Registration Dossier

Administrative data

Description of key information

Skin irritation

In a GLP compliant K1 in vitro skin irritation study conducted according to OECD Guideline 439 and EU Method B.46, T003422 was observed to be non-irritating to skin. Based on these results, T003422 should not be classified as a skin irritant according the criteria of the CLP regulation (EC) No 1272/2008.

Eye irritation

In a K1 Bovine Corneal Opacity and Permeability (BCOP) test, performed according to OECD guideline 437 and EU method B.47, T003422 did not induce ocular irritation. No classification is required for eye irritation or serious eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 2012-05-22 to 2012-05-28
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Non-GLP study according to OECD Guideline 439.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
no
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RT003422PFP031
- Expiration date of the lot/batch: 2017-03-01 (expiry date)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ca.20 °C), dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, the solid test item was applied directly on top of the tissue.
Test system:
human skin model
Remarks:
of human-derived epidermal keratinocytes
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: not applicable
Justification for test system used:
The EPISKIN Skin Irritation Test-42 hours using human epidermis skin constructs supplied by SkinEthic Laboratories, Lyon, France, has been accepted as a replacement to the in vivo test, Draize Skin Irritation Test, by the European Centre for the Validation of Alternative Methods .
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small model (0.38 cm²) supplied by SkinEthic Laboratories, Lyon France
- Tissue batch number(s): 12-EKIN-021
- This model is a three-dimensional human epidermis model, which consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. After 13 days in culture a multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum has formed.
- On receipt, the kit contents were checked and the inserts with tissues on agarose were stored at room temperature until use. The kit was used within the expiry date indicated by the supplier (expiry date: 2012-05-28). The maintenance medium was pre-warmed to 37º C. The tissues were removed from the agar and placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 24 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
- 10 ± 2mg of test item was dispensed over each tissue using glass weighing boats. The tissues were wetted with 5 µL of distilled water prior to application of the test substance.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 5% CO2 in air.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period of 15 ± 0.5 minutes at room temperature, each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 ± 1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After 42 ± 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
At the end of 3 hours ± 5 minutes, all the inserts were blotted on absorbent paper. The tissue was removed from the insert using a biopsy punch and the epidermis was separated from the collagen matrix using forceps. The epidermis and the collagen matrix were placed together in a micro tube.
When all the tissues had been punched, the tissues (epidermis and collagen matrix) were vortexed with 500 µL of acidic isopropanol (0.04 N HCl final concentration).
The tissues were then extracted by storing at 2-8 ºC, protected from light, for 70 hours.
After formazan extraction, duplicate 200 µL aliquots of the extractant from each micro tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.
- The viability of each tissue was expressed as a percentage of the mean negative control value.
- Test for reduction of MTT by test item: It is possible that a test substance may reduce MTT, resulting in the production of a blue colour without any involvement of cellular mitochondrial dehydrogenase. Since the test item is rinsed off the tissue before the MTT assay, this is usually avoided. The MTT reducing capability of the test item, JNJ-42808415-AAA, was investigated. The test item, JNJ-42808415-AAA, was evaluated for its colour or ability to become coloured in contact with water (simulating a tissue humid environment). There was no change in the test item, JNJ-42808415-AAA/MTT solution or the water control/MTT solution after three hours incubation in the dark. The test item did not interacted with the MTT.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability (≥ 19.5%): 21.8 +/-0.3% (CV =1.2%)
- Barrier function (IC50 determination, ≥ 1.5 mg/mL): 1.7 mg/mL
- Morphology: well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination:
- on blood of the same donors, the following was verified: the absence of HIV1 and 2 antibodies, the absence of hepatitis C antibodies, the absence of hepatitis B antigen HBs;
- on epidermal cells of the same donors, the following was verified: the absence of bacteria, fungus and mycoplasma.
- Expiration date: 2012-05-028

