Registration Dossier

Administrative data

Description of key information

skin irritation (OECD 404): irritating
eye irritation (OECD 437): non-corrosive; (OECD 492): not irritating

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:
08 - 15 Oct 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No information on purity was given. The reversibility of effects was observed only up to 7 days after removal of the patches.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
1981
Deviations:
yes
Remarks:
no information on purity; 7-day observation period
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approx. 12 - 16 weeks
- Weight at study initiation: 2.14 - 2.76 kg
- Housing: individually in suspended metal cages
- Diet: Rabbit Diet (A.W. Tindall Limited, Holbeach, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 20
- Humidity (%): 60 - 68
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 mL
Duration of treatment / exposure:
4 h
Observation period:
7 days
Reading time points: 1, 24, 48 and 72 h and 7 days
Number of animals:
6
Details on study design:
TEST SITE
- Area of exposure: back
- Type of wrap: The treated skin was covered with a gauze patch (2.5 cm x 2.5 cm), which was held in place with two lengths of adhesive strapping (sleek) in the form of a cross. To prevent the animals from interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset (TUBIGRIP).

REMOVAL OF TEST SUBSTANCE
- Washing: The skin was cleaned of residual test substance by gentle swabbing with cotton wool soaked in ether.
- Time after start of exposure: 4 h

SCORING SYSTEM: Draize scoring system
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
other: mean after 24/48/72 h
Score:
2.33
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Remarks on result:
other: desquamation was observed on Day 7
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
other: mean after 24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Remarks on result:
other: desquamation was observed on Day 7
Irritation parameter:
erythema score
Basis:
animal: #3 and #6
Time point:
other: mean after 24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Remarks on result:
other: desquamation was observed on Day 7
Irritation parameter:
erythema score
Basis:
animal: #4 and #5
Time point:
other: mean after 24/48/72 h
Score:
2.67
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Remarks on result:
other: desquamation was observed on Day 7
Irritation parameter:
edema score
Basis:
animal #2
Time point:
other: mean after 24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
edema score
Basis:
animal: #1 and #6
Time point:
other: mean after 24/48/72 h
Score:
0.67
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal: #3, #4 and #5
Time point:
other: mean after 24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritant / corrosive response data:
All test sites showed barely perceptible or well defined erythema with or without minimal oedema 1 h after removal of the patches. Similar reactions were noted after 24 h. By the 48 h observation moderate erythema had developed at 3 test sites while barely perceptible or well defined erythema persisted at the remaining 3 test sites. Minimal oedema was also seen at 5 test sites at this time. Barely perceptible to moderate erythema with or without minimal oedema persisted at all test sites at the 72 h reading. A final observation at Day 7 showed a reduction in the level of reaction when barely perceptible erythema with desquamation was noted at all 6 test sites.

1. Result of the irritation study.

Observation time

Rabbit no.

1

2

3

4

5

6

Erythema

Edema

Erythema

Edema

Erythema

Edema

Erythema

Edema

Erythema

Edema

Erythema

Edema

1 h

2

1

1

0

1

0

2

1

1

1

2

1

24 h

2

1

1

0

2

1

2

1

2

1

2

1

48 h

3

1

1

0

2

1

3

1

3

1

2

1

72 h

2

0

1

0

2

1

3

1

3

1

2

0

7 days

1

0

1

0

1

1

1

1

1

1

1

0

Mean value

24 + 48 + 72 h

2.33

0.67

1.00

0.00

2.00

1.00

2.67

1.00

2.67

1.00

2.00

0.67

Interpretation of results:
other: Skin Irrit. Cat 2 according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the skin irritation study the test substance caused slight to moderate skin irritation (mean erythema values over 24, 48 and 72 h: 2.33, 1.0, 2.0, 2.67, 2.67, 2.0; mean edema values over 24, 48 and 72 h: 0.67, 0.0, 1.0, 1.0, 1.0, 0.67), which was not fully reversible after 7 days. Thus the test subtance is considered as Skin Irrit. Cat. 2 (H315) according Regulation EC (no) 1272/2008
Executive summary:

