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Description of key information

Skin irritation
An in vitro skin corrosion, an in vitro skin irritation and an in vivo skin irritation study are used in a weight of evidence approach.

In a K1 in vitro skin corrosion study according to OECD Guideline 431 and EU Method B.40, T003642 was not corrosive to the skin based on the criteria of the CLP regulation (EC) No 1272/2008.

In addition, in a K1 in vitro skin irritation study according to OECD guideline 439 and EU Method B.46, the test item is irritant and should be classified category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and the negative in vitro skin corrosion test.

In addition, in a K1 in vivo skin irritation study in New Zealand White rabbits according to OECD Guideline 404 and EU Method B.4, no evidence for skin irritation was noted for T003642, and is therefore not classified based on the criteria of the CLP Regulation (EC) No 1272/2008.

Eye irritation

An in vitro eye irritation study and an in vivo eye irritation study are used in a weight of evidence approach.

In a K1 Bovine Corneal Opacity and Permeability test, performed according to OECD guideline 437 and EU Method B.47, T003642 is not irritating or corrosive to eyes.

In a K1 in vivo eye irritation study, according to OECD Guideline 405 and EU Method B.5, T003642 did not cause any irreversible eye irritating reactions.

Therefore, T003642 should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and the criteria of the CLP Regulation (EC) No 1272/2008.

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:
weight of evidence
Study period:
From 2016-03-14 to 2016-03-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented GLP study performed according to OECD guideline 431 and EU method B.40 BIS.
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0020709377
- Expiration date of the lot/batch: 2016-08-31 (retest date)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
-Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a liquid: Not applicable, the liquid test item was applied undiluted.
Test system:
human skin model
Remarks:
model of human-derived epidermal keratinocytes
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200) supplied by MatTek Corporation, Ashland MA, U.S.A
- Tissue batch number(s): Lot no.: 23662 kit DD and EE
- The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM medium (Dulbecco’s Modified Eagle’s Medium) (supplemented DMEM medium, serum-free supplied by MatTek Corporation) per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test item was applied.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature during 3-minute exposure and 37°C during 1-hour exposure
- Temperature of post-treatment incubation (if applicable): 37°C
- All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 67 - 86%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.3°C).
- 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, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 µl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT medium: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
- The DMEM medium was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.
- Test for the interference with the MTT endpoint: A test item may interfere with the MTT endpoint if it is coloured 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.
- Test for colour interference by the test item: the test item was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured substances in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, 50 μl of the test item or 50 µl Milli-Q water as a negative control were added to 0.3 ml Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
- Test for reduction of MTT by the test item: the test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 μl of the test item was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative ontrol, sterile Milli-Q water was tested concurrently. At the end of the exposure time it was checked if a blue / purple colour change was observed.

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

DECISION CRITERIA
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non-corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
Duration of treatment / exposure:
3 minutes and 1 hour
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
4 replicates per test item, negative control and positive control: 2 for the 3-minute exposure and 2 for the 1-hour exposure
Irritation / corrosion parameter:
% tissue viability
Remarks:
Mean tissue viability
Run / experiment:
test item after 3-minute application
Value:
127
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
individual values: 132 and 122
Irritation / corrosion parameter:
% tissue viability
Remarks:
Mean tissue viability
Run / experiment:
test item after 1-hour application
Value:
125
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
individual values: 127 and 123
Irritation / corrosion parameter:
other: optical density
Remarks:
Mean absorption
Run / experiment:
test item after 3-minute application
Value:
2.256
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
individual values: 2.350 and 2.163
Irritation / corrosion parameter:
other: optical
Remarks:
Mean absorption
Run / experiment:
test item after 1-hour application
Value:
2.357
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Individual values: 2.402 and 2.312
Irritation / corrosion parameter:
other: coefficient of variation
Run / experiment:
test item after 3-minute application
Value:
8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: coefficient of variation
Run / experiment:
test item after 1-hour application
Value:
3.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
3-minute application:
- viability:
negative control: 100 (107 and 93)
positive control: 8 (8 and 8)
- coefficient of variation between tissue replicates
negative control: 13.5
positive control: 4.1
- mean optical density:
negative control: 1.777 +/- 0.182
positive control: 0.141 +/- 0.004

1-hour application:
- viability:
negative control: 100 (103 and 97)
positive control: 7 (6 and 7)
- coefficient of variation between tissue replicates:
negative control: 5.4
positive control: 7.7
- mean optical density:
negative control: 1.887 +/- 0.074
positive control: 0.124 +/- 0.007

Results/Other Effects
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.

Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.

Acceptance of results:
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 8% (8 and 8%) and 7% (6 and 7%) after 1 hour exposure.
In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 14%, indicating that the test system functioned properly.
Interpretation of results:
GHS criteria not met
Conclusions:
It is concluded that the test is valid and that the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in the report.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2016-05-03 to 2017-05-09
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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0020709377
- Expiration date of the lot/batch: 2016-08-31 (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
- Final preparation of a liiquid: not applicable, the test item was applied undiluted
Test system:
human skin model
Remarks:
model 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:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm²), SkinEthic Laboratories, Lyon France
- Tissue batch number(s): Lot no.: 16-EKIN-018
- 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 prewarmed Maintenance Medium for 24 hours at 37°C.
-The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five μl of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.

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 81 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.7 - 37.1°C).
- 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 phosphate buffered saline 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 tissues 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 transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated 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 70 hours. 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. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.
- Test for reduction of MTT by test item: The test item was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (project 512941). Because 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.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability (≥ 19.5): 22.3+/-0.3% (CV=1.4%)
- Barrier function (IC50 determination, ≥ 1.5 mg/mL): 2.5 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: 2016-05-09

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 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 tissues 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 MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL

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

POSITIVE CONTROL (5% SDS)
- Amount(s) applied (volume or weight): 25 µL, re-spread after 7 minutes contact time
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:
Test item after 15 min treatment
Value:
35
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
SD: +/- 6 / range: 29 to 42
Irritation / corrosion parameter:
other: Mean absoption
Run / experiment:
Test item after 15 min treatment
Value:
0.287
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
SD: +/-0.052 / range: 0.245 to 0.346
Other effects / acceptance of results:
mean tissue viability (percentage of control):
negative control: 100 +/- 16
positive control: 24 +/- 1

mean optical density:
negative control: 0.832 +/- 0.132
positive control: 0.199 +/- 0.010

Interpretation:
All viability of all replicates was within one category.

Results:
Since the mean relative tissue viability for the test item was below 50% it is considered to be an irritant.

Acceptance of results:
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 24% (23 to 25%). 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 16%, 1% and 6% for the negative, positive control and the test item respectively, indicating that the test system functioned properly.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
It is concluded that this test is valid and that JNJ-54582034-AAA (T003642) is irritant in the in vitro skin irritation test under the experimental conditions described in this report and should be classified category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and the negative in vitro skin corrosion test (project 512941).
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2003-05-21 to 2003-02-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
Himalayan
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology KG, branch Lohndorf, D-24601 Lohndorf/Post Wankendorf
- Age at study initiation: approx. 4 months
- Weight at study initiation: 2.3 - 2.5 kg
- Housing: Singly in cages with dimensions of 425 mm x 600 mm x 380 mm
- Diet (e.g. ad libitum): ssniff@ K-H V2333 (ssniff Spezialdiaten GmbH, D-59494), ad libitum
- Water (e.g. ad libitum): Drinking water was offered daily, ad libitum
- Acclimation period: at least 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
other: The surrounding untreated skin served as a control.
Amount / concentration applied:
TEST MATERIAL:
- Amount(s) applied (volume or weight with unit): 0.5 mL
Duration of treatment / exposure:
4 h
Observation period:
6 days
Reading time points: 1, 24, 48 and 72 h, 4, 5 and 6 days
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: dorsal skin
- % coverage: approx. 6 cm²
- Type of wrap if used: semi-occlusive dressing (non-irritating tape)

OBSERVATION TIME POINTS
(indicate if minutes, hours or days)
60 minutes, 24, 48, 72 hours and 4 to 6 days after patch removal.

SCORING SYSTEM:
- Method of calculation: Draize scoring system
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 6 days
Irritation parameter:
erythema score
Basis:
animal: #2-3
Time point:
24/48/72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 6 days
Irritation parameter:
edema score
Basis:
animal: #1-2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 6 days
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 6 days
Irritant / corrosive response data:
An erythema (grade 1) was observed in animal nos. two and three 48 and 72 hours after patch removal, in animal no. 2 until 5 days after patch removal.
An oedema (grade 1) was observed in animal no. three 48 and 72 hours after patch removal.
There were no systemic intolerance reactions.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study, the test material caused only slight and fully reversible skin irritating reactions. The individual mean values for erythema/eschar and edema from gradings at 24, 48 and 72 h were 0.0/0.7/0.7 and 0.0/0.7/0.7 in each of the three animals, respectively. Therefore, the material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not skin irritating.
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:
weight of evidence
Study period:
From 2016-03-22 to 2016-04-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented GLP study according to OECD guideline 437.
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 (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0020709377
- Expiration date of the lot/batch: 2017-08-31 (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: not applicable, the test item was applied undiluted

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.

