Registration Dossier

Administrative data

Description of key information

Not skin irritating

Eye irrit, 2 (H319), according to the CLP regulation (EC) 1272/2008

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:
October from 05 to 12, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
adopted May 12th, 1981
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd.
- Age at study initiation: 15 weeks males, 16 weeks females.
- Weight at study initiation: 2.6 kg males, 2.8 kg females.
- Housing: individually in stainless steel cages equipped with an automatic cleaning and drinking system.
- Diet: pelleted standard Kliba 341, ad libitum.
- Water: community tap water from Itingen, ad libitum.
- Acclimation: four days under test conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C.
- Humidity: 40 - 70 %.
- Air changes: 10-15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent light/12 hours dark.
- Other: music during the light period, air-conditioned room.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Preparation: the test article was applied moistened with bi-distilled water.
- Amount applied: 0.5 g.
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
2 males and 1 female.
Details on study design:
TEST SITE
- Area of exposure: approximately 24 hours before treatment, the dorsal fur was clipped with an electric clipper, exposing an area of approximately 100 square centimeters (10 cm x 10 cm). The skin of the animals was examined once, 1 day prior to test article administration. Only those animals with no signs of skin injury or irritation were used in the test. On test day the test article was applied to approx. 6 cm2 of the intact skin of the clipped area.
- Type of wrap if used: treated area was covered with a 3.0 cm x 3.0 cm patch of surgical gauze. The gauze was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with an elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: the skin was flushed with lukewarm tap water.
- Time after start of exposure: 4 hours.

OBSERVATIONS
- Viability/Mortality and clinical signs: daily.
- Body Weights: at the start of acclimatization, day 1 of test (application day) and at termination of observation.

SCORING SYSTEM
The skin reaction was assessed at 1, 24, 48 and 72 hours after the removal of the dressing, gauze patch and test article. The corrosive properties of the test article and the staining of the treated skin area related to the test article were described and recorded.
The dermal irritation scores were evaluated in conjunction with the nature and reversibility or otherwise of the responses observed. The individual scores do not represent an absolute standard for the irritant properties of a material, and they are viewed as reference values.

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
Maximum possible score 4

EDEMA FORMATION
No edema................................................................................................................ 0
Very slight edema (barely perceptible).................................................................. 1
Slight edema (edges of area well defined by definite raising)................................ 2
Moderate edema (raised approximately 1 mm)........................................................ 3
Severe edema (raised more than 1 mm and extending beyond area of exposure).4
Maximum possible score 4
Maximum cumulative score: 8
Irritation parameter:
erythema score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 2.3
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 2.3
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Test item showed a primary irritation score of 0.00 when applied to intact rabbit skin. Local signs (mean values from 1 hour to 72 hours) consisted of grade 0.00 erythema and grade 0.00 edema.
A black coloration on the back was observed in all animals from 1 to 72 hours.
No destructions or irreversible alterations of the treated skin were observed; no corrosive effect was evident on the skin.
Other effects:
CLINICAL SIGNS AND VIABILITY / MORTALITY
No acute clinical symptoms were observed in the animals during the test period, and no mortality occurred. Body weight gain of all rabbits was similar. A black coloration on the back was observed in all animals from 1 to 72 hours.

The skin irritation scores for each animal are presented below.

Time period Animal Sex Erythema Edema Cumulative score Mean cumulative score
1 hr 69 M 0 0 0 0.00
70 M 0 0 0
71 F 0 0 0
24 hrs 69 M 0 0 0 0.00
70 M 0 0 0
71 F 0 0 0
48 hrs 69 M 0 0 0 0.00
70 M 0 0 0
71 F 0 0 0
72 hrs 69 M 0 0 0 0.00
70 M 0 0 0
71 F 0 0 0

The mean scores for skin irritation as a mean of 24, 48 and 72 hours are presented below.

