Registration Dossier

Administrative data

Description of key information

Skin irritation (OECD 404): irritating
Eye irritation (OECD 437): not irritating

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:
13 - 20 Nov 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No information on purity was given. Observation period was only 7 days.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
1981
Deviations:
yes
Remarks:
no data on test substance purity given, post-observation time not long enough to confirm supposed reversibility
GLP compliance:
yes
Test material information:
Composition 1
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: 2.19-3.00 kg
- Housing: animals were housed individually in suspended metal cages
- Diet: Rabbit Diet (A.W. Tindall Limited, Holbeach, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 45-50
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
other: unchanged (100% test substance) and absolute ethanol (25, 5 and 1% test substance)
Controls:
other: Untreated sites of the same animal served as control.
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 mL
- Concentration: 1, 5, 25 and 100%
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:
3
Details on study design:
TEST SITE
- Area of exposure: area on the back (four suitable test sites were selected)
- Type of wrap: The test substance was applied to the skin and covered with a patch which consisted of a 2.5 cm square of surgical gauze two layers thick. The loaded patch was held in contact with one of the selected test sites with two lengths of Sleek adhesive strapping (Smith & Nephew Limited) in the form of a cross. Additionally, the trunk 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:
mean
Remarks:
of 3 animals
Time point:
other: mean after 24/48/72 h
Score:
3
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Remarks on result:
other: 100% test substance
Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: mean after 24/48/72 h
Score:
2.7
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: 100% test substance
Irritation parameter:
edema score
Basis:
animal #2
Time point:
other: mean after 24/48/72 h
Score:
3.3
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: 100% test substance
Irritation parameter:
edema score
Basis:
animal #3
Time point:
other: mean after 24/48/72 h
Score:
4
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: 100% test substance
Irritant / corrosive response data:
100% test substance:
The test substance produced severe cutaneous irritation in all rabbits. Well-defined erythema with severe oedema extending beyond the treatment site was noted at all treated skin sites 1 h after removal of the patches. Moderate to severe erythema with slight to severe oedema extending beyond the test sites continued to be noted at all treated skin sites up to 72 h after treatment. Observation on Day 7 revealed very slight erythema in 2 animals. Hyperkeratinisation and desquamation was noted in all treated skin sites at this time.

25% test substance:
The test substance produced distinct cutaneous irritation in all rabbits. Well-defined erythema with slight to severe oedema extending beyond the test sites was noted at all treated skin sites 1 hour after removal of the patches. The erythema increased slightly at the 24-hour reading to show moderate to severe erythema at one treated site and well-defined erythema in a further two treated skin sites. The erythema continued and well-defined erythema was noted in 2/3 treated skin sites at the 72-hour observation; barely perceptible erythema was noted in the remaining test site. The oedema gradually regressed and very slight to slight oedema was noted in 2/3 treated skin sites at the 72-hour observation. Hyperkeratinisation was noted in 2/3 treated skin sites on Day.7. All test sites showed desquamation extending beyond the test site at this time.

5% test substance:
The test substance produced minimal cutaneous irritation in 3/3 rabbits. Barely perceptible erythema with minimal oedema was noted at all treated skin sites 1 h after removal of the patches. The erythema quickly regressed and all skin sites appeared normal at subsequent observations.

1% test substance:
The test substance produced minimal cutaneous irritation in 2/3 rabbits. Barely perceptible erythema with minimal oedema was noted at 2/3 treated skin sites 1 h after removal of the patches. The erythema quickly regressed and all skin sites appeared normal at subsequent observations.

Table 1. Results of the skin irritation study following 4 h exposure with undiluted test substance.

Observation time

Rabbit no.

1

2

3

Erythema

Edema

Erythema

Edema

Erythema

Edema

1 h

3

4

3

4

3

4

24 h

3

4

3

4

3

4

48 h

3

2

3

4

3

4

72 h

3

2

3

2

3

4

7 days

1HD

0

1HD

0

0HD

0

Mean value
24 + 48 + 72 h

3.0

2.7

3.0

3.3

3.0

4.0

H: hyperkeratinisation

D: desquamation

 

Table 2. Results of the skin irritation study following 4 h exposure with test substance at concentration of 25% (v/v) in absolute ethanol.

Observation time

Rabbit no.

1

2

3

Erythema

Edema

Erythema

Edema

Erythema

Edema

1 h

2

2

2

4

2

4

24 h

2

1

2

2

3

4

48 h

2

1

2

1

2

4

72 h

1

0

2

1

2

2

7 days

0D

0

0HD

0

0HD

0

Mean value
24 + 48 + 72 h

1.7

0.7

2.0

1.3

2.3

3.3

H: hyperkeratinisation

D: desquamation

Table 3. Results of the skin irritation study following 4 h exposure with test substance at concentration of 5% (v/v) in absolute ethanol.