The skin irritation potential of the test substance was determined by an in vivo skin irritation test in rabbits according to OECD Guideline 404 and in compliance with GLP (1985). Treatment with test substance caused slight to moderate skin irritation (mean erythema values over 24, 48 and 72 h: 2.33, 1.0, 2.0, 2.67, 2.67, 2.0; mean edema values over 24, 48 and 72 h: 0.67, 0.0, 1.0, 1.0, 1.0, 0.67), which was not fully reversible after 7 days. Therefore, the test substance is considered to possess an irritant potential.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
8 - 11 Oct 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
no experimental 48 h reading performed, no data on purity given
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
no experimental 48 h reading performed, no data on purity given
Qualifier:
according to
Guideline:
other: Federal Register 1973, Volume 38, No. 187, 1500.41
Deviations:
not specified
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approx. 12 - 16 weeks
- Weight at study initiation: 2.14 - 2.76 kg
- Housing: individually in suspended metal cages
- Diet: Rabbit Diet (A.W. Tindall Limited, Holbeach, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 20
- Humidity (%): 60 - 68
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
occlusive
Preparation of test site:
other: clipped, the right side was abraded, the left site remained intact
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Applied volume: 0.5 mL
Duration of treatment / exposure:
24 h
Observation period:
72 h
Reading time points: approx. 24 and 72 h after test substance application
Number of animals:
6
Details on study design:
TEST SITE
- Area of exposure: back
- Type of wrap: The treated skin was covered with a surgical gauze patch (2.5 cm x 2.5 cm), which was held in place with two lengths of Sleek adhesive strapping in the form of a cross. In order to ensure completely occlusive conditions a length of thin rubber sheeting (10x35 cm) was wrapped around the torso of each rabbit and then secured by an elasticated corset (Tubigrip).

REMOVAL OF TEST SUBSTANCE
- Washing: The skin was cleaned of residual test substance by gentle swabbing with cotton wool soaked in ether.
- Time after start of exposure: 24 h

SCORING SYSTEM: Draize scoring system

Local reactions of animals with abraded skin were not taken into account for hazard assessment.
Irritation parameter:
erythema score
Basis:
animal: #1, #3 and #6
Time point:
other: mean after 24/48/72 h
Score:
2
Max. score:
4
Reversibility:
no data
Remarks on result:
other: no 48 h data are available; for calculation of mean scores, the 48 h values were assumed to be the same as those at either 24 h or 72 h, whichever is greater (worst case assumption)
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
other: mean after 24/48/72 h
Score:
1
Max. score:
4
Reversibility:
no data
Remarks on result:
other: no 48 h data are available; for calculation of mean scores, the 48 h values were assumed to be the same as those at either 24 h or 72 h, whichever is greater (worst case assumption)
Irritation parameter:
erythema score
Basis:
animal: #4 and #5
Time point:
other: mean after 24/48/72 h
Score:
2.67
Max. score:
4
Reversibility:
no data
Remarks on result:
other: no 48 h data are available; for calculation of mean scores, the 48 h values were assumed to be the same as those at either 24 h or 72 h, whichever is greater (worst case assumption)
Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: mean after 24/48/72 h
Score:
0.67
Max. score:
4
Reversibility:
no data
Remarks on result:
other: no 48 h data are available; for calculation of mean scores, the 48 h values were assumed to be the same as those at either 24 h or 72 h, whichever is greater (worst case assumption)
Irritation parameter:
edema score
Basis:
animal #2
Time point:
other: mean after 24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: no 48 h data are available; for calculation of mean scores, the 48 h values were assumed to be the same as those at either 24 h or 72 h, whichever is greater (worst case assumption)
Irritation parameter:
edema score
Basis:
animal: #3, #4 and #5
Time point:
other: mean after 24/48/72 h
Score:
1.67
Max. score:
4
Reversibility:
no data
Remarks on result:
other: no 48 h data are available; for calculation of mean scores, the 48 h values were assumed to be the same as those at either 24 h or 72 h, whichever is greater (worst case assumption)
Irritation parameter:
edema score
Basis:
animal #6
Time point:
other: mean after 24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
no data
Remarks on result:
other: no 48 h data are available; for calculation of mean scores, the 48 h values were assumed to be the same as those at either 24 h or 72 h, whichever is greater (worst case assumption)
Irritant / corrosive response data:
Extension of the skin reactions beyond the site of application was noted at the intact and abraded sites of all 6 rabbits at the 24 and 72 h readings.
Well defined erythema with minimal or slight oedema was seen at 5 intact and 5 abraded test sites at the 24 h reading. Barely perceptible erythema was noted at the remaining intact and abraded site at this time.
At the 72 h reading barely perceptible or well-defined erythema with or without minimal oedema persisted at 4 intact and 4 abraded test sites. Moderate erythema with minimal oedema had developed at the remaining 2 intact and 2 abraded test sites by this observation.
Desquamation was apparent at 1 abraded test site at the 24 h reading and at 2 intact and 1 abraded test sites at the 72 h reading.

