Registration Dossier

Administrative data

Description of key information

Skin irritation

In a GLP compliant K1 in vivo skin irritation study in New Zealand White rabbits conducted according to OECD Guideline 404 and EU Method B.4, T003066 was observed to be non-irritating to rabbit skin. Based on these results, T003066 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, T003066 did not induce ocular irritation. No classification is required for eye irritation or serious eye damage based on 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 irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2009-03-18 to 2009-03-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Young adult New Zealand White Rabbit, SPF from Harlan Laboraotries B.V.
- Age when treated: 15 weeks (male), 15-16 weeks (females)
- Weight when treated: 2513 g (male), 2646 - 2878 g (females)
- Housing: standard laboratory conditions; individually housed in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks and haysticks were provided for gnawing.
- Diet (e.g. ad libitum): ad libitum, pelleted standard Provimi Kliba 3418
- Water (e.g. ad libitum): ad libitum, community tap water
- Acclimation period: 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%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): automatically controlled light cycle of 12 hrs light and 12 hrs dark, music during the daytime light period

IN-LIFE DATES: From: 2009-03-18 To: 2009-03-30
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Remarks:
purified
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5g
- Concentration (if solution): not applicable (solid)

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
at 1, 24, 48 and 72 hours after exposure (after removal of dressing, gauze patch and test item)
Number of animals:
3 (1 male, 2 females)
As it was suspected that the test item might produce irritancy, a single animal (female) was treated first. As neither a corrosive effect nor a severe irritant effect was observed after the 4-hour exposure, the test was completed using the two remaining animals for an exposure period of four hours.
Details on study design:
TEST SITE
- Area of exposure: left flank
- % coverage: 2.5 cm x 2.5 cm
- Type of wrap if used: on the day of treatment, 0.5 g of T003066 was placed on a surgical gauze patch 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 dressing was removed and skin was flushed with lukewarm tap water to clean the application site so that any reactions (erythema) were clearly visible at that time.
- Time after start of exposure: 4 hours

SCORING SYSTEM: numerical scoring system listed in the Commission regulation No. 440/2008 B.4
Irritation parameter:
erythema score
Basis:
mean
Remarks:
male 63
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
erythema score
Basis:
mean
Remarks:
female 64
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
erythema score
Basis:
mean
Remarks:
female 65
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
edema score
Basis:
mean
Remarks:
male 63
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
edema score
Basis:
mean
Remarks:
female 64
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
edema score
Basis:
mean
Remarks:
female 65
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritant / corrosive response data:
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). No staining produced by the test item of the treated skin was observed. Neither alterations of the treated skin were observed nor were corrosive effects evident on the skin.
Other effects:
- no mortality occurred
- no clinical signs were observed during the course of the study
- the body weights of all rabbits were considered to be within the normal range of variability
- no necropsy was performed at the end of the study
Interpretation of results:
GHS criteria not met
Conclusions:
Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 2001) and the criteria of the CLP Regulation (EC) No
1272/2008, T003066 is considered to be 'non-irritating' to rabbit skin.
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
September 18, 2008-September 19, 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented study performed according to OECD Guideline 431 and EU Method B40 bis, with no deviations impacting the results of the study.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
Council Regulation (EC) No 440/2008 B40 bis
Deviations:
no
GLP compliance:
not specified
Species:
other: three-dimensial human epidermis model
Strain:
other: not applicable
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Epi-200 kits and MTT-100 assay diluent were purchased from MatTek Corporation, Ashland, MA U.S.A.
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 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10mm ∅).
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.
Type of coverage:
open
Vehicle:
water
Remarks:
Deionized
Controls:
other: see below
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): wetted with 50 µl deionized water
Duration of treatment / exposure:
3 minutes and 1 hour, in duplicate
Observation period:
Not relevant for this type of test.

Number of animals:
Duplicate EpiDerm™ tissues were treated with the test item, positive, and negative controls for 2 different treatment intervals
Details on study design:
Treatment:
After pre-incubation of EpiDerm™ tissues was completed (1 hour 15 minutes for the 1 hour treatment and for the 3 minutes treatment) medium was replaced by 0.9 mL fresh assay medium in all four 6-well plates. 50 µL deionised water (negative control) were added into the first insert atop the EpiDerm tissue. The procedure was repeated with the second tissue. It was proceeded with test item and the positive control in the same manner until all tissues of the same treatment interval were dosed. The 6-well plates were placed into the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).