Animal Sex Erythema Edema Cumulative
 score
Mean Cumul.
Score (24/48/72)
69 M 0.00 0.00 0.00 0.00
70 M 0.00 0.00 0.00
71 F 0.00 0.00 0.00
Interpretation of results:
other: not classified, according to the CLP Regulation (EC) No 1272/2008
Conclusions:
The substance is not a skin irritant.
Executive summary:

The irritancy potential of the test material to the skin of New Zeland white rabbit was assessed, according to the OECD Guideline 404. The intact skin of the rabbits was exposed for 4 hours to the substance, in a semi-occlusive dressing. Animals were examined for signs of erythema and edema and the responses were scored at 1 hour following the patch and substance removal/washing, and then 24, 48 and 72 hours later.

No acute clinical symptoms were observed and no mortality occurred. A black coloration on the back was observed in all animals from 1 to 72 hours, but no signs of irritations were observed. Test item showed a primary irritation score of 0.00 when applied to intact rabbit skin. Local signs (mean values from 1 hour to 72 hours) consisted of grade 0.00 erythema and grade 0.00 edema.

Conclusion

The substance is not 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 vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
November from 15 to 22, 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: readings was difficult and in some cases not feasible, due to substance staining
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
adopted February 24th, 1987
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: PI PETIT (El Masnou, Barcelona, Spain).
- Age at study initiation: approximately 14 weeks.
- Weight at study initiation: 2.3 - 2.5 kg.
- Housing: housed individually in stainless steel cages (48 x 59 x 37 cm), with a grille floor, placed on racks.
- Diet: standard rabbit diet UAR 112, ad libitum.
- Water: an automatic drinking system, ad libitum.
- Acclimation period: at least 7 days.
- Health check: both eyes of each animal were examined before treatment in order to reject any animal showing eye alterations.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 ± 2 °C, sometimes reaching 16 and 22 °C.
- Humidity: 40 - 70 %, sometimes reaching levels of 28 - 39 % and 75 - 80 %.
- Photoperiod: 12 hour light/12 hour dark.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
- Amount applied: 0.1 ml
- Application: the test substance was instilled, undiluted, in a single application.
Duration of treatment / exposure:
24 hours
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
3 males
Details on study design:
APPLICATION OF TEST MATERIAL
The animal was immobilized and the product was poured into the right conjunctival sac, made perfectly accessible by care fully pulling out the lower lid. After application of the test substance, the lids of the treated eye were held closed for approximately one second.

REMOVAL OF TEST SUBSTANCE
- Washing: the treated eye was washed with 0.9 % w/v sodium chloride solution, after Colirusi Fluoresceina application.
- Time after start of exposure: 24 hours.

TOOL USED TO ASSESS SCORE
Followving the reading carried out 24 hours after treatment, Colirusi Fluoresceina was applied to the treated eye of each animal.

SCORING SYSTEM
The degree of eye irritation was evaluated for each rabbit 1, 24, 48 and 72 hours after treatment, following the guideline.
All the measures carried out at each 24, 48 and 7 hour examination for each type of lesion are used respective mean values.

- Corneal Irritation
Opacity: degree of density (densest area used for assessment)
No ulceration or opacity............................................................................................................................................... 0
Scattered or diffuse areas of opacity (apart from slight loss of normal luster), details of iris clearly visible............. 1
Easily discernible translucent area, details of iris slightly blurred................................................................................2
Opalescent area, details of iris not visible, countour of pupil barely discernible.........................................................3
Opaque cornea, iris not discernible through the opacity............................................................................................. 4

- The area of corneal opacity was recorded according to the following scale:
A quarter or less, but not zero ......................1
More than a quarter, less than half................2
More than half, less than three quarters........3
More than three quarters, up to whole area...4

- Iridic Irritation
Normal............................................................................................................................................................................0
Deeply marked folds, congestion, swelling, moderate hyperemia around corena or injection, one or more
of the aforementioned, iris conserving reaction to light................................................................................................ 1
No reaction to light, hemorrhage, serious damage (any or all of the above)................................................................ 2