Table 1. Results of skin irritation study (intact skin).

Observation time

Rabbit no.

1

2

3

4

5

6

Erythema

Edema

Erythema

Edema

Erythema

Edema

Erythema

Edema

Erythema

Edema

Erythema

Edema

24 h

2

1

1

0

2

2

2

2

2

2

2

2

48 h

No experimental data available; for calculation of mean scores, the 48 h values were assumed to be the same as those at 48 h (worst case assumption)

72 h

2

0

1

0

2

1

3

1

3

1

2

0

 

Table 2. Calculation of mean scores (intact skin).

 

Rabbit no.

1

2

3

4

5

6

Erythema

Edema

Erythema

Edema

Erythema

Edema

Erythema

Edema

Erythema

Edema

Erythema

Edema

Mean value

24 + 48* + 72 h*

2

0.67

1

0

2

1.67

2.67

1.67

2.67

1.67

2

1.33

*No 48 h data are available: for calculation of mean scores, the 48 h values were assumed to be the same as those at either 24 or 72 h, whichever is greater (worst case assumption).

Interpretation of results:
other: Skin Irrit. Cat 2 acording to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the skin irritation study the test substance induced moderate skin irritation, which was not fully reversible after 3 days. Thus, the test subtance is considered as Skin Irrit. Cat. 2 (H315) according Regulation EC (no) 1272/2008.
Executive summary:

In a supporting study, the skin irritation potential of the test substance was determined by an in vivo skin irritation test in rabbits according to a testing protocol similar to OECD Guideline 404 and in compliance with GLP (1985). Treatment with test substance caused moderate skin irritation, which was not fully reversible after 3 days. Therefore, the test substance is considered to possess an irritant potential.

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:
weight of evidence
Study period:
23 - 25 Sep 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
human
Strain:
other: EpiOcular™
Details on test animals or tissues and environmental conditions:
TEST MODEL (EpiOcular™ Kit)
- Source: MatTek Corporation, Ashland, USA
- Lot No.: 21572

TEST METHOD
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. Irritant materials are identified by their ability to damage the underlying cell layers which is determined through a decrease in cell viability as determined by MTT reduction.

ADAPTATION TO CELL CULTURE CONDITIONS
1.0 mL assay medium (37 °C) was aliquoted into 6-well plates. The inserts with EpiOcular™ tissues were transferred aseptically into the plates and pre-incubated at standard culture conditions for 1 h. Afterwards, the medium was replaced by 1 mL fresh assay medium and the EpiOcular™ tissues were incubated at standard culture conditions overnight (18 h). After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca²+ Mg²+ free DPBS. The tissues were incubated at standard culture conditions for 30 min.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1.5
- CO2 gas concentration (%): 5 ± 0.5
- Humidity (%): 95
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Applied volume: 50 µL

POSITIVE SUBSTANCE
- Substance: methyl acetate
- Applied volume: 50 µL

NEGATIVE CONTROL
- Substance: deionised water
- Applied volume: 50 µL
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
120 min
Number of animals or in vitro replicates:
The test was performed in duplicate for each treatment and control group.
Details on study design:
TEST SITE
- Area of exposure: 0.6 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the treatment time, the test substance was removed by extensively rinsing the tissues with Ca²+Mg²+ free DPBS in clean beakers.
- Post-treatment incubation period: 2 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, a volume of 300 µL MTT solution was added to each well for 3 h at standard culture conditions. After removal of the MTT solution, wells were rinsed three times with Ca²+Mg²+ free DPBS. Extraction of the formazan product was carried out in 2 mL isopropanol. At the end of the extraction period the optical density (OD) was measured.

EVALUATION OF RESULTS
1) The mean OD value of the blank control wells (ODBlk) for each experiment were calculated.
2) The ODBlkfrom each OD value of the same experiment (blank corrected values) were subtracted.
3) The mean value of the two aliquots for each tissue (= corrected test substance OD) were calculated.
4) The mean value of the two relating tissues for each control and test substance (= corrected mean OD) were calculated. For further calculations only the corrected mean negative control OD value was needed.
5) The corrected OD value of the negative control corresponds to 100% viability.
6) The percent viability of each of the two relating tissues for each control and test substance relative to the negative control (100% control) were calculated.
Viability (%) = [(corrected test substance OD) / (corrected mean negative control OD)] x 100

EVALUATION CRITERIA
A test substance leading to a tissue viabilty > 60% (relative to the negative control tissue viability) is considered as non-irritating.
A test substance leading to a tissue viabilty < 60% (relative to the negative control tissue viability) is considered as irritating.

