Registration Dossier

Administrative data

Description of key information

Skin irritation:

In a K2 in vivo skin irritation study in New Zealand Whtie Rabbits according to a method equivalent to OECD Guideline 404 (van Ravestyn, 1985), T000749 was observed to be slightly irritating to the skin and should be classified as irritating to the skin (Category 2) 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 and EU method B.47, T000749 did not induce occular irritation.

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:
1985-10-01 to 1985-10-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Comparable to OECD Guideline 404 study with acceptable restrictions.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Principles of method if other than guideline:
This study follows the guidelines published in the Federal Register Vol. 43 No. 163 Part IV, EPA July 26, 1979.
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Remarks:
Albino
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Laboratory in-house stock colony
- Age at study initiation: No data
- Weight at study initiation: 4.35 - 4.45 kg
- Housing: Housed in a wire cage in compliance with AALAC regulations.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: No data


ENVIRONMENTAL CONDITIONS: no data


IN-LIFE DATES: No data
Type of coverage:
open
Preparation of test site:
other: intact or abraded
Vehicle:
not specified
Controls:
other: the back of the rabbits with intact skin
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): undiluted

VEHICLE: no data
Duration of treatment / exposure:
Single dose for 6 hour exposure
Observation period:
after 24 hours, 2, 3, 4, 5, 6, 7, 10 and 14 days, etc. until reactions proved to be reversible.
Number of animals:
Two males
Details on study design:
TEST SITE
- Area of exposure: The skin at the right side of the back was abraded, whereas the left side was left intact. A definite sample was applied for each side to an area of 1" x 1" skin square on the intact and on the abraded skin.
- % coverage: No data
- Type of wrap if used: Not applicable.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): The materials were flushed and washed with handwarm water and gently dried.
- Time after start of exposure: after 6 hours


SCORING SYSTEM:
The reactions were evaluated in both the group with intact skin and the group with abraded skin after 24 hours, 2, 3, 4, 5, 6, 7, 10 and 14 days, etc. until reactions proved to be reversible. The Primary Irritation Index (PII) and Classification was according to Draize and evaluated after 24 and 72 hours of application.
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.67
Max. score:
4
Reversibility:
not fully reversible within: 13 days
Remarks on result:
other: Intact skin
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
not fully reversible within: 13 days (score 1 on days 7, 10 and 13)
Remarks on result:
other: Intact skin
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 13 days
Remarks on result:
other: Intact skin
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
not fully reversible within: 13 days
Remarks on result:
other: Intact skin
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.67
Max. score:
4
Reversibility:
not fully reversible within: 13 days
Remarks on result:
other: abraded skin
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
not fully reversible within: 13 days
Remarks on result:
other: abraded skiin
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 13 days
Remarks on result:
other: abraded skin
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
not fully reversible within: 13 days
Remarks on result:
other: abraded skin
Irritant / corrosive response data:
T 749 resulted in a slight irritation to the skin of the rabbits.
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the conditions of this study, the test item resulted in a slight irritation to the skin of the rabbits. Based on the results of this study and the criteria of the CLP Regulation, the substance is classified as irritating to the skin based on persisting inflammation observed until day 14 which is reflected in the scoring of erythema and edema until day 14.
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 conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2016-03-22 to 2016-03-22
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
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
Principles of method if other than guideline:
The study procedures were also in compliance with the following guidelines:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P, Dukic M, Alix D and Sina J F, Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: A15JB2997
- Expiration date of the lot/batch: 2016-10-21 (expiry 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

FORM AS APPLIED IN THE TEST : Light yellow, light brown liquid
Species:
other: bovine eyes
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Source: bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Bovine eyes were used as soon as possible but within 4 hours after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.

- Preparation of corneas: the eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
Vehicle:
unchanged (no vehicle)
Controls:
other: negative control (physiological saline) and positive control (ethanol)
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL

NEGATIVE CONTROL
- Amount applied: 750 µL

POSITIVE CONTROL
- Amount applied: 750 µL
Duration of treatment / exposure:
Corneas were incubated for 10 ± 1 minutes at 32 ± 1°C
Duration of post- treatment incubation (in vitro):
The opacity determination was performed after 120 ± 10 minutes of incubation at 32 ± 1°C following 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:
CORNEA SELECTION AND OPACITY READING
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
The medium from the anterior compartment was removed and 750 μL of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

OPACITY MEASUREMENT
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (l = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to:
opacity = ((I0/I)-0.9894)/0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea before/after test item treatment.
The change of opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test item treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test item treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

APPLICATION OF SODIUM FLUORESCEIN
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) was evaluated.

