Registration Dossier

Administrative data

Description of key information

The test item is not considered to be irritating to skin and not considered to be irritating to eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Oct 24-26, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Council Regulation (EC) No 761/2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: reconstructed skin produced by SkinEthic Laboratories
Source strain:
other: adult human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
Vehicle:
water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: in vitro skin model RHE
- Tissue batch number(s): 12022A1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: a minimum of 25 mL PBS; excess PBS was removed by gently shaking the inserts and blotting the bottom with the blotting paper
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/L
- Incubation time: 3 hours
- Spectrophotometer: plate spectrophotmeter
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD > 0.7 (acceptance criteria), OD = 1.116 (result)
- Barrier function: 4 h < ET50 < 9 h (acceptance criteria), 4.5 h (result)
- Morphology: Well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum. At least 4 viable cell layers present. Absence of significant histological abnormalities. (acceptance criteria), confirmed, 5/6 cell layers (result)

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- Method of calculation used: A direct interaction with the MTT is detected if the test material solution clearly turns blue or purple.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability after 42 minutes exposure is less than or equal to 50%
- The test substance is considered to be not irritating to skin if the viability after 42 minutes exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 10 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS solution
Duration of treatment / exposure:
42 min (+/- 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (+/- 1 hour)
Number of replicates:
3
Irritation / corrosion parameter:
other: Optical density
Run / experiment:
Run 1/tissue 1
Value:
1.479
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: Optical density
Run / experiment:
Run 1/tissue 2
Value:
1.426
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: Optical density
Run / experiment:
Run 1/tissue 3
Value:
1.081
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1/mean value
Value:
87.71
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions reported, the test material is not irritanting to skin.
Executive summary:

The in vitro study was performed to assess the irritation potential of the test material by means of the Human Skin Model Test. The test consisted of a topical exposure of the test item to a human reconstructed model followed by a cell viability test. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential.
Triplicates of the human skin model RHE (Reconstructed Human Epidermis model) were treated either with the test item, the negative or the positive control for 42 minutes. 16 µL of either the negative control (PBS-buffer) or the positive control (5% sodium dodecylsulphat solution) were applied to each tissue. Before adding the test item, 10 µL of deionised water was spread to the epidermis surface to improve further contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to each tissue.
Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system.
After treatment with the negative control the absorbance values reached the required acceptability criterion of a mean optical density (OD) > 1.2 and < 2.5 for the treatment interval thus showing the quality of the tissues.
The tissue viability after treatment with the test item was higher than 50 % (mean viability: 87.7 %). Therefore, the test item is not considered to possess an irritant potential.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Nov 29, 2012 - Apr 08, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
An in vivo study was conducted for a non-EU country.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: about 14-15 weeks
- Weight at study initiation: 2,72 kg (mean)
- Housing: separately in special rabbit cage (manufacturer: Becker; type K99/30 KU, floor area about 5400 cm2, a shelter with an integrated sitting board of about 1820 cm2; overall height: 60 cm)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: more than 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 19 °C
- Air changes (per hr): --
- Photoperiod (hrs dark / hrs light): 12/12 hours

IN-LIFE DATES: From: day 1 To: day 8
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
water
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g
- Concentration (if solution): diluted with some droplets of aqua pro injectione

Duration of treatment / exposure:
4 h
Observation period:
The first examination of the treated skin sites followed 1 hour after removal of the patch. Thereafter, examinations were performed after 24, 48, and 72 hours and then daily up to experimental day 8.
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: 6 cm2
- % coverage: 100
- Type of wrap if used: self-adhesive fabric mull

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no
- Time after start of exposure: 4 h

SCORING SYSTEM: Skin changes at the application sites were evaluated according to the DRAIZE-, OECD-and EEC recommendations.
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
The initial test with one animal and the confirmatory test with two further animals showed no signs of irritation after treatment with the test item at all timepoints investigated.
Other effects:
- Other adverse local effects: none
- Other adverse systemic effects: All animals survived the observation period. The body weight gain of the treated rabbits was inconspicous.
Interpretation of results:
GHS criteria not met
Conclusions:
No skin irritating potential could be detected. According to CLP, the test material should not be classified as a skin irritant.
Executive summary:

The study was performed similar to the OECD Guideline for Testing of Chemicals, No. 404 and the recommendations of DRAIZE (1959).
To test for primary skin irritation, 0.5 g of the test material was mixed with some drops of Aqua pro injectione to ensure good contact with the skin. Afterwards the test material was spread onto 6 cm2 patches and applied to the intact skin of previously shaven rabbits for a 4 hours period under occlusive conditions. The study was started as an initial test with one animal and followed by the confirmatory test with two further animals. The first examination of the treated skin sites followed 1 hour after removal of the patches. Thereafter, examinations were performed daily for further 7 days.

Under the conditions of the present study no signs of irritation were seen.

Evaluation of all animals (mean score)

Day (after treatment)
1
(1 hour)
2
(24 hours)
3
(48 hours)
4
(72 hours)
Erythema 0 0 0 0
Edema 0 0 0 0

No skin irritating potential could be detected. According to CLP, the test material should not be classified as a skin irritant.

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:
weight of evidence
Study period:
September 24, 2012 - November 6, 2012
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 Ocular Corrosives and Severe Irritants)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) No. 1152/2010 amending, for the purpose of its adaption to technical progress, Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
PREPARATION OF THE TEST MATERIAL
The test item was prepared as a 20% (w/v) suspension in a 0.9% sodium chloride solution. The stability in the vehicle was not investigated. The test item preparation was administered within <1 hour after preparation.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL: 750 µL (i.e. 150mg/750µL)

NEGATIVE / VEHICLE CONTROL: 750 µL

Designation: 0.9% sodium chloride solution
Supplier: B. Braun Melsungen AG
Batch: 11461014
Released until: October 2014



POSITIVE CONTROL: 750 µL

Designation: Art. 814223 (Imidazole)
Supplier: Merck KGaA
Batch: S5419523
Released until: August 31, 2014

Imidazole was dissolved with 0.9% sodium chloride solution to a concentration of 20% (w/v)
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
in vitro: triplicate design
Irritation parameter:
in vitro irritation score
Run / experiment:
Run 1 / Experiment 1
Value:
4.2
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.0 and, thus, within two standard deviations of the current historical mean of the negative control (IVIS: -0.7 – 3.9). After treatment with the positive control (20% Imidazole) the calculated IVIS was 87.3 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS 78.9 – 143.0). Therefore, the study fulfilled the validity criteria.

Opacity
Permeability
IVIS
per cornea
per group
(mean value)
SD
Negative control
0.9% NaCl Solution
0.388
0.009
0.528
1.0
0.5
1.479
0.003
1.524
0.865
0.002
0.900
Positive control
20% Imidazole solution
48.736
3.032
94.223
87.3
6.4
59.846
1.445
81.518
63.016
1.549
86.247
Test item
Test Item
4.629
0.007
4.740
4.2
0.6
3.500
0.006
3.597
4.154
0.006
4.250


Interpretation of results:
study cannot be used for classification
Conclusions:
The IVIS obtained after treatment with the test item was 4.2. According to the OECD Guideline 437, no prediction can be made if the IVIS score is > 3 <= 55.
Executive summary:

This in vitro study was performed to assess the ocular corrosive or severe irritant potential by means of the BCOP (Bovine Corneal Opacity and Permeability Assay). Therefore, the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test material as a 20% suspension in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% Imidazole was used.

Three corneas were used per group (test material, negative and positive control group).

After a first opacity measurement of the fresh bovine corneas, 750 µL of the suspended test material, positive or negative control were applied on the corneas and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase the test material, the positive, and the negative control were rinsed from the corneas and the opacity was measured again.

After the opacity measurements, the permeability of the corneas was determined by application of 1 mL of a fluorescein solution for 90 minutes at 32 ± 1 °C. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically.