- Conjuctival Irritation
Redness (refers to palpebral and bulbar conjunctivae, cornea and iris).
Normal blood vessels................................................................................................................................................... 0
Some clearly hyperemic blood vessels (injected)........................................................................................................ 1
Diffuse, crimson color, individual vessels difficult to discern...................................................................................... 2
Diffuse deep red........................................................................................................................................................... 3

- Edema: lids and/or nictitating membranes
No swelling.................................................................................................................................................................... 0
Some swelling above normal (includes nictitating membranes).....................................................................................1
Obvious swelling with partial eversion of lids............................................................................................................... 2
Swelling wi th lids about half closed............................................................................................................................. 3
Swelling with lids more than half closed....................................................................................................................... 4
Irritation parameter:
cornea opacity score
Basis:
animal: 2/3
Time point:
24/48/72 h
Score:
1
Reversibility:
not fully reversible within: 7 days, in one animal
Irritation parameter:
iris score
Basis:
animal: 3/3
Time point:
24/48/72 h
Remarks on result:
not determinable
Remarks:
due to the coloring from test substance
Irritation parameter:
conjunctivae score
Basis:
animal: 1/3
Time point:
24/48/72 h
Score:
2.33
Reversibility:
not fully reversible within: 7 days
Remarks on result:
not determinable
Remarks:
in 2 out of 3 animals
Irritation parameter:
chemosis score
Basis:
animal: 2/3
Time point:
24/48/72 h
Score:
< 2
Reversibility:
not fully reversible within: 7 days, in one animal
Remarks on result:
other: in the 3rd animal reaction were not recorded within 7 days
Irritant / corrosive response data:
In the course of the first 24 hours after administration, all the animals showed edernatic lesions ranging from swelling of the lids (grade 1) to more than half closed lids (grade 4).
Discharge was also observed ranging from light to moistening of the lids and surrounding hair, affecting a considerable area around the eye and injected vessels in the bulbar conjunctivae.
24 hours following administration, all the animals showed diffuse opacity of the cornea (grade 1) ranging from an affected area of one quarter (grade 1) to near totality of the cornea (grade 4). Due to the colouring produced by the test substance in the eyes of the animals, diffused crimson vessels (grade 3) were observed in the palpebral conjunctivae of only one animal and none of the animal's irises could be clearly observed .
2 , 3 and 4 days following administration, observed ocular injuries were seen to be subsiding.
7 days post-administration, one animal showed vessels of the palpebral conjunctivae clearly more injected than normal (grade 1). Also, another animal showed nearly half closed, swollen lids (grade 3) and diffuse opacity of the cornea (grade 1) which occupied less than one quarter of the total area (grade 1).
Other effects:
CLINICAL SIGNS
The behaviour and physical condition of the rabbits were normal throughout the Study.

The eye irritation scores for each animal are presented in the table below.

Animal N. Reaciton 1 h 24 hrs 48 hrs 72 hrs 4 d 7 d Mean 24/48/72 hrs
3989 Cornea Density - 1 1 1 1 0 1.00
Area - 4 3 2 1 0 -
Iris - - - - - - -
Conjunctiva Hyperemia - - - - - - -
Oedema 3 1 1 1 1 0 0.33
4101 Cornea Density - 1 0 0 0 0 0.33
Area - 1 0 0 0 0 -
Iris - - - - 0 0 -
Conjunctiva Hyperemia - 3 2 2 2 1 2.33
Oedema 2 2 1 1 1 0 1.33
3993 Cornea Density - 1 1 1 1 1 1.00
Area - 4 3 2 2 1 -
Iris - - - - - - -
Conjunctiva Hyperemia - - - - - - -
Oedema 3 4 4 4 4 3 4.00

- : non quantifiable due to the colouring from test substance.