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): >/=99.9% Ethanol
Duration of treatment / exposure:
Corneas were incubated for 10 ± 1 minutes at 32 ± 1°C
Duration of post- treatment incubation (in vitro):
After 10 ± 1 minutes of treatment, the corneas were thoroughly rinsed to remove test item and incubated for 120 ± 10 minutes at 32 ± 1°C with fresh medium followed by opacity measurement. The permeability measurement of the corneas was performed following the opacity measurement after the incubation period of 90 minutes ± 5 minutes.
Number of animals or in vitro replicates:
Three corneas were selected at random for each treatment group
Details on study design:
SELECTION AND 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.

After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM.

TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (99.9% Ethanol) or test item 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 10 ± 1 minutes at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium Life Technologies) and thereafter with cMEM. 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea will be read against a cMEM filled chamber, and the initial opacity reading thus determined will be recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group

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.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
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.

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.

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 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
Remarks:
mean
Run / experiment:
First Test
Value:
2.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Range of IVIS score of test substance
Remarks:
-0.2 to 5.1
Irritation parameter:
cornea opacity score
Remarks:
mean
Run / experiment:
First Test
Value:
1.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Range of corneal opacity score of test substance
Remarks:
-0.2 to 5.0
Irritation parameter:
other: permeability value
Remarks:
mean
Run / experiment:
First Test
Value:
0.036
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Range of permeability value of test substance
Remarks:
0.002 to 0.096
Irritation parameter:
in vitro irritation score
Remarks:
mean
Run / experiment:
Second Test
Value:
0.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Range of IVIS score of test substance
Remarks:
-0.6 to 0.6
Irritation parameter:
cornea opacity score
Remarks:
mean
Run / experiment:
Second test
Value:
0.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Range of corneal opacity
Remarks:
-0.6 to 0.6
Irritation parameter:
other: permeability value
Remarks:
mean
Run / experiment:
Second Test
Value:
0.002
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Range of permeability value
Remarks:
-0.001 to 0.005
Other effects / acceptance of results:
mean in vitro irritancy score (range): First Test
negative control: 1.2 (0.3 to 1.9)
positive control: 68.9 (61 to 72.9)

mean opacity scores (range): First Test
negative control: 0.9 (0.2 to 1.5)
positive control: 28.2 (23.0 to 32.1)

mean permeability scores (range): First Test
negative control: 0.015 (0.008 to 0.029)
positive control: 2.711 (2.096 to 3.332)

mean in vitro irritancy score (range): Second Test
negative control: -1.1 (-2.1 to -0.1)
positive control: 35.3 (33.5 to 38.7)

mean opacity scores (range): Second Test
negative control: -1.2 ( -2.1 to -0.1)
positive control: 19.7 (18.1 to 20.6)

mean permeability scores (range): Second Test
negative control: 0.002 (0.002 to 0.003)
positive control: 1.039 (0.870 to 1.371)

Other effects:
Since in the first test the IVIS scores of the test item treated replicates were spread over 2 categories a repeat test was performed.

First test-The corneas treated with the positive control were turbid after the 10 minutes of treatment. The corneas were translucent (with spot for 2 out of 3) after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium

Second test-The corneas treated with the positive control were turbid after the 10 minutes of treatment. The corneas were clear after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium.

Interpretation:
Since in the first test the IVIS scores of the test item treated replicates were spread over 2 categories a repeat test was performed.
Since in both tests the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Aceptance of results:
In the first test, the negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that thenegative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the negative control was <3. The mean in vitro irritancy score of the positive control (Ethanol) was 69 (61 to 73) and was within two standard deviations of the current historical positive control mean. 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.