The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

PERMEABILITY DETERMINATIONS
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

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

INTERPRETATION
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
test item after 10 minutes of treatment
Value:
0.8
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: -1.2 to 2.5
Irritation parameter:
cornea opacity score
Run / experiment:
test item after 10 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 opacity range: 61.1 to 2.5
Irritation parameter:
other: cornea permeability score
Run / experiment:
test item after 10 minutes of treatment
Value:
-0.005
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: test item permability range: -0.011 to 0.002
Other effects / acceptance of results:
NEGATIVE CONTROL:
mean in vitro irritancy score: 1.2 (0.3 to 1.9)
mean opacity score: 0.9 (0.2 to 1.5)
mean permeability score: 0.015 (0.008 to 0.029)

POSITIVE CONTROL:
mean in vitro irritancy score: 68.9 (61.0 to 72.9)
mean opacity score: 28.2 (23.0 to 32.1)
mean permeability score: : 2.711 (2.096 to 3.332)

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

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

The mean in vitro irritancy score of the negative control was <3. 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 (Ethanol) was 68.9 (61.0 to 72.9) 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.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.8 (-1.2 to 2.5) after 10 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:
key study
Study period:
2004-01-30 to 2004-1-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Only a study summary was available for review, which provided limited details on the test substance, tested animals, methodological details and results.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The ocular irritancy potential of the test material was assessed using the Rabbit Enucleated Eye Test (REET). This method involved the application of the test material onto the cornea of the enucleated eye.
GLP compliance:
no
Remarks:
This study was conducted in facility operating to GLP within the UK national GLP monitoring progr amme, but the study report has not been audited by the QA Unit. No formal claim of GLP compliance i s made for this study.
Specific details on test material used for the study:
no data
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS:
- Five enucleated eyes, obtained from the New zealnd white strain of rabbit.

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): The eyes were maintained at a temeprature of 32°C +/-1.5°C within the superfusion apparatus.

Vehicle:
not specified
Controls:
yes, concurrent negative control
other: 0.9% sodium chloride
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL of the test material was applied onto the cornea of each eye.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): no data
- Concentration (if solution): 0.9% sodium chloride
- Lot/batch no. (if required): no data
Duration of treatment / exposure:
single application for 240 min
Number of animals or in vitro replicates:
Three enucleated eyes were treated with the test item, two enucleated eyes were treated as control.
Details on study design:
REMOVAL OF TEST SUBSTANCE:
- Washing (if done): no

OBSERVATION TIME POINTS
- corneal opacity and corneal epithelium condition: 60, 120, 180 and 240 min
- fluorescein uptake: 240 min
- corneal swelling: 60, 120 and 240 min


SCORING SYSTEM:
- Method for Evaluation of Ocular Irritation by Slit-Lamp Biomicroscopic Examination (McDonald Shadduck Score System)
- The scoring scheme measures the severity of corneal cloudiness and the area of the cornea involve d. Severity of corneal cloudiness is graded as follows:
0 = Normal cornea. Appears with the slit-lamp as having a bright grey line on the epithelial surface and a bright grey appearance of the stroma.
1 = Some loss of transparency. Only the anterior half of the stroma is involved as observed with an optical section of the slit-lamp. The underlying structures are clearly visible with diffuse illumination, although some cloudiness can be readily apparent with diffuse illumination.
2 = Moderate loss of transparency. In addition to involving the anterior stroma, the cloudiness extends all the way to the endothelium. The stroma has lost its marble-like appearance and is homogeneously white. With diffuse, illumination, underlying structures are clearly visible.
3 = Involvement of the entire thickness of the stroma. With optical section, the endothelial surface is still visible. However, with diffuse illumination the underlying structures are just visible.
4 = Involvement of the entire thickness of the stroma. With the optical section cannot clearly visualise the endothelium. With diffuse illumination, the underlying structures cannot be seen.
The surface of the cornea relative to the area of cloudiness is divided into five grades from 0 to 4:
0 = Normal cornea with no area of cloudiness
1 = 1 to 25% area of stromal cloudiness
2 = 26 to 50% area of stromal cloudiness
3 = 51 to 75% area of stromal cloudiness
4 = 76 to 100% area of stromal cloudiness
FLUORESCEIN - The use of fluorescein is a valuable aid in defining epithelial damage for fluorescein staining. The a rea can be judged as a 0 to 4 scale using the same terminology as for corneal cloudiness. The intensity of fluorescein staining can be divided into a 0 to 4 scale:
0 = Absence of fluorescein staining
1 = Slight fluorescein staining confined to a small focus. With diffuse illumination the underlying struc tures are clearly visible, although there is some loss of detail.
2 = Moderate fluorescein staining confined to a small focus. With diffuse illumination the underlying str uctures are clearly visible, although there is some loss of detail.
3 = Marked fluorescein staining. Staining may involve a larger portion of the cornea. With diffuse illu mination underlying structures are barely visible but are not completely obliterated.
4 = Extreme fluorescein staining. With diffuse illumination the underlying structures cannot be seen.