Observation time

Rabbit no.

1

2

3

Erythema

Edema

Erythema

Edema

Erythema

Edema

1 h

1

0

1

1

1

0

24 h

0

0

0

0

0

0

48 h

0

0

0

0

0

0

72 h

0

0

0

0

0

0

7 days

0

0

0

0

0

0

Mean value
24 + 48 + 72 h

0

0

0

0

0

0

 

Table 4. Results of the skin irritation study following 4 h exposure with test substance at concentration of 1% (v/v) in absolute ethanol.

Observation time

Rabbit no.

1

2

3

Erythema

Edema

Erythema

Edema

Erythema

Edema

1 h

0

0

1

1

1

1

24 h

0

0

0

0

0

0

48 h

0

0

0

0

0

0

72 h

0

0

0

0

0

0

7 days

0

0

0

0

0

0

Mean value
24 + 48 + 72 h

0

0

0

0

0

0

Interpretation of results:
other: Skin Irrit. Cat. 2 at 100% test substance
Remarks:
according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of this skin irritation study in rabbits the test substance at concentration of 100% was irritating to the skin. Concentrations of 1, 5 and 25% of the test substance were considered not irritating to skin.
Executive summary:

A skin irritation study was performed according to OECD 404 and GLP. The primary irritation test in rabbits was performed at 1, 5, 25 and 100 % in Ethanol.

The mean values for 24-72 hours in 3 rabbits for 1% and 5% in Ethanol were all 0.0. The mean values in 3 rabbits for 25% in Ethanol were for erythema 1.7, 2.0 and 2.3, for edema 0.7, 1.3 and 3.3.

Mean values for the pure test item at 24-72 hours in 3 rabbits: Erythema 3.0, 3.0, 3.0; edema 2.67, 3.33, 4.0, not fully reversible after 7 days. The post-observation time was only 7days and was not long enough to confirm supposed reversibility.

Under the conditions of this skin irritation study in rabbits the test substance at concentration of 100% was irritating to the skin. Concentrations of 1, 5 and 25% of the test substance were considered not irritating to skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
18 Jan 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Test material information:
Composition 1
Species:
other: cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in Hank's Buffered Salt Solution (HBSS) at ambient temperature.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL

POSITIVE SUBSTANCE
- Amount applied in the test: 0.75 mL
- Purity: 99%

NEGATIVE CONTROL
- Amount applied: 0.75 mL
Duration of treatment / exposure:
10 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
triplicates for each treatment and control group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
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.

QUALITY CHECK OF THE ISOLATED 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.

TREATMENT METHOD:
The cornea holder consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. 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 substance 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.

REMOVAL OF TEST SUBSTANCE:
The test substance was rinsed off from the application side with saline.

- POST-EXPOSURE INCUBATION: 2 h in a vertical position

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (OP_KiT opacitometer, Electro Design, France).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microplate reader (Versamax Molecular Devices) at 490 nm.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55 no prediction can be made.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean value of 3 corneae
Value:
0.03
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
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 fullfilling the criteria as severe irritating/corrosive.
Relative to the negative control, the test substance did not cause an increase of the corneal opacity or permeability.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control resulted in an IVIS which was within two standard deviations of the current historical mean.
- Acceptance criteria met for positive control: The positive control resulted in opacity and permeability values that were less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Table 2. 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

0

0.00

0.061

0.064

0.92

0.96

0

0.068

1.02

0

0.063

0.95

Positive

control

63*

1.420*

79.10

79.95

55*

1.376*

59.08

59*

1.393*

76.89

Test substance

0*

0.023*

0.35

0.03

-1*

0.026*

-0.61

0*

0.023*

0.35

*: corrected values

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the BCOP assay the test substance was not irritating to the eye. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.03.
Executive summary:

A Bovine Corneal Opacity and Permeability (BCOP) test was performed according to OECD 437.

The in vitro irritancy score (IVIS) was 0.03 (threshold for classification ≥55.1 or ≤ 3). The test item did not cause an increase of the corneal opacity or permeability and is therefore not 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

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). In this study the test substance at concentration of 100% was irritating to the skin. Concentrations of 1, 5 and 25% of the test substance were considered not irritating to skin.

 

Eye

The eye irritation potential of the test substance was determined in a bovine corneal opacity and permeability (BCOP) test according to OECD Guideline 437 and in compliance with GLP (2016). In this study the test substance was not irritating to the eye.

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.