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDI CTION: 1

INTERPRETATION
- A test item is considered to be irritant in the skin irritation test (category 2) if: the relative mean tissue viability of three individual tussues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered to be non-irritant in the in vitro skin irritation test (no category) if the relative mean tissue viability after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
When the result of the test is irritant and a tiered testing strategy is employed for classification of skin irritation/corrosivity, additional testing for skin corrosivity (not part of this study) should be considered.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg

NEGATIVE CONTROL (DPBS)
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL (5% SDS)
- Amount(s) applied (volume or weight): 10 µL
Duration of treatment / exposure:
15 +/- 0.5 minutes (the positive control was re-spread after 7 minutes application time)
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 replicates per test item together with negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean tissue viability
Run / experiment:
1
Value:
96.5
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: range: 88.9 to 102.6% SD: +/- 7.0
Irritation / corrosion parameter:
other: optical density mean
Run / experiment:
1
Value:
0.697
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: range: 0.65 to 0.774 SD: +/-0.047
Other effects / acceptance of results:
mean tissue viability (percentage of control):
negative control: 108.3 +/- 16.0
positive control: 25.2 +/-5.5

mean optical density:
negative control: 0.723 +/- 0.106
positive control: 0.182 +/- 0.036

Results:
The test item was checked for possible direct MTT reduction and colour interference. There was no change in the test item, JNJ-42808415-AAA/MTT solution or the water control/MTT solution after three hours incubation in the dark at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. The test item had not interacted with the MTT. The test item, JNJ-42808415-AAA/water solution and water control were colourless after the 15 minute shaking period. The test item, JNJ-42808415-AAA, had not shown any potential for colouring water.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 96.5 % (88.9 to 102.6%). Since the mean relative tissue viability for the test item was above 50% it is considered to be a non-irritant.

The pH of the test item, JNJ-42808415-AAA, was diluted to 10% w/v with distilled water to obtain an aqueous solution for pH measurement. The pH of the test item, measured using pH indicator paper, was approximately 7.0.

The percentage mean viability of the positive control was 25.2 ± 5.5 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18. The mean absorbance of the triplicate negative control values was 0.723 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 16.0 which was below the maximum value of 18.
Interpretation of results:
GHS criteria not met
Conclusions:
It was concluded that the test item, JNJ-42808415-AAA, with a mean tissue viability of 96.5±7.0%, was predicted as non-irritant to the skin.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2016-03-07 to 2016-03-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented GLP study performed according to OECD guideline 439 and EU method B.46. There were no deviations from the study plan.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15AB0305
- Expiration date of the lot/batch: 2017-01-23 (retest date)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, the solid test item was applied directly on top of the skin tissue.
Test system:
human skin model
Remarks:
of human-derived epidermal keratinocytes
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: not applicable
Justification for test system used:
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 skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC)
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2) supplied by SkinEthic Laboratories, Lyon, France.
- Tissue batch number(s): 16-EKIN-010
- 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.
- On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewa rmed Maintenance Medium for 24 hours at 37°C.
- The skin was moistened with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and the solid test item (18.2 to 31.5 mg) was added into 12-well plates on top of the skin tissues.

TEMPERATURE USED FOR TEST SYSTEM - Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 71 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 - 37.2C).
- Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with ph osphate buffered saline (PBS) to remove residual test item. 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 t is sues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After incubation, cell culture inserts were dried carefully to remove excess medium and were transfer red into a 12-wells plate prefilled with 2 mL MTT-medium (0.3 mg/mL). The tissues were in cubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplic ate 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 t issues. Skin irritation potential of the test item was classified according to remaining cell viability foll owing exposure of the test item.
- Test for reduction of MTT by test item: A test item may interfere with the MTT endpoint if it is coloure d and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed. The test item was checked for possible direct MTT colour interference before the study was started. Because solutions did not turn blue / purple and a blue / purple pre cipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability (≥ 19.5%): 22.3 +/-0.7% (CV =3.1%)
- Barrier function (IC50 determination, ≥ 1.5 mg/mL): 2.3 mg/mL
- Morphology: well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination:
- on blood of the same donors, the following was verified: the absence of HIV1 and 2 antibodies, the absence of hepatitis C antibodies, the absence of hepatitis B antigen HBs;
- on epidermal cells of the same donors, the following was verified: the absence of bacteria, fungus and mycoplasma.
- Expiration date: 2016-03-16