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein
Irritation parameter:
cornea opacity score
Remarks:
mean of 3 eyes
Run / experiment:
60 minutes
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: 26 to 50% area of stromal cloudiness
Irritation parameter:
cornea opacity score
Remarks:
mean of 3 eyes
Run / experiment:
120 minutes
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: Normal cornea with no area of cloudiness
Irritation parameter:
cornea opacity score
Remarks:
mean of 3 eyes
Run / experiment:
180 minutes
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: Normal cornea wtih no area of cloudiness
Irritation parameter:
cornea opacity score
Remarks:
mean of 3 eyes
Run / experiment:
240 minutes
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: Normal cornea with no area of cloudiness
Irritation parameter:
fluorescein retention score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: all scores were 0
Irritation parameter:
percent corneal swelling
Run / experiment:
60 minutes
Value:
10.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation parameter:
percent corneal swelling
Run / experiment:
120 minutes
Value:
15.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation parameter:
percent corneal swelling
Run / experiment:
240 minutes
Value:
14.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Other effects / acceptance of results:
negative control eyes:
- corneal opacity: 0 (mean of 2 eyes)

- corneal epithelium condition: normal (2 eyes)
- fluorescein uptake: 0 (mean of 2 eyes)
- corneal swelling: 4.7 at 60 min post dosing, 5.0 at 120 minutes post dosing and 4.6 at 240 minutes post dosing

Corneal epithelium condition was normal at all timepoints.
Interpretation of results:
GHS criteria not met
Conclusions:
Following assessment of the data for all endpoints the test material was considered unlikely to have the potential to cause severe ocular irritancy in vivo.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

van Ravestyn (1985) investigated acute dermal irritation of T000749 in New Zealand White rabbits (2 males after 6 hours of exposure to 0.1 mL of test item). Skin reactions were recorded after 24 hours, 2, 3, 4, 5, 6, 7, 10 and 14, etc. days until reactions proved to be reversible. Under the conditions of this study, the test item resulted in a slight irritation to the skin of the rabbits. Based on the results of this study and the criteria of the CLP Regulation (EC) No 1272/2008, the test item is classified as irritating to the skin based on persisting inflammation observed until day 14 which is reflected in the scoring of erythema and edema until day 14.

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

Eye irritation:

Eurlings (2016) investigated eye irritation in an in vitro bovine corneal opacity-permeability (BCOP) assay. 750 µl of undliuted test item was applied on the top of 3 corneas for 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 treated with T000749 were slightly translucent after the 10 minutes of treatment. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.8 (-1.2 to 2.5) after 10 minutes. Since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage based on this study.

In addition, a rabbit enucleated eye test (REET) was performed by Sanders (2004) to assess the ocuar irritancy potential of T000749. Three enucleated eyes, obtained from the New Zealand White strain of rabbit, were treated with 0.1 ml of T000749. Corneal opacity (60, 120, 180 and 240 minutes after application), corneal swelling (60, 120 and 240 minutes after application) and fluorescein uptake (240 minutes after application) were observed and scored. No indication of irritation was noted. The test item was not considered to have the potential to cause severe occular irritancy in vivo.

The BCOP study is considered the key result for assessing the eye irritation endpoint. According to Chapter R.7a: Endpoint specific guidance Version 5.0 - December 2016 (R.7.2.11.2), data obtained from non-validated suitable in vitro tests can only be used according to the criteria set out in section 1.4 of Annex XI to the REACH Regulation, i.e. only positive results can be accepted in a weight of evidence approach. As the result of the non-validated REET test was negative, this study was added to the dossier as supporting evidence and the newly conducted and validated BCOP study is selected as key study.

Justification for classification or non-classification

Skin irritation:

According to the in vivo acute dermal irritation study, slight skin irritation was noted for T000749. Based on the results of this study and the criteria of the CLP Regulation (EC) No 1272/2008, the test item is classified as irritating to the skin (category 2) based on persisting inflammation observed until day 14 which is reflected in the scoring of erythema and edema until that day.

Eye irritation:

According to the in vitro eye irritation study (BCOP), T000749 induced no ocular irritation. Although the outcome of the BCOP study doesn't trigger a classification for eye irritation, the substance is classified as eye irritant category 2, based on the skin irritation that was observed and persistent for 14 days in the study of van Ravestyn (1985).