In the repeat test, 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 negative control was <3. The mean in vitro irritancy score of the positive control (Ethanol) was 35 (34 to 39) and was within two standard deviations of the current historical positive control mean but < 55. 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 of results:
GHS criteria not met
Conclusions:
Since in both tests the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2003-05-21 to 2003-02-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
Himalayan
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology KG
branch Lohndorf
D-24601 Lohndorf/Post Wankendorf
- Age at study initiation: approx. 4.5 months
- Weight at study initiation: 2.3 kg
- Housing: Singly in cages with dimensions of 425 mm x 600 mm x 380 mm
- Diet (e.g. ad libitum): ssniff® K-H V2333 (ssniff Spezialdiaten GmbH, D-59494 Soest), ad libitum
- Water (e.g. ad libitum): Tap water was offered daily ad libitum.
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ±15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: The untreated eye served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Duration of treatment / exposure:
Single instillation
Observation period (in vivo):
4 days
Reading time points: 1, 24, 48 and 72 h and 4 days
Number of animals or in vitro replicates:
3
Details on study design:
SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: hand-slit lamp / fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal: #1-3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 4 days
Irritation parameter:
iris score
Basis:
animal: #1-3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 4 days
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
chemosis score
Remarks:
Chemosis
Basis:
animal: #1-3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
Corneal opacity (grade 1) was observed in all animals 24 to 72 hours after instillation.
The fluorescein test performed 24 hours after instillation revealed corneal staining in all animals (1 /4 of the surface).
Conjunctival redness (grade 1) was observed in animal no. one 24 to 72 hours after instillation.
The irises were not affected by instillation of the test item.
Other effects:
- Other observations: There were no systemic intolerance reactions.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study, the test material caused only mild and fully reversible eye irritating reactions. The individual mean values from gradings at 24, 48 and 72 h were 1.0 for corneal opacity, 0.0 for iritis and chemosis in all 3 animals, and 1.0/0.0/0.0 for conjunctival redness in each animal, respectively. The observed effects were fully reversible within 4 days post-instillation. Therefore, the material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not eye irritating.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

Eurlings (2016a) investigated skin corrosion according to the OECD Guideline 431 and EU Method B.40 BIS. After 3 minutes and 1 hour of exposure to 25 µL of test item, 127% and 125% mean tissue viability was observed, respectively. Based on the results, the test item is not corrosive to the skin.

Eurlings (2016b) also investigated skin irritation according to the OECD Guideline 439 and EU Method B.46. After 15 minutes of exposure to 25 µL of test item, 35% mean tissue viability was observed. Based on the results, the test item is irritant in the in vitro skin irritation test category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and the negative in vitro skin corrosion test (Eurlings, 2016a).

An in vivo skin irritation study in Himalayan rabbits was performed according to OECD Guideline 404 and EU Method B.4 (2003). Under the conditions of the study, the test material caused only slight and fully reversible skin irritating reactions. The individual mean values for erythema/eschar and edema from gradings at 24, 48 and 72 h were 0.0/0.7/0.7 and 0.0/0.7/0.7 in each of the three animals, respectively. Therefore, the material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not skin irritating.

Eye irritation

Eurlings (2016c) investigated eye irritation in an in vitro bovine corneal opacity-permeability (BCOP) assay. 750 µl of T003642 was applied for 10 minutes. The test item did not induce ocular irritation through both endpoints, resulting in a mean in a mean in vitro irritancy score of after 240 minutes. No classification is required for eye irritation or serious eye damage.

An in vivo eye irritation study in Himalayan rabbits was performed according to OECD Guideline 405 and EU Method B.5 (2003). Under the conditions of the study, the test material caused only mild and fully reversible eye irritating reactions. The individual mean values from gradings at 24, 48 and 72 h were 1.0 for corneal opacity, 0.0 for iritis and chemosis in all 3 animals, and 1.0/0.0/0.0 for conjunctival redness in each animal, respectively. The observed effects were fully reversible within 4 days post-instillation. Therefore, the material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not eye irritating.

Justification for classification or non-classification

Skin irritation/corrosion

The test item was demonstrated to be an irritant to skin in an in vitro study performed according to OECD Guideline 439. However, in an vitro skin corrosion study and an in vivo skin irritation study, the test item demonstrated not to be corrosive or irritating to the skin.

Also, data from an in vitro eye irritation study and an in vivo eye irritation study showed no classification is required for eye irritation or serious eye damage and the eyes are more sensitive than skin. In conclusion, the test item should not be classified as irritant or corrosive to the skin according to the criteria of the CLP regulation (EC) No 1272/2008.

Eye irritation

Based on the negative results in both the in vitro and in vivo eye irritation study with T003642 and the criteria of the CLP Regulation (EC) 1272/2008, T003642 should not be classified for eye irritation or corrosion.