Registration Dossier

Administrative data

Description of key information

Skin irritation:

In a K1 in vivo skin irritation study in New Zealand White Rabbits according to OECD Guideline 404 and EU Method B.4, T003063 was not irritating to the skin based on 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, T003063 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 vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-03-23 to 2009-03-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented GLP study according to OECD guideline 404 en EU Method B.4.
Justification for type of information:
The study was performed in 2009 to investigate further the results obtained in the in vitro test. The report was finalized in 2015.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 507652
- Expiration date of the lot/batch: 2009-04-03 (retest date)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (20 +/- 5°C), light portected
- Stability under test conditions: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was used as delivered by the sponsor and was moistened with approximately 0.5 mL of purified water before application.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: young adult New Zealand White rabbits, SPF; from Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst, The Nertherlands
- Age when treated: 15 weeks (male), 15 weeks (females)
- Weight when treated: 2631 grams (male), 2831 - 2852 grams (females)
- Housing: Standard Laboratory Conditions; individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (Harlan Laboratories Ltd., Füllinsdorf) and haysticks 4642 (batch no. 69/08, Provimi Kliba AG) were provided for gnawing.
- Diet (e.g. ad libitum): pelleted standard Provimi Kliba 3418 rabbit maintenance diet, ad libitum
- Water (e.g. ad libitum): Community tap water from Füllinsdorf, ad libitum
- Acclimation period: 5 days (from 2009-03-18 to 2009-03-22) under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 30 - 70% (values above 70% during cleaning process possible)
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12, automatically controlled light cycle, music during the daytime light period

IN-LIFE DATES: From: 2009-09323 To: 2009-03-26
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Remarks:
purified
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g/animal, as delivered by the sponsor
- The pH of the test item was measured before the study initiation date in a 1 % (w/w) solution in purified water and was found to be 5.
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
1 male and 2 females
Details on study design:
TEST SITE
- Area of exposure: left flank, 100 cm² (10 cm x 10 cm)
- % coverage: no data, ca. 2.5 cm x 2.5 cm
- Type of wrap if used: 0.5g of T003063 on a surgical gauze patch was held in contact with the skin by means of an adhesive hypoallergenic aerated semi-occlusive dressing and a restrainer bandage wrapped around the abdomen.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the skin was flushed with lukewarm tap water to clean the application site.
- Time after start of exposure: 4 hours after treatment

SCORING SYSTEM:
- The skin reaction was assessed according to the numerical scoring system listed in the Commission Regulation (EC) No. 440/2008, B.4, at approximately 1, 24, 48 and 72 hours after exposure (removal of the dressing, gauze patch and test item).
Irritation parameter:
erythema score
Basis:
mean
Remarks:
male 60
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
mean
Remarks:
male 60
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
other: scaling
Basis:
mean
Remarks:
male 60
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
mean
Remarks:
female 61
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
mean
Remarks:
female 61
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
other: scaling
Basis:
mean
Remarks:
female 61
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
mean
Remarks:
female 62
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
mean
Remarks:
female 62
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
other: scaling
Basis:
mean
Remarks:
female 62
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
Irritation:
- The test item did not elicit any skin reactions at the application site of any animal at any of the observation times (all scores 0).

Corrosion:
- Neither alterations of the treated skin were observed nor were corrosive effects evident on the skin.
Other effects:
Mortality/Viability:
- No mortality occurred.

Clinical signs:
- No clinical signs were observed during the course of the study.

Body Weights:
- The body weights of all rabbits were considered to be within the normal range of variability.

Pathology:
- Macroscopic findings: no necropsy was performed at the end of the study.

Coloration:
- No staining produced by the test item of the treated skin was observed.
Interpretation of results:
GHS criteria not met
Conclusions:
Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 2001), T003063 is considered to be “not irritating” to rabbit skin. According to the CLP classification, the test item should not be classified.
Endpoint:
skin irritation: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 2008-09-18 to 2008-09-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented non-GLP study performed according to OECD Guideline 431
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Council Regulation (EC) No 440/2008 B40 bis, dated 30 May, 2008
Deviations:
no
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 507652
- Expiration date of the lot/batch: 2009-04-03
- Purity: 100.4 %
- 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: the test material was not crushed or grinded in a mortar with pestle since this did not improve the consistency.
- Final preparation of a solid: about 25 mg of test material were wetted with 50 µL deionised water. The test item was spread to match the size of the tissue.
Test system:
human skin model
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: - Source: Epi-200 kits and MTT-100 assays diluent were purchased from MatTek Corporation.
- Tissue batch number(s): 10564 kit h
- The EpiDerm tissue 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 06 cm2) are cultured on specially prepared cell cultures inserts (Millicells, 10 mm diameter).
- EpiDerm tissues were shipped at 4°C on medium-supplemented agarose gels in a 24-well plate and reached Harlan CCR GmbH on June 25, 2008. Tissues were stored at 4°C until June 26, 2008 prior to use. On day of receipt EpiDerm tissues were kept in the refrigerator until use. 1 - 1.5 hours before starting the assay, tissues were transferred to 6-well plates with assay medium, which was immediately replaced before the test was started.
- 1-1.5 hours before dosing, EpiDerm™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed assay medium. Two 24-well plates were prepared as holding plates containing 300 μL assay medium. The holding plates were pre-warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until use.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 3 times with phosphate buffered saline (PBS)
- Observable damage in the tissue due to washing: no data

