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Administrative data

Description of key information

  • Skin irritation

In an acute dermal irritation/corrosion study in New Zealand White rabbits similar to OECD Guideline 404 and EU method B.4, no evidence for skin irritation was noted for T001036.

  • Eye irritation

Based on the results of an in vitro eye irritation study using bovine eyes, according to OECD Guideline 437 and EU method B.47 and the criteria of the CLP Regulation (EC) No 1272/2008, 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
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-10-01 to 2004-09-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study is similar to OECD Guideline 404 (Acute Dermal Irritation / Corrosion) and EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion). The study was non-GLP and limited information was given on the test substance, test animals and methodology.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
no
Remarks:
This study was conducted in a facility operating to Good Laboratory Practice within the UK national GLP monitoring programme. No formal claim of GLP compliance is 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 and environmental conditions:
TEST ANIMALS: no data

ENVIRONMENTAL CONDITIONS: no data

IN-LIFE DATES: no data
Type of coverage:
semiocclusive
Preparation of test site:
not specified
Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g
- Concentration (if solution): no data

VEHICLE: no data
Duration of treatment / exposure:
single 4-hour application
Observation period:
Skin reactions were recorded 1, 24, 48 and 72 hours after administration.
Number of animals:
three male rabbits
Details on study design:
TEST SITE
- Area of exposure: no data
- % coverage: no data
- Type of wrap if used: no data


REMOVAL OF TEST SUBSTANCE
- Washing (if done): no data
- Time after start of exposure: no data


SCORING SYSTEM:
Scored according to the Draize scale.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
other: based on 24 and 72 hour readings
Score:
0
Max. score:
8
Reversibility:
other: not applicable
Remarks on result:
other: Non-Irritant
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 158
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 168
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 175
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 158
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 168
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 175
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritant / corrosive response data:
No evidence of skin irritation was noted.
Other effects:
no data
Interpretation of results:
GHS criteria not met
Conclusions:
No evidence of skin irritation was noted. 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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2015-06-01 to 2015-06-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study according to OECD guideline 437 and EU method B.47. No deviations were recorded.
Qualifier:
according 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:
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 described in the study are also in compliance with the following documents:
- 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: A14JB3442
- Expiration date of the lot/batch: 30 September 2016 (retest date)
- Purity test date: 18 Februay 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated on TSDS
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not indicated

OTHER SPECIFICS: Correction factor is 1
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.

- 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.

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
First test
- Amount(s) applied (volume or weight with unit): 368.5 to 398.3 mg, applied directly on the corneas in such a way that the cornea was completely covered
Based on a solubility test in which the test item formed a non-homogenous suspension with lumps (treated with vortex and ultrasonic waves) in physiological saline, the test item was used as delivered by the sponsor and added pure on top of the corneas

Second test
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 20% (w/w) non-homogeneous suspension with lumps of test item prepared in physiological saline

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µl
- Lot/batch no. (if required): no data
- Purity: no data

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 20% (w/v)
Duration of treatment / exposure:
Corneas were incubated for 240 ± 10 minutes at 32 ± 1°C.
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.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

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

APPLICATION DOSE AND EXPOSURE TIME
368.5 to 398.3 mg test item in the first test
20% (w/w) test item in the second test
240 ± 10 minutes

TREATMENT METHOD:
In the first test, 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 (368.5 to 398.3 mg). In the second test, the medium from the anterior compartment was removed and 750 µl of either the negative control, positive control
(20% (w/v) Imidazole solution) or 20% (w/w) test item as requested by the sponsor was introduced onto the epithelium of the cornea. The holder was slightly rotated,
with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position
for 240 ± 10 minutes at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
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 (OP-KIT, MC2, Clermont-Ferrand, France). 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 3 were not used.
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: The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in 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)
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.
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:
1
Value:
-1.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
2
Value:
-1.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
First test:
The individual in vitro irritancy scores for the negative controls ranged from -0.7 to 1.4. The individual positive control in vitro irritancy scores ranged from 130.6 to 137.8. 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 -2.7 to -0.7 and permeability values ranging from -0.019 to -0.013. 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 -2.9 to -0.9 after 240 minutes of treatment with the test item.

Second test:
The individual in vitro irritancy scores for the negative controls ranged from -0.4 to 0.5. The individual positive control in vitro irritancy scores ranged from 151.3 to 158.8. 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 -1.3 to -0.3 and permeability values ranging from -0.017 to 0.004. 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 -1.6 to -0.4 after 240 minutes of treatment with the test item.

In the first test, the negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that 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 134.6 (130.6 to 137.8) 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.

In the second test, the negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative
control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 153.9 (151.3 to 158.8) 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.

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.1 (-1.6 to -0.4) after 240 minutes of treatment.

Since the test item induced an IVIS ≤ 3 in both tests, no classification is required for eye irritation or serious eye damage.
Interpretation of results:
GHS criteria not met
Conclusions:
Since the test item induced an IVIS ≤ 3 in both tests, no classification is required for eye irritation or serious eye damage, according to the criteria of the CLP Regulation (EC) No 1272/2008.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Sanders (2004) investigated acute dermal irritation of T001036 in 3 male rabbits (after 4 hours of exposure to 0,5 g of test item). Skin reactions were recorded 1, 24, 48 and 72 hours after administration and scored according to the Draize scale.

No evidence of skin irritation was noted. The test item did not meet the criteria for classification as irritant or corrosive to the skin according to the criteria of the CLP Regulation (EC) No 1272/2008.

The in vitro skin irritation study has been waived because adequate data from an in vivo skin irritation study are available.

Eye irritation:

Eurlings (2015) investigated the eye hazard potential of the test item by an in vitro bovine corneal opacity-permeability (BCOP) assay. This K1 study was selected as key study. 368.5 to 398.3 mg of T001036 (as supplied) was applied for 240 minutes in the first test and 750 µl of a 20% (w/w) non-homogenous suspension with lumps of T001036 in physiological saline in the second test. 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). In the first test, the test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.6 (-2.9 to -0.9) after 240 minutes of treatment. In the second test, a mean in vitro irritancy score of -1.1 (-1.6 to -0.4) was calculated after 240 minutes of treatment. Since the test item induced an IVIS≤3 in both tests, no classification is required for eye irritation or serious eye damage. In both tests, the acceptability criteria were met and it was concluded that the test conditions were adequate and the test system functioned properly.

In addition, a rabbit enucleated eye test was performed by Sanders (2004) to assess the ocular irritancy potential of T001036. 3 enucleated eyes of New Zealand White rabbits were treated with 0.1 mL (approx. 80 mg) (undiluted) of T001036. Corneal opacity, corneal swelling and fluorescein uptake were observed at 60, 120, 180 and 240 minutes after application and scored. No indication of irritation was noted. The test item was considered unlikely to have the potential to cause severe ocular irritancy in vivo.

Justification for classification or non-classification

Skin irritation:

According to the in vivo acute dermal irritation study no evidence for skin irritation was noted for T001036. 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:

In the in vitro eye irritation study (BCOP) and rabbit enucleated eye test, T001036 showed no irritating effects. No classification is required for eye irritation or serious eye damage.