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDI CTION: 1

INTERPRETATION
- A test item is considered to be irritant in the skin irritation test (category 2) if: the relative mean tissue viability of three individual tussues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered to be non-irritant in the in vitro skin irritation test (no category) if the relative mean tissue viability after 15 minutes of exposure to the test item 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 ITEM
- Amount applied: 18.2 to 31.5 mg

NEGATIVE CONTROL (PBS)
- Amount applied: 25 μl

POSITIVE CONTROL (5% SDS)
- Amount applied: 25 μl
Duration of treatment / exposure:
15 +/- 0.5 minutes (the positive control was re-spread after 7 minutes contact time)
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 replicates per test item together with negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean tissue viability
Run / experiment:
1
Value:
94
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: range: 85 to 106% SD: +/-10.9
Irritation / corrosion parameter:
other: optical density
Remarks:
mean
Run / experiment:
1
Value:
1.11
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: range 0.997 to 1.248 SD: +/-0.128
Other effects / acceptance of results:
mean tissue viability (percentage of control):
negative control: 100 +/- 8.1
positive control: 19 +/- 6.9

mean optical density:
negative control: 1.176 +/- 0.096
positive control: 0.227 +/- 0.081

Interpretation: The viabilities of all replicates were within one category.

Results:
The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compa red to the negative control tissues was 94% (85 to 106%). Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 19% (15 to 27%). The absolute mean OD570 of the negative control tissues was within 0.6 and 1.5. The standard deviation values of the percentage viability of three tissues treated identically were 8%, 7% and 11% for the negative, positive control and the test item respectively, indicating that the test system functioned properly.
Interpretation of results:
GHS criteria not met
Conclusions:
It is concluded that this test is valid and that the test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.
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:
From 2016-03-10 to 2016-03-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented GLP study according to OECD guideline 437 and EU method B.47. Two deviations were recorded, that did not affect the integrity of the study. 1) The negative control opacity and permeability were not corrected for the mean negative control opacity and permeability respectively: since it is the optimized procedure to not correct the negative control values for the mean negative control this does not influence the study results. 2) One negative control treated eye was excluded due to a permeability value which was outside the historical control data: since the two remaining control eyes show an acceptable response and the outcome of the test item is clearly not influenced by the exclusion of the negative control eye this does not impact the study outcome.
Reference:
Composition 0
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
Principles of method if other than guideline:
The study procedures were also in compliance with the following guidelines:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P, Dukic M, Alix D and Sina J F, Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15AB0305
- Expiration date of the lot/batch: 2017-01-23 (retest date)
- Purity: 99.7% (w/w)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: the solid test item was applied directly
Species:
other: bovine eyes
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Bovine eyes were used as soon as possible but within 4 hours after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.

- Preparation of corneas: the eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). 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.
Vehicle:
unchanged (no vehicle)
Controls:
other: negative control (physiological saline) and positive control (20% (w/v) Imidazole solution in physiological saline)
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 343.0 to 380.9 mg

NEGATIVE CONTROL
- Amount applied: 750 µl

POSITIVE CONTROL
- Amount applied: 750 µl
- Concentration: 20% (w/v) imidazole solution
Duration of treatment / exposure:
Corneas were incubated for 240 ± 10 minutes at 32 ± 1°C
Observation period (in vivo):
After 240 ± 10 minutes of treatment, opacity was measured with an opacitometer. The permeability measurement of the corneas was performed after the incubation period of 90 minutes ± 5 minutes following the opacity measurement.
Number of animals or in vitro replicates:
Three corneas were selected at random for each treatment group
Details on study design:
CORNEA SELECTION AND OPACITY READING
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 an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
The medium from the anterior compartment was removed and 750 μl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered. 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. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

OPACITY MEASUREMENT
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (l = 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 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea before/after test item treatment.
The change of 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 positive control or test item treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test item 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.

