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Description of key information

Skin irritation: moderate irritant (OECD 404, GLP study)

Eye irritation: irritating (OECD 405, GLP study)

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:
2 June 1993 to 23 June 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study.
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
EC B.4
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Broekman Institute, Someren, The Netherlands.
- Age at study initiation: Approx. 12-13 weeks
- Weight at study initiation: 2385-2591 grams
- Housing: Individually in labelled cages with perforated floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands).
- Diet (e.g. ad libitum): Standard laboratory rabbit diet (LKK—20, pellet diameter 4mm, Hope Farms, Woerden, The Netherlands) approx. 100 gram per day.
- Water (e.g. ad libitum): Free access to tap-water diluted with decalcified water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): relative humidity of 55%
- Air changes (per hr): Air-conditioned with 15 air changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was 12 hours artificial fluorescent light and 12 hours dark per day

Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Remarks:
None
Amount / concentration applied:
0.5 gram
Duration of treatment / exposure:
4 hours
Observation period:
- Skin reactions: assessed at approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days afterthe removal of the dressings and test substance.
- Viability/mortality/toxicity- assessed twice daily throughout duration of study.
- Bodyweight was recoreded on day of treatment (prior to application)
Number of animals:
3 male rabbits
Details on study design:
TEST SITE:
Approximately 24 hours before treatnent, the dorsal fur was shaved with electric clippers, exposing an area of approximately 150 square centimeters (10x15 cm2). Prior to test substance administration, the animals were physically examined and the shaved area of skin to be treated wasnormal. On test day 1, 0.5 gram of the test substance was applied to the intact skin of the shaved area on one flank, using a Scotchpak-non-woven patch (2x3 cm, 3M, St.Paul, U.S.A.) mounted on Micropore tape (3M, St. Paul, U.S.A.). The contralateral flank was similarly prepared (but without test substance) to act as a procedural control. The dressing was wrapped around the abdomen and secured with an elastic bandage (Coban, 3M, St. Paul, U.S.A.).

REMOVAL OF TEST SUBSTANCE:
Four hours after the application of the test substance, the dressing was removed and the remaining test substance removed using a tissue moistened with tap-water and subsequently a dry tissue.

SCORING SYSTEM:
ERYTHEMA AND ESCHAR FORMATION
No erythema - 0
Very slight erythema (barely perceptible) - 1
Well defined erythema - 2
Moderate to severe erythema - 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) - 4
OEDEMA FORMATION
No oedema - 0
Very slight oedema (barely perceptible) - 1
Slight oedema (edges of area well defined by definite raising) - 2
Moderate oedema (raised approximately 1 rum) - 3
Severe oedema (raised more than 1 mm and extending beyond area of exposure) - 4
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
other: overall at 24, 48 and 72 h
Score:
2
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
other: overall at 24, 48 and 72 h
Score:
1.7
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
other: overall at 24, 48 and 72 h
Score:
1.7
Irritation parameter:
erythema score
Max. score:
4
Remarks on result:
other: Max. duration: d; Max. value at end of observation period: 1 (related to all animals)
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
other: overall at 24, 48 and 72 h
Score:
2.3
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
other: overall at 24, 48 and 72 h
Score:
3
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
other: overall at 24, 48 and 72 h
Score:
3
Irritation parameter:
edema score
Max. score:
3
Remarks on result:
other: Max. duration: d; Max. value at end of observation period: 1 (related to all animals)
Irritant / corrosive response data:
Reversibility of any observed effect: Changes not fully reversible within 21 days
Other effects:
Erythema was difficult to score, due to brown staining of the treated skin in all animals, caused by the test substance

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred
Interpretation of results:
moderately irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance resulted in a primary irritation index of 4.5 (moderately irritating) when applied to the intact rabbit skin.
Executive summary:

In a GLP compliant, guideline Acute Toxicity: Dermal Irritation/Corrosion test the test substance resulted in a primary irritation index of 4.5 (moderately irritating) when applied to the intact rabbit skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June 1993 to 30 June 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Broekman Institute, Someren, The Netherlands.
- Age at study initiation: Approx. 12 weeks
- Weight at study initiation: 2208 - 2468 grams
- Housing: Individually in labelled cages with perforated floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands).
- Diet (e.g. ad libitum): Standard laboratory rabbit diet (LKK-20, pellet diameter 4 mm, Hope Farms, Woerden, The Netherlands) approx. 100 g per day.
- Water (e.g. ad libitum): Free access to tap-water diluted with decalcified water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): relative humidity of 55%
- Air changes (per hr): Air-conditioned with 15 air changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was 12 hours artificial fluorescent light and 12 hours dark per day

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
-amount applied: 0.1 ml
Duration of treatment / exposure:
Prior to test substance administration, the animals were physically examined and the eyes were found to be normal.