Interpretation of results:
study cannot be used for classification
Conclusions:
The substance is considered to be an eye irritant to rabbits.
Executive summary:

The substance was assessed for eye irritation potential to rabbits, according to the OECD guideline 405. The right eye of three male rabbits was treated with the substance (non diluted) and after 24 hours the eyes were washed.

In the course of the first 24 hours after administration, the substance induced edematic lesions, ranging from some swelling above normal of the lids (grade 1) to more than halfclosed lids (grade 4). Also observed were injected vessels in the bulbar conjunctivae of all animals and discharge ranging from mild to moistening of the lids and surrounding hair, affecting a considerable area around the eye. All the animals showed opacity of the cornea (grade 1) ranging from an affected area of one quarter (grade 1) to near totality of the cornea (grade 4). Due to the colouring produced by the test substance in the palpebral conjunctivae and iris of some of the animals, diffused crimson vessels (grade 3) were observed in only one animal. 2, 3 and 4 days following administration, observed ocular injuries had begun to subside.

In the reading carried out 7 days post-administration, one animal showed vessels clearly more injected than normal (grade 1) in the palpebral conjunctivae. Also, another animal showed nearly half closed, swollen lids (grade 3) and opacity of the cornea (grade 1) which occupied less than one quarter of the total area (grade 1).

Conclusion

The substance is considered to be an eye irritant to rabbits.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
October from 18 to October 25, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
adopted February 24th, 1987
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd.
- Age at study initiation:15 weeks males, 16 weeks females.
- Weight at study initiation: 2.6 - 2.8 kg males, 2.1 kg females.
- Housing: individually in stainless steel cages equipped with an automatic cleaning and drinking system.
- Diet: pelleted standard Kliba 341, rabbit maintenance diet, ad libitum.
- Water: community tap water from Itingen, ad libitum.
- Acclimation: four days under laboratory conditions after veterinary examination.
- Health check: the eyes of the animals were examined once, one day prior to test article administration. Only those animals with no signs of ocular injury or irritation were used on the test.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 10-15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent light/12 hours dark.
- Other: music during the light period, room was air-conditioned.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
- Amount applied: 0.1 g
- Application: the test article was applied undiluted as delivered by the sponsor.
Observation period (in vivo):
72 hours after administration
Number of animals or in vitro replicates:
2 males and 1 female
Details on study design:
APPLICATION OF TEST MATERIAL
The test article was placed in the conjunctival sac of the left eye of each 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 article.

REMOVAL OF TEST SUBSTANCE
- Washing: eyes were not rinsed.

OBSERVATIONS
- Viability/Mortality and clinical signs: daily.
- Body Weights: at the start of acclimatization, day 1 of test (application day) and at termination of observation.
- Sacrifice: all rabbits were euthanized by an intravenous injection of T61 (Hoechst/AG) into the ear vein and discarded.

TOOL USED TO ASSESS SCORE: eye examinations were made with a slit-lamp 30 SL/M and a Varta Cliptrix diagnostic-lamp.

SCORING SYSTEM
The eyes of each animal were examined for ocular irritation 1, 24, 48 and 72 hours after administration. The irritation was assessed according to the following numerical scoring system. The corrosive properties of the test article and the staining of the treated eye were described and recorded.
To calculate the irritation effect all scores of each animal at each of the following reading times (24, 48, 72 hours) were used in calculating the respective mean values (Council Directive 83/467/EEC, July 1983 and 67/548/EEC, May 1987, Brussels, Belgium).
The ocular irritation scores were evaluated in conjunction with the nature and reversibility or otherwise of the responses observed.