ACCEPTABILITY OF THE ASSAY
1. The negative control OD is > 0.8 and < 2.5
2. The mean relative viability of the positive control is below 60% of the negative control viability.
3. The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test substance). This applies also to the killed controls (substance and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.
Irritation parameter:
other: cell viability (%)
Remarks:
mean values of 2 tissues
Run / experiment:
30 min exposure
Value:
100.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
The optical pre-experiment (colour interference pre-experiment) to investigate the test substance’s colour change potential in water or isopropanol did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test substance with MTT-reagent did not show blue colour.

ACCEPTANCE OF RESULTS:
All acceptance criteria were met.

Table 1. Results after 30 min incubation time

Test group

Absorbance*

Mean absorbance of 2 tissues*

Rel. absorbance (%)**

Absolute value of the difference of the rel. absorbance (%) Tissue 1 and 2

Rel. absorbance (% of negative control)**

Tissue 1

Tissue 2

Tissue 1

Tissue 2

Negative control

1.568

1.653

1.611

97.4

102.6

5.3

100.0

Positive control

0.107

0.114

0.110

6.6

7.1

0.5

6.9

Test substance

1.675

1.563

1.619

104.0

97.0

7.0

100.5

* Mean of two replicate wells after blank correction

** Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

Interpretation of results:
other: CLP/EU GHS criteria not met, no calssification according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the conducted test, the test substance did not exhibit irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.
Executive summary:

An in vitro eye irritation test using a human cornea model according to OECD 492 was conducted in compliance with GLP. Since the viability value of the test substance exposed tissues did not decrease below 60% (100.5%), the test substance is not considered to possess an eye irritating potential.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22 July 2015
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:
2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Odenwaldschlachthof Brensbach, Brensbach, Germany
- Donor animals: at least 9 month old
- Time interval prior to initiating testing: The corneae were isolated after delivery of the eyes and directly used in the BCOP test on the same day.
- Transport medium and temperature conditions: The isolated eyes were transported in Hank's Buffered Salt Solution (HBSS) at ambient temperature.

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation, pigmentation, opacity): yes
- Dissection of the eyes and treatment: The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each cornea was mounted in a specially designed cornea holder.
- Description of the cornea holder: The cornea holder consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively.
- Test medium used in the cornea holder: The incubation medium consisted of MEM, supplemented with 1.1 g/500 mL sodium bicarbonate, 5 mL/500 mL L-glutamine, 5 mL penicillin/streptomycin (500 U penicillin, and 500 µg streptomycin per 5 mL). Immediately before starting the test, MEM was supplemented with 1% fetal calf serum.
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: At the end of the equilibration period, the basal opacity was determined (t0). Each cornea with a value of the basal opacity >7 was discarded.

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission passing through the cornea via an opacitometer.
- Specification of the device: OP_KiT opacitometer (Electro Design, France)
Vehicle:
unchanged (no vehicle)
Controls:
other: number of eyes for the negative control: 3; number of eyes for the positive control: 3
Amount / concentration applied:
TEST MATERIAL
- Applied volume: 0.75 mL

POSITIVE SUBSTANCE
- Substance: 2-ethoxyethanol
- Applied volume: 0.75 mL
- Purity: 99%

NEGATIVE CONTROL
- 0.9% NaCl (w/v) solution in deionised water (saline)
Duration of treatment / exposure:
10 min at 32 ± 1 °C
Number of animals or in vitro replicates:
number of eyes for the test item: 3
Details on study design:
TEST CONDITIONS
- Short description of the method used: The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on the top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. The anterior compartment received the test item or the controls on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath for 10 minutes.

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test item was rinsed off from the application side with saline.

