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Administrative data

Description of key information

Skin irritation/corrosion (OECD 404): not 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:
11 - 15 Nov 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Five different test substance concentrations and the vehicle were tested on each animal.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
1992
Deviations:
yes
Remarks:
Five different test substance concentrations and the vehicle were tested on each animal.
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
29 Dec 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Danish Accreditation Fund (DANAK); National Board of Health, Medicines Division, Denmark
Species:
rabbit
Strain:
other: Albino Mol:Russian
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: MØllegaard Breeding and Research Centre, Lille Skensved, Denmark
- Weight at study initiation: 2.1 - 2.2 kg
- Housing: individually in PPO/HIPS (Noryl ®) cages (floor area: 2576 cm²) with perforated floor
- Diet: pelleted complete rabbit diet (Altromin 2123), ad libitum
- Water: domestic quality drinking water acidified with hydrochloric acid to pH 2.5, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 55 ±15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
other: ethanol 96% and diethyl phthalate (DEP) 1:1
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5 mL
- Concentrations: 1, 5, 10, 25 and 100%

VEHICLE
- Amount(s) applied: 0.5 mL
- Concentration: 50% ethanol (96%) and 50% diethyl phthalate (DEP)
Duration of treatment / exposure:
4 h
Observation period:
72 h
reading time points: 1, 24, 48 and 72 h
Number of animals:
4 females
Details on study design:
TEST SITE
- Area of exposure: 10 x 10 cm on the dorsal area of the trunk divided into six test sites: two anterior located test sites, two centrally located test sites and two posterior located test sites
- Type of wrap if used: The gauze patches were secured with 2.5 cm wide adhesive tape and fixed with Scanpor tape (5 cm width). Thereafter Nobifix (6 cm width) was loosely wound round the trunk.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After removal of the tape and patches, the treated skin was cleaned with lukewarm water.
- Time after start of exposure: 4 hrs

OBSERVATION TIME POINTS
reading time points: 1, 24, 48 and 72 h

SCORING SYSTEM:
- Method of calculation: according to the method described by Draize (Draize et al., 1944)
Irritation parameter:
erythema score
Basis:
animal: #1, #2 and #4
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: test substance concentrations: 1, 5, 10, 25 and 100%
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: test substance concentration: 100%
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Remarks on result:
other: test substance concentrations: 1 and 5%
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.67
Max. score:
4
Reversibility:
fully reversible within: 72 h
Remarks on result:
other: test substance concentrations: 10 and 25%
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: test substance concentrations: 1, 5, 10, 25 and 100%
Irritant / corrosive response data:
Very slight erythema was observed after 24 h in one rabbit at the test sites exposed to the vehicle and the test substance at concentrations of 1, 5, 10 and 25%, and in the same rabbit after 48 h at the test sites exposed to 10% and 25% test substance concentrations. All effects were fully reversible within 72 h after dosing. No effects occurred the test site exposed to 100%.

Table 1. Results of skin irritation study.

 

 

Erythema Score at hours

Edema Score

Rabbit no.

Test

substance concentration [%]

1

24

48

72

mean (24/48/72 h)

1

24

48

72

mean (24/48/72 h)

1

100

0

0

0

0

0

0

0

0

0

0

25

0

0

0

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

0

0

0

5

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

Vehicle

0

0

0

0

0

0

0

0

0

0

2

100

0

0

0

0

0

0

0

0

0

0

25

0

0

0

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

0

0

0

5

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

Vehicle

0

0

0

0

0

0

0

0

0

0

3

100

0

0

0

0

0

0

0

0

0

0

25

0

1

1

0

0.67

0

0

0

0

0

10

0

1

1

0

0.67

0

0

0

0

0

5

0

1

1

0

0.33

0

0

0

0

0

1

0

1

0

0

0.33

0

0

0

0

0

Vehicle

0

1

0

0

0.33

0

0

0

0

0

4

100

0

0

0

0

0

0

0

0

0

0

25

0

0

0

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

0

0

0

5

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

Vehicle

0

0

0

0

0

0

0

0

0

0

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the Acute Dermal Irritation/Corrosion Test in rabbits the test substance does not possess any skin irritating potential.
Executive summary:

The primary skin irritant effect was investigated according to OECD Guideline 404, "Acute Dermal Irritation/Corrosion", 1992 and GLP. The study was extended to investigate five different concentrations of the test article and a vehicle control on

each animal. Four rabbits were exposed to different concentrations of the test article at six skin sites. After 4 hours of exposure the test article was removed and the skin was examined 1, 24, 48 and 72 hours after termination of exposure. Under the experimental conditions described in this report, the mean score for erythema and oedema in the four rabbits used were as follows: 0.0, 0.2, 0.2, 0.1, 0.1, 0.1 (100%, 25%, 10%, 5%, 1% and vehicle) and 0.0, 0.0, 0.0, 0.0, 0.0, 0.0 (100%, 25%, 10%, 5%, 1% and vehicle), respectively.

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:
13 Mar 2017
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)
Version / remarks:
(July, 2013)
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
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
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 stored in Hank's Buffered Salt Solution (HBSS) containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes.
- 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.
- Indication of any antibiotics used: yes, 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in transport medium
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: 20% (w/v) in 0.9% NaCl in deionised water (saline)

POSITIVE CONTROL
- Amount applied: 0.75 mL
- Concentration: 10% (w/v) benzalkonium in 0.9% NaCl in deionised water (saline)

NEGATIVE CONTROL
- Amount applied: 0.75 mL
- Concentration: 0.9% NaCl in deionised water (saline)
Duration of treatment / exposure:
240 min at 32 ± 1 °C
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. After equilibration for about 1 hour, 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 240 minutes.

NUMBER OF REPLICATES: 3 corneae per test group

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

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 (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS), IVIS = opacity value + (15x OD490 value)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as serious eye damage and labelled Category 1 according to CLP/GHS.
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
Remarks:
mean value of 3 corneae
Run / experiment:
240 min
Value:
0.16
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.28). The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 116.83) corresponding to a classification as serious eye damaging (CLP/GHS (Cat 1)).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative 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.
- Acceptance criteria met for postive control: The positive control resulted in an IVIS which was within two standard deviations of the current historical mean.

Table 2. Results after 240 min incubation.

Test group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

 

 

Mean

 

Mean

Negative Control

0

0.33

0.048

0.063

0.72

1.28

1

0.068

2.02

0

0.073

1.10

Positive Control

112.67*

0.080*

113.87

116.83

117.67*

0.114*

119.38

115.67*

0.106*

117.26

Test substance

-1.33*

0.003*

-1.29

0.16

0.67*

0.040*

1.27

-0.33*

0.056*

0.51

*corrected values

Interpretation of results:
GHS criteria not met
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 0.16.
Executive summary:

BCOP assay was performed according to OECD 437 and GLP. After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item, the positive and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 0.16. No classification is required according to Regulation (EC) 1272/2008.

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 (1996). 4 rabbits were each treated with the test substance at concentrations of 1, 5, 10, 25% diluted in DEP/ethanol (1:1) and 100% under semiocclusive dressing for 4 h. The vehicle was applied as control. Treatment with the vehicle and test substance at concentrations of 1, 5, 10 and 25% caused slight skin irritation in one animal (mean erythema values over 24, 48 and 72 h: 0.33, 0.33, 0.67, 0.67, 0.67 and 0.00; edema score: 0.0), which was fully reversible within 72 h. The other three animals did not show any signs of irritation (erythema and edema scores over 24, 48 and 72 h: 0.00).Therefore, the test subsatance is not considered to be irritating to the skin.

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 (2017). Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.16. Thus, the test substance is not considered to be irritant to the eye.

Justification for classification or non-classification

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