Registration Dossier

Administrative data

Description of key information

Skin irritation:

In a K2 in vivo skin irritation study in New Zealand White rabbits according to the OECD guideline N°404 and the EU Method B.4, no evidence for skin irritation was noted for T001159.

Eye irritation:

In a K1 Bovine Corneal Opacity and Permeability (BCOP) test, performed according to OECD guideline 437 and EU method B.47, T001159 did not induce occular 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
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-04-07 to 2005-04-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Non-GLP study according to OECD guideline 404 and EU Method B.4
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
Inadequate documentation
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
Inadequate documentation
GLP compliance:
no
Remarks:
The 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 the 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:
0.5 g
Duration of treatment / exposure:
4 hours
Observation period:
72 hours, with observations at 1, 24, 48, and 72 hours
Number of animals:
3
Details on study design:
TEST SITE: no data

REMOVAL OF TEST SUBSTANCE: no data

SCORING SYSTEM:
Draize System 0-4
From Draize J H (1959) “Dermal Toxicity” in Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Assoc. of Food and Drug Officials of the US, Austin, Texas p47
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
72 h
Score:
0
Max. score:
8
Remarks on result:
other: Non-irritant
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 46
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 48
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 49
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 46
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 48
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 49
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 at any time at any test site.
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance was found to be non-irritant to rabbit skin.
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 2015-06-22 to 2015-06-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 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: M14IB5308
- Expiration date of the lot/batch: 2015-09-01 (retest date)
- Purity test date: no data
- Certificate of analysis release date: 2015-04-23

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
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A 20% (w/v) suspension of the test item was prepared in physiological saline. The suspension was treated with ultrasonic waves.
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.

- 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.

- 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 (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. Three corneas were selected at random for each treatment group.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20% (w/v) in physiological saline

VEHICLE
- Amount applied: 750 µL

POSITIVE CONTROL
- Amount applied: 750 µL
- Concentration: 20% (w/v) imidazole solution
Duration of treatment / exposure:
Corneas were incubated for 240 ± 10 minutes at 32 ± 1°C.
Observation period (in vivo):
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:
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 (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. Three corneas were selected at random for each treatment group.

TREATMENT OF CORNEAS
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/v) test item was introduced onto the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions 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.

OPACITY MEASUREMENT
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.

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.

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 240 minutes of treatment
Value:
-0.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
test item IVIS range: -1.6 to 1.4
Irritation parameter:
cornea opacity score
Run / experiment:
test item after 240 minutes of treatment
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: test item opacity range: -1.3 to 1.7
Irritation parameter:
other: cornea permeability score
Run / experiment:
test item after 240 minutes of treatment
Value:
-0.014
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: test item permeability score range: -0.017 to 0.012
Other effects / acceptance of results:
The corneas treated with the positive control were turbid after the 240 minutes of treatment. Two corneas were clear and one cornea was flecked on the edge after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium.

NEGATIVE CONTROL:
mean in vitro irritancy score (range): 0.0 (-0.4 to 0.5)
mean opacity scores (range): 0.0 (-0.3 to 0.7)
mean permeability scores (range): 0.000 (-0.011 to 0.017)

POSITIVE CONTROL:
mean in vitro irritancy score (range): 153.9 ( 151.3 to 158.8)
mean opacity scores (range): 106.0 (92.7 to 115.7)
mean permeability scores (range): 3.195 (2.373 to 3.935)

- 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 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 test conditions were adequate and that the test system functioned properly.

Interpretation:
The IVIS of all replicates was within one category.
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 -0.2 (-1.6 to 1.4) 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 conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

Sanders (2005) investigated acute dermal irritation of T001159 in New Zealand White rabbits (3 males 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.

There were no signs of irritation for any animal during the 72-hour observation period the following the 4-hour exposure period. The test item did not meet the criteria for classification as irritant or corrosive according to EU labelling regulations Commission Directive 2001/59/EC.

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 (2015) investigated eye irritation in an in vitro bovine corneal opacity-permeability (BCOP) assay. 750 µl of a 20% (w/v) suspension of the 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 treated with T001159 were clear after 240 minutes of treatment. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.2 (-1.6 to 1.4). Since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

In addition, a rabbit enucleated eye test (REET) was performed by Sanders (2005) to assess the ocuar irritancy potential of T001159. Three enucleated eyes, obtained from the New Zealand White strain of rabbit, were treated with 0.1 ml of T001159 (approx 74 mg). 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.

The test item had no effect on corneal opacity and corneal epithelium condition at any time. There was no fluorescein uptake by any eye at any time observed, and only minimal corneal swelling (7.1 - 10.5%). 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 for classification purposes.

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 T001159. 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, T001159 induced no ocular irritation. The test item did not meet the criteria for classification and no classification is required for eye irritation of serious eye damage according to the criteria of the CLP regulation (EC) No 1272/2008.