APPLICATION OF SODIUM FLUORESCEIN
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) 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 (Sigma-Aldrich Chemie GmbH, Germany). 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.

PERMEABILITY DETERMINATIONS
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 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 was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

ELECTRONIC DATA CAPTURE
Observations/measurements in the study were recorded electronically using the following programme: Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

INTERPRETATION
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 item induced irritation through only one of the two endpoints.

The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
Test item after 240 minutes of treatment
Value:
-0.8
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Remarks:
Test item IVIS range: -2.7 to 0.9
Irritation parameter:
cornea opacity score
Run / experiment:
Test item after 240 minutes of treatment
Value:
-0.9
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: test item opacity range: -2.6 to 0.9
Irritation parameter:
other: cornea permeability value
Run / experiment:
Test item after 240 minutes of treatment
Value:
0.001
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: test item permeability range: -0.005 to 0.010
Other effects / acceptance of results:
mean in vitro irritancy score (range):
negative control: 0.1 (-0.8 to 0.9)
positive control: 174.1 (153.5 to 195.1)

mean opacity scores (range):
negative control: -0.2 (-1 to 0.7)
positive control: 124.9 (100.1 to 153.0)

mean permeability scores (range):
negative control: 0.014 (0.013 to 0.015)
positive control: 3.281 (2.806 to 3.562)

The corneas treated with the positive control were turbid after the 240 minutes of treatment. The corneas were clear after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium.

The mean 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. Since one of the eyes showed a permeability value which was outside the historical control data range, this eye was excluded from analysis. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 174 (153 to 195) and within the historical positive control data range. Furthermore the opacity and permeability values of the positive control were within two standard deviations of the current historical mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation:

The IVIS of all replicates was within one category.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.8 (-2.7 to 0.9) after 240 minutes of treatment. Since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 2012-03-05 to 2012-03-05
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Non-GLP-study, carried out based on the current OECD guideline 437 (2009) and in conformity with the EEC validation study (contract ref. B91/B4-3081/013188, 1991; Gautheron et al., 1994) with some modifications (Vanparys et al., 1993).
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
no
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
RT003422PF031
- Expiration date of the lot/batch:
no data
- Purity test date:
no data


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Stored at room temperature in a closed and labeled container protected from light.
- Stability under test conditions:
no data
- Solubility and stability of the test substance in the solvent/vehicle:
The test item was insoluble in the vehicle as a 20 % (w/w) formulation.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Tested as a 20 % (w/w) suspension
Species:
other: Freshly isolated bovine cornea
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Local slaughterhouse
- Bovine (<12 months) eyes were excised by a slaughterhouse veterinarian and collected in a plastic jar containing one liter of Hank’s Balanced Salt Solution (HBSS, Sigma). Medium storage and transportation of eyes to the laboratory was performed at room temperature. The eyes were used within 24 hours after killing the animals.



Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.750 ml
- Concentration (if solution): 20% (w/w) in vehicle

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.750 ml
- Concentration (if solution): 0.9% (w/v) NaCl in water