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: no data
- Incubation time: 3 hours
- After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and wells were rinsed three times with PBS. Inserts were transferred into new 24 well plates. The inserts were immersed into extractant solution by gently pipetting 2 mL extractant solution (isopropanol) into each insert. The level rised above the upper edge of the insert, thus completely covering the tissue from both sides. The 24 well plate was sealed to inhibit isopropanol evaporation. The formazan salt was extracted for approx. 18 hours 35 minutes with shaking at room temperature.

After the extraction period was completed for both treatment intervals the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. 24 well plates were placed on a shaker for approx. 15 minutes until solution was homogeneous in colour.
Per each tissue 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate, both from the 3 minutes exposure and from the 1 hour exposure. OD was read in a microplate reader (Versamax® Molecular Devices, D-85737 Ismaning) at 570 nm without reference filter. Mean values were calculated from the 3 wells per tissue.

- To check MTT reducing capability a solution of MTT in DMEM (1.0 mg/mL) was prepared and each about 50 mg of the test item were added to 1 mL MTT
medium. If the mixture turned blue/purple after about 1 hour at room temperature, the test material would have been presumed to have reduced the MTT.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA: no data

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

PREDICTION MODEL / DECISION CRITERIA
- The mean OD value obtained for the duplicate tissues per test item were used to calculate a percentage viability relative to the negative control, which was arbitrarily set at 100%.

- The test item is considered to be corrosive to skin:
(1) if the viability after 3 minutes exposure is less than 50%, or
(2) if the viability after 3 minutes exposure is greater or equal than 50% and the viability after 1 hour exposure is less than 15%.

The test item is considered to be non-corrosive to skin:
(1) if the viability after 3 minutes exposure is greater or equal than 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): about 25 mg

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 tissues pet 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 of 2 tissues
Run / experiment:
3-minute application
Value:
91.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 2 tissues
Run / experiment:
1-hour application
Value:
95.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: absorbance 570 nm
Remarks:
mean of 2 tissues
Run / experiment:
3-minute application
Value:
1.526
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: range of 2 tissues: 1.612-1.709
Irritation / corrosion parameter:
other: absorbance 570 nm
Remarks:
mean of 2 tissues
Run / experiment:
1-hour application
Value:
1.576
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: range of 2 tissues: 1.537-1.616
Other effects / acceptance of results:
3-minute application:
- viability (percentage of control):
negative control: 100.0
positive control: 20.2
- mean absorbance 570 nm (range):
negative control: 1.660 (1.612 - 1.709)
positive control: 0.335 (0.329 - 0.341)

1-hour application:
- viability (percentage of control):
negative control: 100.0
positive control: 11.5
- mean absorbance 570 nm (range):
negative control: 1.646 (1.543 - 1.749)
positive control: 0.189 (0.185 - 0.193)

Direct MTT reduction:
- Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Results:
- After treatment with the test item T003063 the relative absorbance values did not decrease relevantly, both after 3 minutes and after 1 hour treatment (91.9% and 95.8%).

Acceptance of results:
- After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD > or = 0.8 for both treatment intervals thus showing the quality of the tissues.
- Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control both for the 3 minutes treatment interval (20.2%) and for the 1 hour treatment interval (11.5%) thus ensuring the validity of the test system.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of the study, T003063 is observed to be non corrosive to the skin.
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 2016-02-22 to 2016-02-23
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:
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. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M14KB4863
- Expiration date of the lot/batch: 2017-11-10 (retest date)
- Purity: 100.5%
- 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
- Solubility and stability of the test substance in the solvent/vehicle: Since no workable suspension of the test item in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, the test item was applied undiluted





Species:
other: Freshly isolated bovine cornea
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Vitelco, -'s Hertogenbosch, The Netherlands
- Bovine eyes were used as soon as possible but within 4 hours after slaughter. Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. 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): 365.2 to 385.3 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 0.9% (w/v)

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 20% (w/v) Imidazole


Duration of treatment / exposure:
Corneas were incubated for 240 ± 10 minutes at 32 ± 1°C
Duration of post- treatment incubation (in vitro):
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:
3 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 MC2 (Clermont-Ferrand, France) 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 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 (365.2 to 385.3 mg).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test 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.