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Time of determination: After the test item was rinsed off from the application side, the corneae were incubated for further 2 h in a vertical position, followed by a second opacity reading (t130).
- Specification of the device: OP_KiT opacitometer, Electro Design, France

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: After the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by sodium fluorescein solution. Corneae were incubated again in horizontal position for 90 min in a water-bath at 32 ± 1 °C. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with a spectrophotometer as optical density at 490 nm (OD490).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (0.4% (w/v); dissolved in HBSS)
- Incubation time: 90 min at 32 ± 1 °C
- Treatment for measuring: Complete medium from the posterior compartment was removed, well mixed, transferred into a 96 well plate and OD490 was determined.
- Specification of the spectrophotometer: Versamax Molecular Devices
Irritation parameter:
in vitro irritation score
Remarks:
mean value of 3 tissues
Run / experiment:
10 min incubation
Value:
5.27
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.
The positive control (2-ethoxyethanol) showed clearly increased opacity and distinctive permeability of the corneae fulfilling the criteria for classification as severe irritating/corrosive.

Table 1: Results after 10 min incubation time.

Test group

Opacity value =
Difference (t130-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

 

Mean

 

Mean

 

 

Negative

control

1

0.33

0.061

0.069

1.92

1.37

0

0.070

1.05

0

0.076

1.14

Positive

control

69.67*

0.629*

79.10

71.69

41.67*

1.161*

59.08

59.67*

1.148*

76.89

Test substance

4.67*

0.108*

6.29

5.27

1.67*

0.195*

4.59

4.67*

0.017*

4.92

*corrected values

Interpretation of results:
other: non-corrosive according to Regulation (EC) No 1272/2008
Conclusions:
The eye damaging potential of the test susbtance was assessed in the Bovine Corneal Opacity and Permeability Test (BCOP) according to OECD 437. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 5.27. According to OECD Guideline 437 no prediction on the irritation potential can be made but the test substance does not have to be classified as serious eye irritant (Eye Dam. Cat. 1).
Executive summary:

The eye damaging potential of the test susbtance was assessed in a BCOP test according to OECD 437. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 5.27. According to OECD Guideline 437 no prediction on the irritation potential can be made, but the test substance does not have to be classified as serious eye irritant (Eye Dam. Cat. 1).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin

The skin irritation potential of the test substance was determined by an in vivo skin irritation test in rabbits according to OECD Guideline 404 and in compliance with GLP (1985). 6 New Zealand White rabbits were treated with 0.5 mL of the undiluted test substance under semi-occlusive dressing for 4 h. Treatment with test substance caused slight to moderate skin irritation (mean erythema values over 24, 48 and 72 h: 2.33, 1.0, 2.0, 2.67, 2.67, 2.0; mean edema values over 24, 48 and 72 h: 0.67, 0.0, 1.0, 1.0, 1.0, 0.67), which was not fully reversible after 7 days. Therefore, the test substance is considered to possess an irritant potential.

In a supporting study, the skin irritation potential of the test substance was determined by an in vivo skin irritation test in rabbits according to a testing protocol similar to OECD Guideline 404 and in compliance with GLP (1985). 6 New Zealand White rabbits were treated with 0.5 mL of the undiluted test substance under occlusive dressing for 4 h. Since no 48-h observation had been performed, for calculation of the scores, the 48-h scores were assumed to be the same as those at either 24 or 72 h, whichever was greater. Treatment with test substance caused moderate skin irritation (mean erythema values over 24, 48 and 72 h: 2.0, 1.0, 2.0, 2.67, 2.67 and 2.0; mean edema values over 24, 48 and 72 h: 0.67, 0.0, 1.67, 1.67, 1.67 and 1.33), which was not fully reversible after 3 days. Therefore, the test substance is considered to possess an irritant potential.

Eye

The eye irritation potential of the test substance was determined by a bovine corneal opacity and permeability (BCOP) test according to OECD Guideline 437 and in compliance with GLP (2015). Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 5.27 (threshold for serious eye damage: IVIS >55). Thus, no prediction on the irritation potential can be made but the test substance does not have to be classified as serious eye damage (Eye Dam. Cat. 1).

To exclude irritating properties towards the eyes, an in vitro eye irritation test using a human cornea model according to OECD Guideline 492 and in compliance with GLP was conducted (2015). Since the viability value of the test substance exposed tissues did not decrease below 60% (100.5%), the test substance is not considered to possess an eye irritating potential.

In conclusion, based on available results, the test substance is not considered to be irritant to the eyes.

Justification for classification or non-classification

The available data on skin irritation meet the criteria for classification as Skin Irrit. Cat. 2 (H315) according to Regulation (EC) 1272/2008.

The available data on eye irritation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.