After the end of the treatment interval the first insert was removed immediately from the 6-well plate. Using a wash bottle the tissue was gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the insert and blotting the bottom with blotting paper. The insert was placed in the prepared holding plate. It was proceeded with test item and the positive control in the same manner until all EpiDerm™ tissues were dosed.

MTT Assay:
The MTT concentrate was prepared on the day of testing and diluted with the MTT diluent. The remaining MTT solution was stored in the dark at 4 °C for later use on the same day (not until next day). Two 24-well plates were prepared before end of the tissue prewarming period. 300 µL of the MTT solution were added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until use.

After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to the MTT-plates. 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.

Some test chemicals may reduce MTT, which will result in a blue colour without any involvement of cellular mitochondrial dehydrogenase. Although in the present assay the test chemicals were rinsed off and the DMEM medium beneath the tissues was changed before contact with MTT medium, some amount of a test chemical may be released by the tissues into the MTT medium and directly reduce the MTT, which would be interpreted as
"tissue viability". 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. No colour change could be observed in the present study.

Interpretation of results:
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%.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute
Value:
103.4
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour
Value:
97.6
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
After treatment with the test item T003066 the relative absorbance values both after 3 minutes and after 1 hour treatment were not decreased (103.4% and 97.6%). Both are well above the threshold of 50% for 3 minutes treatment and above 15% for 1 hour treatment. Therefore, the test item is not considered corrosive.
After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD ≥ 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 to 20.2% for the 3 minutes treatment interval and 11.5% for the 1 hour treatment interval thus ensuring the validity of the test system.
Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, under the experimental conditions reported, the test item T003066 was observed not to be corrosive to 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 2016-08-01 to 2016-08-02
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.
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
Principles of method if other than guideline:
The study procedures were also in compliance with the following guidelines:
- European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.47
“Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants ". Official Journal of the European Union No. L324; Amended by EC No. 1152/2010 No. L142, 09 December 2010.
- 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)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16CB1581
- Expiration date of the lot/batch: 2019-04-04
- Certificate of analysis release date: 2016-04-07

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: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none

FORM AS APPLIED IN THE TEST: White powder
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 applied: 319 to 358 mg

NEGATIVE CONTROL
- Amount applied: 750 µL

POSITIVE CONTROL
- Amount applied: 750 µL
- Concentration (if solution): 20% (w/v) imidazole solution
Duration of treatment / exposure:
Corneas were incubated for 240 ± 10 minutes at 32 ± 1°C
Duration of post- treatment incubation (in vitro):
The opacity measurement was performed after the exposure period. 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:
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.

NEGATIVE CONTROL USED
physiological saline (Eurovet Animal Health, Bladel, The Netherlands)

POSITIVE CONTROL USED
20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 µL of the negative control and 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.

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 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.
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
Run / experiment:
Test item after 240 minutes of treatment
Value:
-0.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Test item IVIS range: -0.7 to 0.5
Irritation parameter:
cornea opacity score
Run / experiment:
Test item after 240 minutes of treatment
Value:
-0.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: test item corneal opacity score range: -1.0 to -0.8
Irritation parameter:
other: permeability value
Run / experiment:
Test item after 240 minutes of treatment
Value:
0.019
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: test item permeability value range: 0.008 to 0.036
Other effects / acceptance of results:
mean in vitro irritancy score (range):
negative control: -0.2 (-0.8 to 0.9)
positive control: 158.7 (140.0 to 187.0)

mean opacity scores (range):
negative control: -0.4 (-1.0 to 0.6)
positive control: 121.5 (107.4 to 149.1)

mean permeability scores (range):
negative control: 0.014 (0.011 to 0.016)
positive control: 2.485 (2.136 to 2.792)

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.

Interpretation:
The IVIS of all replicates was within one category.