METHODS FOR MEASURED ENDPOINTS
-CORNEAL OPACITY: Opacity determinations will be 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.
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 compa rtment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Nafluor escein/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 e ach 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 ra nge (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 c alculate 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 of 3 eyes
Run / experiment:
1
Value:
-0.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: range of IVIS score of test item: -2.6 to 1.0
Irritation parameter:
cornea opacity score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
-0.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: range of corneal opacity score of test item: -2.6 to 0.8
Irritation parameter:
other: permeability score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
0.006
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: range of permeability value of test item 0.000 to 0.018
Other effects / acceptance of results:
mean in vitro irritancy score (range):
negative control: -1.9 (-2.2 to -1.3)
positive control: 131.4 (117.3 to 156.0)

mean opacity scores (range):
negative control: -2.0 (-2.4 to -1.4)
positive control: 111.9 (97.4 to 130.7)

mean permeability scores (range):
negative control: 0.010 (0.008 to 0.013)
positive control: 1.299 (0.883 to 1.684)

The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with the test item showed opacity values ranging from -2.6 to 0.8 and permeability values ranging from 0.000 to 0.018. 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. Hence, the in vitro irritancy scores ranged from -2.6 to 1.0 after 240 minutes of treatment with the test item.

Acceptance of results
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 131 (117 to 156) 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 of results:
GHS criteria not met
Conclusions:
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 2008-09-11 to 2008-09-11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented non-GLP study performed according to OECD guideline 437.
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
Qualifier:
according to
Guideline:
other: INVITTOX (UK) protocol no. 98 "The bovine Corneal Opacity and Permeability Assay", dated February 1994
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd, UK , Procedure Details, April 1997
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Deviations:
no
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 507652
- Expiration date of the lot/batch:2009-04-03
- Purity: 100.4%
- 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
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:
Species:
other: Freshly isolated bovine cornea
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Odenwaldschlachthof Brensbach, D-64395 Brensbach
- Freshly isolated bovine eyes were collected from the abattoir. Excess tissue was removed from the excised eyes and they were contained and transported in Hank's BSS supplemented with streptomycin / penicillin at room temperature. The bovine eyes were stored in the refrigerator at about 2 - 8 °C until preparation of the corneae on the same day.

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): approximately 100 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 1.0 mL
- Concentration (if solution): 0.9% (w/v)

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 1.0 mL
- Concentration (if solution): 10% (w/v)
- Lot/batch no. (if required): 036K0208

Duration of treatment / exposure:
240 minutes (± 5 minutes)
Observation period (in vivo):
Opacity measurement was performed after treatment. The permeability determination was performed after incubation for about 90 minutes in a water-bath at 32 ± 2 °C following opacity measurement.
Number of animals or in vitro replicates:
3 corneas per treatment group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- All eyes were carefully examined macroscopically for defects. Those representing defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefuly removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneae used in the experiment were collected in Hank's BSS supplemented with streptomycin/penicillin and checked finally with a view box for defects. The corneae were stored individually in a minimum volume of 5 mL. The preservation medium was composed of Medium 199 supplemented with Lglutamine, Na-bicarbonate and Taurine. Shortly before use, Dextran was added.
- Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete minimum essential medium (cMEM). The posterior compartment was filled first to
return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 2 °C in a water-bath.
At the end of the incubation period, the medium was removed from both compartments and replaced with fresh cMEM.

TREATMENT METHOD
Fresh cMEM was placed in the posterior compartment, while the anterior compartment received the test item (approx. 100 mg) or negative or positive control at a volume of 1.0 mL each on the surface of the corneae and was incubated at 32 ± 2 °C in the waterbath in a vertical position. The incubation time lasted 240 minutes (± 5 minutes).

REMOVAL OF TEST SUBSTANCE
After the test item was rinsed off from the application side by changing cMEM several times, fresh cMEM was replaced in both compartments and opacity was measured (t240).

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea. After recording the basal opacity of all corneae, the mean value of all corneae were taken and any cornea deviating from this by more than 3 units, also –3 units is possible, was discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.
Medium was completely removed from the anterior compartment and replaced by the test item, positive or negative control. The anterior compartment was plugged. The cornea was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test item and was incubated in a water-bath at 32 ± 2 °C for 240 minutes. Afterwards, the opacity was measured again (t240).
The change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
The mean corrected opacity value of each treatment group was calculated from the individual corrected opacity values of the treated corneae for each treatment condition.
- Corneal permeability: Following to the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (v/v) fluorescein solution. Corneae were incubated again in a horizontal position for about 90 minutes in a water-bath at 32 ± 2 °C. Medium from the posterior compartment was removed with a 5 mL-syringe, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
The corrected OD490 value of each treated cornea of positive control corneae was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.
The mean corrected permeability values of each treatment group was calculated from the individual corrected permeability values of the treated corneae for each treatment condition.

