0.018

-

0.021

-

20% Imidazol

 

4

1.132

1.143

1.105

1.116

0.015

0.948

0.200

1.138

1.111

1.160

1.133

5

0.767

0.754

0.740

0.727

0.012

0.743

0.716

0.753

0.726

6

1.006

1.028

0.979

1.001

0.023

1.028

1.001

1.051

1.024

20%

Test item

7

-0.001

-0.001

-0.028

-0.028

0.001

-0.020

0.010

0.000

-0.027

-0.002

-0.029

8

0.018

0.018

-0.009

-0.009

0.001

0.019

-0.008

0.017

-0.010

9

0.002

0.003

-0.025

-0.024

0.001

0.003

-0.024

0.003

-0.024

SD : standard deviation

OD : optical density

In vitro irritancy score (IVIS)

 

Cornea No.

Opacity

Permeability

IVIS

Per Cornea

Per Group

Mean

SD

0.9% NaCl

1

-0.398

0.001

-0.383

-0.139

0.396

2

-0.597

0.061

0.318

3

-0.637

0.019

-0.352

20% Imidazol

4

68.113

1.116

84.853

89.425

10.926

5

70.623

0.727

81.528

6

86.879

1.001

101.894

20% Test item

7

5.125

-0.028

4.705

4.130

0.574

8

3.692

-0.009

3.557

9

4.488

-0.024

4.128

Conclusions:
Under the present test conditions, the test item tested in the in vitro BCOP test method, had an IVIS value of 4.130, which is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently no prediction concerning irritant or corrosive potential of the test item can be made.
Executive summary:

The purpose of this study was to determinea possible potency of the test item of being 'ocular corrosive and severe irritant'employing an in vitro system.The Bovine Corneal Opacity and Permeability Assay (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine corneain vitro. In this test method, possible damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability inisolated corneas from bovine eyes.

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber.The measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of the test item.

Three corneas were used for each treatment group (test item, solvent control and positive control). The solid test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% test item as recommended in the test guideline 437 for non-surfactant solids. 0.9% NaCl solution was used as the solvent control and 20% Imidazole in 0.9% NaCl solution as the positive control item.

The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was 240 minutes. The optical density (OD) was measured at a wavelength of 490 nm.

The acceptance criteria of validity were fulfilled in this test.

Following treatment with test item a mean opacity value of 4.435 ± 0.718 and a mean permeability value of -0.020 ± 0.010 compared to the solvent control were determined. An IVIS of 4.130 ± 0.574 was calculated. The calculated IVIS of 4.130 is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently no prediction concerning irritant or corrosive potential of the test item can be made.


Conclusion

Under the present test conditions, the test item tested in the in vitro BCOP test method, had an IVIS value of 4.130, which is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently no prediction concerning irritant or corrosive potential of the test item can be made.

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Under the present test conditions test item tested at two exposure periods of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin (LPT, 2016).

Additionally, the test item tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin. Hence, the test item did not show irritant properties and is therefore not classified as irritant (UN GHS no category) (LPT, 2016).

Under the present test conditions, the test item tested in the in vitro BCOP test method, had an IVIS value of 4.130, which is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, identifying test substances as inducing serious eye damage (UN GHS Category 1) (LPT, 2016). Consequently no prediction concerning irritant or corrosive potential of the test item can be made.

Justification for classification or non-classification

Based on the assessment of two in vitro studies (skin corrosion and skin irritation) and according to criteria of EC Regulation 1272/2008 the test item is neither corrosive nor irritating to skin and therefore must not be classified.

Based on the available data no unequivocal prediction concerning eye irritant or corrosive potential of the test item can be made. By analogy to structural similar substances, the test item is classified as irritating to eyes (Eye Irrit. Cat. 2; H319 "Causes serious eye irritation.").