Discussion:
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 159 (140 to 187) 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:
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.6 (-0.7 to -0.5) 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:
experimental start and completion date: September 11, 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Well documented, non-GLP study performed according to internationally accepted guidelines and recommendations: INVITTOX (UK) protocol no. 98 "The Bovine Corneal Opacity and Permeability Assay", dated February 1994 and BCOP Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.
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: BCOP Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Deviations:
no
GLP compliance:
no
Species:
other: bovine eyes
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Freshly isolated bovine eyes were collected from the abattoir
Collection of bovine eyes:
Excess tissue was removed from the excised eyes and they were contained and transported in Hank's BSS supplemented with streptomycin/penicilin at room temperature. The eyes were used immediately after delivery to the laboratory.
Preparation of corneae:
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully 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 listed above.
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, and the basal opacity was determined (t0). For measurement, the posterior compartment was plugged and the anterior compartment was unplugged.

Use of preserved corneae:
The bovine eyes were fetched from the slaughterhouse and stored in the refrigerator at about 2 - 8 °C until preparation of the corneae on the same day. The cornea of the freshly delivered eye was removed as described earlier and inserted in pre-cooled preservation medium. The corneae were stored individually in a minimum volume of 5 mL. The preservation medium was composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine. Shortly before use, Dextran was added.
Vehicle:
unchanged (no vehicle)
Controls:
other: negative (3 corneae) and positive (3 corneae) control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg
Duration of treatment / exposure:
240 minutes (± 5 minutes)
Observation period (in vivo):
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).
In the second step of the assay, permeability of the cornea possibly caused by the test item, was determined. Fresh cMEM medium was added to the posterior compartment and 1 mL of a Na-fluorescein solution, 0.5 % dissolved in Dulbecco’s phosphate-buffered saline, was placed in the anterior compartment. Corneae were incubated again in a horizontal position for further 90 minutes at 32 ± 2 °C in the water-bath. The optical density of an aliquot of the mixed medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).
Number of animals or in vitro replicates:
Negative control: 3 corneae
Positive control 3 corneae
Test item: 3 corneae
Details on study design:
Opacity measurement:
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).

Permeability determination:
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.

Criteria for determination of a valid test:
The test was acceptable if the positive control caused an at least moderate effect.

Evaluation of results:
- Opacity
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.
- Permeability
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.

In vitro score calculation:
The following formula was used to determine 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 the 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.
Irritation parameter:
in vitro irritation score
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
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.
The test item T003066 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.
Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item T003066 is not considered to be an eye irritant (in vitro score 0.00).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin irritation

Rached (2015) investigated acute dermal irritation/corrosion in 1 male and 2 female New Zealand White rabbits after 4 hours of exposure to 0.5 g of test item. Based on the results, the test item did not provoke any skin reactions at the application site of any animal (all scores 0). No staining produced by the test item of the treated skin was observed. Neither alterations of the treated skin were observed nor were corrosive effects evident on the skin.

In addition, Heppenheimer (2009) investigated skin corrosion in an In vitro skin corrosion study. After 3 minutes and 1 hour of exposure to 25 mg of test item 103.4% and 97.6% mean tissue viability was observed, respectively. Both are well above the threshold of 50% for 3 minutes treatment and above 15% for 1 hour treatment. Based on the results, the test item was predicted as a non-irritant to the skin. 

The GLP compliant in vivo skin irritation study of 2015 is considered the key result for assessing the skin irritation endpoint, since the in vitro skin corrosion study of 2009 was non-GLP compliant. According to the REACH legislation (art 13.4) newtests (since 2008) must be performed in compliance with GLP. As the result of the non-GLP compliant in vitro skin corrosion study was negative, this result was added to the dossier as supporting evidence and the GLP-compliant 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. 319.0 to 358.0 mg of test item was applied on the top of 3 corneas for 240 minutes +/- 10 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 -0.6 (-0.7 to -0.5) 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 Heppenheim (2008). 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 T003066 a mean IVIS of 0.00 was recorded. Based on the results, the test item is not considered to be an eye irritant.

The BCOP study is considered the key result for assessing the eye irritation endpoint. According to the REACH legislation (art. 13.4) ne tests (since 2008) must be performed in compliance with GLP. As the result of the non-GLP in vitro skin corrosion study was negative, this study was added to the dossier as supporting evidence and the GLP compliant BCOP study is selected as key study for classification purposes.

Justification for classification or non-classification

Skin irritation:

According to the in vivo skin irritation study of 2015, T003066 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, T003066 did not induce ocular irritation and should not be classified for eye irritation or serious eye damage according to the criteria of the CLP regulation (EC) No 1272/2008.