SCORING SYSTEM/ In Vitro Irritancy Score (IVIS)
In vitro score calculation:
The following formula was used to determin the in vitro score of the negative control:
In vitro score = opacity value + (15 x OD490 value)

The following formula was used to determine the in vitro score of the positive control and test item:
In vitro score = corrected opacity value + (15 x corrected OD490 value)

The in vitro score was calculated for each individual treatment and positive control cornea. The mean in vitro score value of each treated group was calculated from the individual in vitro score values.

Depending on the score obtained, the test item was classified into on of the following categories:
0 - 3: non eye irritant
3.1 - 25: mild eye irritant
25.1 - 55: moderate eye irritant
55.1 - 80: severe eye irritant
> 80.1: very severe eye irritant
Irritation parameter:
in vitro irritation score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: permeability score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
0.01
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: range: 0.0023 to 0.0253
Other effects / acceptance of results:
mean in vitro irritancy score (range):
negative control: 2.37
positive control: 118.94

mean opacity scores (range):
negative control: 1 (1-2)
positive control: 104.997 (85.33 - 122.33)

mean permeability scores (range):
negative control: 0.0015 (0.045 - 0.048)
positive control: 0.929 (0.8103 - 1.1463)

The test item T003063 did not cause any opacity or permeability of the corneae compared with the results of the negative control. The calculated in vitro score was 0.00 and therefore, the test item was classified as non eye irritant.
With the negative control (0.9 % NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The in vitro score was calculated as 2.37.
The positive control (10% v/v benzalconium chloride) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant. The in vitro score was calculated as 118.94.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions reported in the study, the test item T003063 is not considered to be an eye irritant.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Rached (2015) investigated acute dermal irritation of T003063 in New Zealand White rabbits (1 male and 2 females after 4 hours of exposure to 0.5 g/animal, of test item). Skin reactions were recorded 1, 24, 48 and 72 hours after exposure (removal of the dressing, gauze patch and test item). The test item did not elicit any skin reactions at the application site of any animal at any of the observation times (all scores 0). Based on the results of this study and the criteria of the CLP Regulation (EC) No 1272/2008, the test item should not be classified as irritating to the skin.

In addition, Heppenheimer (2009) investigated acute dermal corrosion of T003063 according to OECD Guideline 431 and EC Method B40 bis, dated May 30, 2008. The EpiDerm tissue was exposed to about 25 mg of test item for 3 minutes and 1 hour. The mean tissue viability was 91.9% and 95.8%, respectively for 3 minute and 1 hour exposure and the mean absorbance was 1.526 and 1.576, respectively for 3 minute and 1 hour exposure. Based on the results of this study, the test item is observed to be non corrosive to the skin and should not be classified as corrosive to the skin according to the criteria of the CLP Regulation (EC) No 1272/2008.

An in vitro skin irritation study was waived based on the justification that adequate data from an in vivo skin irritation study are available. 

The in vivo skin irritation study is considered the key result for assessing the skin irritation endpoint. According to the REACH legislation (Art 13.4), new tests (since 2008) should be performed in compliance with GLP. As the result of the non-GLP skin corrosion study of 2009 was negative, this study was added to the dossier as supporting evidence an the newly conducted and GLP in vivo skin irritation study 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. 365.2 to 385.3 mg 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 -0.7 (-2.6 to 1.0) 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 Heppenheimer (2008) to assess the ocular irritancy potential of T003063. Approximately 100 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). After 240 minutes of exposure to test item T003063 a mean in vitro irritancy score of 0 was recorded. Since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

The BCOP study of 2016 is considered the key result for assessing the eye irritation endpoint. According to the REACH legislation (Art 13.4), new tests (since 2008) should be performed in compliance with GLP. As the result of the non-GLP BCOP study of 2008 was negative, this study was added to the dossier as supporting evidence an the newly conducted and GLP BCOP study is selected as key study for classification purposes.

Justification for classification or non-classification

Skin irritation:

According to the in vivo acute dermal irritation study no dermal irritation was observed for T003063. The test item did not meet the criteria for classification as irritant or corrosive according to the criteria of the CLP regulation (EC) No 1272/2008.

Eye irritation:

According to the in Bovine Corneal Opacity and Permeabilty (BCOP) test, T003063 induced no ocular irritation. No classification is required for eye irritation or serious eye damage according to the criteria of the CLP regulation (EC) No 1272/2008.