The opacity and permeability assessments were combined to determine an in vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.0 and, thus, within two standard deviations of the current historical mean of the negative control (IVIS: -0.7 – 3.9). After treatment with the positive control (20% Imidazole) the calculated IVIS was 87.3 and, thus, also within two standard deviations of the current historical mean of the negative control (IVIS: 78.9 – 143.0). Therefore, the study fulfilled the validity criteria.

The IVIS obtained after treatment with the test item was 4.2 and, thus, lower than 55.1. According to the OECD Guideline 437, no prediction can be made if the IVIS score is > 3 <= 55.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
March 13-27, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
An in vivo study was conducted for a non-EU country.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld
- Age at study initiation: 28-29 weeks
- Weight at study initiation: 4.38 kg
- Housing: separately in special rabbit cages (manufacturer: Becker; type K99/30 KU)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 20 °C
- Humidity (%): 44 to 59 %.
- Air changes (per hr): --
- Photoperiod (hrs dark / hrs light): 12 / 12 hours

IN-LIFE DATES: From: day 1 To: day 8
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Duration of treatment / exposure:
After instillation, the eyelids were closed for about 1 second.
Observation period (in vivo):
24, 48, and 72 hours, then daily up to day 8 of the experimental part
Number of animals or in vitro replicates:
3 female rabbits
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): no
- Time after start of exposure: --

SCORING SYSTEM: OECD and EEC recommendation

TOOL USED TO ASSESS SCORE: hand-slit lamp / fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
Two animals showed redness of the conjunctivae (score 1) 1 and 6 hours after instillation of the test material.
Other effects:
Lesions and clinical observations: All animals survived the observation period. 1/3 animals showed diarrhea on day 1 and 2, which is considered to be associated to the treatment with the analgetic Buprenorphinhydrochloride and Meloxicam.
Interpretation of results:
GHS criteria not met
Conclusions:
No eye irritating potential could be detected. According to the Classification and Labelling the test material should not be classified as an eye irritant.
Executive summary:

The test material was studied for its eye irritating properties. The test for primary eye irritation was performed initially with one animal and followed by the confirmatory test with two further animals.
For the test of primary eye irritation, the test material was applied into the conjunctival sac of rabbits. The first examination of the eyes followed 1 hour after instillation. The other examinations of the eyes were performed daily for a further 7 days. The GLP study was performed according to OECD TG 405.
No signs of irritation were observed at the cornea or iris.
Overall, the following scores were observed at the timepoints as indicated for each animal:

Animal No Mean score
(24, 48, 72 h)
Maximum value
(24, 48, 72 h)
1 2 3 1 2 3
Cornea  A 0 0 0 0 0 0
Iris  A 0 0 0 0 0 0
Conjunctivae  A 0 0 0 0 0 0
Conjunctivae  B 0 0 0 0 0 0

No eye irritating potential could be detected. According to the Classification and Labelling the test material should not be classified as an eye irritant.


Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

The potential for skin irritation of the test item was investigated in a study according to OECD Guideline 439 as well as in a study according to OECD Guideline 404.

The tissue viability after treatment with the test item was higher than 50 % (mean viability: 87.7 %) in the OECD Guideline study 439. Additionally, no signs of skin irritation were observed in the in vivo skin irritation study according to OECD Guideline 404.

Eye irritation

The potential for eye irritation of the test item was investigated in a study according to OECD Guideline 437 as well as in a study according to OECD Guideline 405.

The IVIS after treatment with the test item was 4.2 and, thus, lower than 55.1 as determined in the OECD Guideline study 437. Based on the decision criteria of the OECD Guideline 437 no prediction can be made. However, no signs of eye irritation/corrosion were observed in the in vivo eye irritation study according to OECD Guideline 405.

Furthermore, a HET-CAM study was performed indicating that the test item is not irritating to eye.

In conclusion, the test item is neither irritating to skin nor to eye.

Justification for classification or non-classification

Suitable and reliable data are available justifying the classification or non-classification of the test item. Based on the study results provided the test item is neither classified for skin irritatio/corrosion nor classified for eye irritation/corrosion according to the Regulation (EC) No 1272/2008.