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.750 ml
- Concentration (if solution): 20% (w/v) Imidazole
Duration of treatment / exposure:
Corneas were incubated for 4 hours at 32°C in a water-bath.
Duration of post- treatment incubation (in vitro):
After 4 hours of treatment, opacity was measured with an opacitometer. The permeability measurement of the corneas was performed after the incubation period of 90 minutes following the opacity measurement.
Number of animals or in vitro replicates:
3 corneas were selected for each treatment group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- Before dissection all eyes were carefully examined and those presenting defects, such as neovascularization, pigmentation or scratches, were discarded. During dissection great care was taken to avoid damage of corneal surfaces (epithelial and endothelial). Selected corneas were dissected with a 2-3 mm rim of sclera for easier handling and stored in a petri-dish containing Hank’s Balanced Salt Solution (HBSS) until use. Corneas were mounted in holders, the endothelial side being applied on the O-ring of the posterior part of the cornea holder. The anterior part of the cornea holder was applied onto the posterior part and held in place with 3 screws. Compartments were filled (the posterior part first) with Minimal Essential Medium (MEM, Sigma) solution. The corneas were incubated for one hour in a water-bath at 32°C.
After incubation of the corneas for one hour in a water-bath at 32°C, the MEM solution was removed and both compartments were refilled with fresh medium. After careful removal of the air-bubbles, background opacity of all corneas was measured with an opacitometer. The opacitometer (OP-KIT, Electro Design, Riom, France) determines the light transmission of a cornea, and displays a numerical opacity value (arbitrary units). Results were written on a specially prepared spreadsheet. Corneas were rejected for use if they showed a background opacity grade greater than three.

TREATMENT METHOD
After background opacity measurement at time zero (t0), medium was removed from the anterior compartment and the test item (20 % (w/w)) suspension was applied in such a way that thecomplete corneal area was covered.The vehicle and positive control formulations were applied to the cornea as 0.75 ml volumes. After closing of the holes with caps, corneas were incubated in a horizontal position for 4 hours at 32°C in a water-bath.

REMOVAL OF TEST SUBSTANCE
After incubation, the vehicle control, the positive control and the test item formulation were removed from the anterior compartment and the epithelium was washed at least 3 times with approximately 4 mL of MEM-solution until the medium was clear. The medium was then removed from the posterior compartment and both compartments were finally refilled with MEM-solution. Then opacity was measured with the opacitometer (at time t240) and the results were written on a specially prepared spreadsheet.

METHODS FOR MEASURED ENDPOINTS
Corneal Permeability
The permeability of the corneas was evaluated immediately after measuring the opacity. The medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% sodium-fluorescein solution (sodium fluorescein was dissolved in Dulbecco's phosphate buffered saline). Corneas were incubated in a horizontal position for 90 minutes at 32°C in a water-bath. After incubation, medium from the posterior chamber was removed, and its optical density (OD) was determined spectrophotometrically at 490 nm. The results on the printout of the spectrophotometer were entered into a specially prepared spreadsheet.

SCORING SYSTEM
Vehicle control:
When background opacity (at time t0) was subtracted from the opacity value measured at time t240, the mean (± SD) corneal opacity of the vehicle control corneas was calculated. Also the mean (± SD) permeability was determined.

Positive control and test item:
Individual corneal opacity (at t240 - t0) and permeability values were corrected with the mean opacity and permeability scores obtained for the vehicle control. Then, the mean (± SD) was calculated of the corrected opacity and permeability values.

Test evaluation criteria:
An in vitro score for the corneal injury was calculated with a formula, which combined results from both opacity and permeability. In vitro score = opacity value + 15 times OD value for permeability. Individual in vitro scores were used to calculate the mean in vitro score.
In vitro scores were sorted into five different classes:
<= 3.0 = non eye irritant
from 3.1 to25.0 = mild eye irritant
from 25.1 to 55.0 = moderate eye irritant
from 55.1 to 80.0 = severe eye irritant
>= 80.1 = very severe eye irritant

Irritation parameter:
in vitro irritation score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
22.4
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: range of IVIS score of test item: 19.1 to 24.9
Irritation parameter:
cornea opacity score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
21.3
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: range of corneal opacity score of test item: 18.0 to 24.0
Irritation parameter:
other: permeability score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
0.071
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: range of permeability value of test item: 0.059 to 0.081
Other effects / acceptance of results:
mean in vitro irritancy score (range):
negative control: 1,2 (0,0 to 3,2)
positive control: 210,1 (187,9 to 247,8)
mean opacity scores (range):
negative control: 1,0 (0 to 3)
positive control: 142,7 (135,0 to 146,0)
mean permeability scores (range):
negative control: 0.017 (0.003 to 0.035)
Positive control: 4,563 (2,794 to 6,920)

The corneas treated with the test item induced a mild increase in opacity (21.3± 3.1) and no increase in permeability (0.071 ± 0.011).