On test day 1, the test substance (0.1 ml) was instilled in the conjunctival sac of an eye of one animal after gently pulling the lower lid away from the
eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control.
Observation period (in vivo):
- Eye reactions: assessed at approximately 1, 24, 48 and 72 hours and 7 days after instillation of the test substance.
- Viability/mortality/toxicity- assessed twice daily throughout duration of study.
- Bodyweight was recorded on day of treatment (prior to instillation)
Number of animals or in vitro replicates:
3 male rabbits
Details on study design:
SCORING SYSTEM:
The following numerical scoring system was used to score the eye lesions:

CORNEA
Opacity: degree of density (area most dense taken for reading)
No ulceration or opacity - 0
Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible - 1
Easily discernible translucent area, details of iris slightly obscured - 2
Nacreous areas, no details of iris visible, size of pupil barely discernible - 3
Opaque cornea, iris not discernible through the opacity - 4
Area of cornea affected
No ulceration or opacity - 0
One quarter or less but not zero - 1
Greater than one quarter, but less than half - 2
Greater than half, but less than three quarters - 3
Greater than three quarters, up to whole area - 4

IRIS
Normal - 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any of these or any combination thereof, iris still reacting to light (sluggish reaction is positive) - 1
No reaction to light, haemorrhage, gross destruction (any or all of these) - 2

CONJUNCTIVAE
Redness (refers to palpebrae and sciera, excluding cornea and iris):
Blood vessels normal - 0
Some blood vessels definitely hyperaemic (injected) - 1
Diffuse, crimson colour, individual vessels not easily discernible - 2
Diffuse beefy red - 3

Chemosis: lids and/or nictitating membrane
No swelling - 0
Any swelling above normal (includes nictitating membrane) - 1
Obvious swelling with partial eversion of lids - 2
Swelling with lids about half closed - 3
Swelling with lids more than half closed - 4

Discharge:
No discharge - 0
Any amount different from normal (does not include small amounts observed in inner canthus of normal animals) - 1
Discharge with moistening of the lids and hairs, just adjacent to lids - 2
Discharge with moistening of the lids and hairs, in a considerable area around the eye - 3


Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
other: overall at 24, 48 and 72 h
Score:
1.7
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
other: overall at 24, 48 and 72 h
Score:
2.7
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
other: overall at 24, 48 and 72 h
Score:
2
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Max. score:
3
Remarks on result:
other: Max. duration: h; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
other: overall at 24, 48 and 72 h
Score:
1
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
other: overall at 24, 48 and 72 h
Score:
1
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
other: overall at 24, 48 and 72 h
Score:
1.3
Irritation parameter:
chemosis score
Max. score:
3
Remarks on result:
other: Max. duration: h; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
other: overall at 24, 48 and 72 h
Score:
0
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
other: overall at 24, 48 and 72 h
Score:
0
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
other: overall at 24, 48 and 72 h
Score:
0
Irritation parameter:
cornea opacity score
Max. score:
0
Remarks on result:
other: Max. duration: 0 h; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
other: overall at 24, 48 and 72 h
Score:
0
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
other: overall at 24, 48 and 72 h
Score:
0
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
other: overall at 24, 48 and 72 h
Score:
0
Irritation parameter:
iris score
Max. score:
1
Remarks on result:
other: Max. duration: 24 h; Max. value at end of observation period: 0 (related to all animals)
Irritant / corrosive response data:
IRRITATION:
Instillation of the test substance into one eye of each of the three male albino rabbits affected the iris in one animal and conjunctivae in all three
animals. The iridic irritation had resolved within 24 hours after instillation in one animal. The irritation of the conjunctivae had resolved within 14 days
in all animals, although lacrimation was still observed in one animal at the final observation.

Treatment of the eyes with 2% fluorescein, 24 and/or 72 hours after test substance instillation revealed no corneal epithelial damage in any of the
animals.

CORROSION:
There was no evidence of ocular corrosion.

REVERSIBILITY OF ANY OBSERVED EFFECTS:
Changes fully reversible within 7 days
Other effects:
No toxic symptoms were observed in the animals during the test period and no mortality occurred.

Brown staining of the outside of the eyelids and the fur on the head and paws of the animals, caused by the test substance, was observed during the study
Interpretation of results:
other: Category 2
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
At the time of testing the test substance was considered mildly irritating to the rabbit eye (Kay and Calandra interpretation of the calculated maximum Draize score: 11). However according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures the test substance is classifed as an eye irritant (Category 2) as a positive response was noted in 2 of the 3 tested animals (conjunctival redness ≥2).
Executive summary:

In a GLP compliant, guideline 'Acute Toxicity: Eye Irritation/Corrosion' test a positive response was noted in 2 of the 3 tested animals (conjunctival redness ≥2). Therefore the test substance is classified as an eye irritant (Category 2) according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H319: Causes serious eye irritation” are therefore required

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

A skin irritation study on the test substance to OECD protocol and to acceptable quality standards resulted in a primary irritation index of 4.5 (moderatley irritating) when appllied to the intact rabbit skin. Therefore the test substance is classified as an skin irritant (Category 2) according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H315: Causes skin irritation” are therefore required.

In a eye irritation test on the test substance to OECD protocol and to acceptable quality standards a positive response was noted in 2 of the 3 tested animals (conjunctival redness ≥2). Therefore the test substance is classified as an eye irritant (Category 2) according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H319: Causes serious eye irritation” are therefore required


Justification for selection of skin irritation / corrosion endpoint:
GLP compliant guideline study with a klimisch score of 1

Justification for selection of eye irritation endpoint:
GLP compliant guideline study with a klimisch score of 1

Justification for classification or non-classification

Skin irritation/corrosion:

In a GLP compliant, guideline Acute Toxicity: Dermal Irritation/Corrosion test the test substance resulted in a primary irritation index of 4.5 (moderately irritating) when applied to the intact rabbit skin.

Eye irritation:

In a GLP compliant, guideline 'Acute Toxicity: Eye Irritation/Corrosion' test a positive response was noted in 2 of the 3 tested animals (conjunctival redness ≥2). Therefore the test substance is classified as an eye irritant (Category 2) according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H319: Causes serious eye irritation” are therefore required