Acceptance of results
In this study, the vehicle control induced a mean opacity, permeability and an in vitro score which fell within our laboratory range of historical control data. The corneas treated with the positive control imidazole (20 % (w/w)) showed a marked increase in opacity and permeability. Imidazole was classified as a very severe eye irritant. Based on these findings, it was concluded that the assay acceptance criteria for the evaluation of the in vitro eye irritating potential of the test item were met.
Interpretation of results:
study cannot be used for classification
Conclusions:
Based on the results, the test item is classified as a mild eye irritant when tested in vitro under the conditions of the test. According to criteria of the CLP Regulation, no prediction can be made on the classification of the test item, since the test item induced an IVIS of 22.4, which is >3 and ≤55.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

Eurlings (2016) investigated skin irritation in a Reconstructed Human Epidermis Test (OECD 439 and EU Method B.46). 18.2 to 31.5 mg of test item was applied directly on top of the tissue for 15 minutes. Cell viability was calculated for each tissue as a percentage of the mean optical density of the negative control tissues. Based on the results, 1.11 mean optical density and 94% mean tissue viability, the test item is a not irritating to the skin. 

In addition, Hubbard (2012) investigated skin irritation in a Reconstructed Human Epidermis Test according to OECD Guideline OECD 439. After 15 minutes of exposure to 10 ± 2 mg of test item, 0.697 mean optical density and 96.5% mean tissue viability was observed. Based on the results, the test item was predicted as a non-irritant to the skin. 

The skin irritation study of 2016 is considered the key result for assessing the skin irritation endpoint. According to the REACH legislation (art 13.4) new tests (since 2008) must be performed in compliance with GLP.

As the result of the non-GLP Reconstructed Human Epidermis Test performed in 2012 was negative, this study was added to the dossier as supporting evidence and the GLP-compliant Reconstructed Human Epidermis Test performed in 2016 is selected as key study for classification purposes.

Eye irritation

Eurlings (2016) investigated eye irritation in an in vitro bovine corneal opacity-permeability (BCOP) assay. 343.0 to 380.9 mg of test item was applied on the top of 3 corneas for 240 minutes. Both opacity and permeability were measured and the resulting objective values were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).  The corneas were clear after the 240 minutes of treatment with the test item. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.8 (-2.7 to 0.9) after 240 minutes. Since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

In addition, an in vitro bovine corneal opacity-permeability (BCOP) assay was performed by Verheyen (2012). 750 µL of undiluted test item was applied on the top of 3 corneas for 240 minutes. Both opacity and permeability were measured and the resulting objective values were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). After 240 minutes of exposure to test item T003422 a mean IVIS of 22.4 (19.1 to 24.9) was recorded. Based on the results, the test item is classified as a mild eye irritant when tested in vitro under the conditions of the test. However, according to criteria of the CLP Regulation, no prediction can be made on the classification of the test item, since the test item induced an IVIS of 22.4, which is >3 and ≤55.

Although the IVIS score in the BCOP performed in 2012 is between 3 and 55, the substance is considered non-irritating to eye based on the result (IVIS < 3) from the more recent study (Eurlings, 2016) performed under GLP according to the OECD guideline.

 

Justification for classification or non-classification

Skin irritation:

According to the in vitro skin irritation study of 2016, T003422 is not a skin irritant. The test item did not meet the criteria for classification as irritant according to the criteria of the CLP regulation (EC) No 1272/2008.

Eye irritation:

According to the In Vitro Bovine Corneal Opacity-Permeability Assay of 2016, T003422 is not an eye irritant or corrosive to eyes and should not be classified for eye irritation or serious eye damage according to the criteria of the CLP regulation (EC) No 1272/2008.