CORNEAL IRRITATION
Opacity: degree of density (densest area used for assessment)
No ulceration or opacity 0
Scattered or diffuse areas of opacity (other than slight dulling of normal luster), details of iris clearly visible 1
Easily discernible translucent area, details of iris siightly obscured 2*
Nacrous area, no details of iris visible, size of pupil barely discernible 3 *
Opaque cornea, iris not discernible through the opacity 4*

IRIDIC IRRITATION
Normal 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperemia, or injection, any of these or combination thereof, iris still reacting to light (sluggish reaction is positive) 1 *
No reaction to light, hemorrhage, gross destruction (any or al 1 of these) 2 *

CONJUNCTIVAL IRRITATION
Redness (refers to palpebral and bulbar conjunctivae, cornea and iris).
Blood vessels normal 0
Some blood vessels definitely hyperemic (injected) 1
Diffuse, crimson color, individual vessels not easily discernible 2 *
Diffuse beefy red 3 *

Chemosis: lids and/or nictitating membranes
No swelling 0
Any swelling above normal (includes nictitating membranes) 1
Obvious swelling with partial eversion of lids 2 *
Swel 1 i ng wi th 1 i ds about hal f cl osed 3 *
Swelling with lids more than half closed 4 *

*Starred figures indicate positive effect
Irritation parameter:
cornea opacity score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 1
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 1
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 2
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 2
Reversibility:
fully reversible within: 48 hrs
Irritant / corrosive response data:
Test item showed a primary irritation score of: 0.67, when applied to the conjunctival sac of the rabbit eye.
In the area of application, black staining of the cornea, conjunctivae, nictitating membrane, sclera and eyelashes by pigment or coloring of the test article was observed in all treated animals until termination of observation (72 hours).
No corrosion of the cornea was observed at any of the measuring intervals.

INDIVIDUAL FINDINGS,
Animal No. 87, male.
After 1 hour: Conjunctivae erythema not visible; edema generally clear visible. Sclera: not visible. Discharge: moderate. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 24 hours: Conjunctivae erythema not visible. Sclera: vessels, generally slight visible. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 48 hours: Conjunctivae erythema not visible. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 72 hours: Conjunctivae erythema not visible. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.

Animal No. 88, male.
After 1 hour: Conjunctivae erythema not visible; edema generally clear visible. Sclera: not visible. Discharge: severe. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 24 hours: Conjunctivae erythema not visible; edema, ventral slight. Nictitating membrane: slight edema. Sclera: vessels, dorsal visible. Discharge: slight. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 48 hours: Conjunctivae erythema not visible. Nictitating membrane: slight edema. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 72 hours: Conjunctivae erythema not visible. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.

Animal No. 89, female.
After 1 hour: Conjunctivae erythema not visible; edema generally clear visible. Sclera: not visible. Discharge: severe. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 24 hours: Conjunctivae erythema not visible; edema, ventral slight. Nictitating membrane: slight edema. Sclera: vessels, ventral slight visible. Discharge: slight. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 48 hours: Conjunctivae erythema not visible. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
After 72 hours: Conjunctivae erythema not visible. Coloration black: cornea, conjunctivae, nictitating membrane, sclera, Iidhair.
Other effects:
VIABILITY / MORTALITY AND CLINICAL SIGNS
No acute clinical symptoms were observed in the animals during the test period and no mortality occured.

BODY WEIGHTS
The body weight gain of all rabbits was similar.

Eye irritation scores for each animal

Animal N. Reaction 1 hr 24 hrs 48 hrs 72 hrs Mean at 24/48/72 hrs
87M Cornea opacity 0 0 0 0 0.00
Iris 0 0 0 0 0.00
Conjunctivae redness 0 0 0 0 0.00
Conjunctivae chemosis 2 0 0 0 0.00
88M Cornea opacity 0 0 0 0 0.00
Iris 0 0 0 0 0.00
Conjunctivae redness 0 0 0 0 0.00
Conjunctivae chemosis 2 1 0 0 0.33
89F Cornea opacity 0 0 0 0 0.00
Iris 0 0 0 0 0.00
Conjunctivae redness 0 0 0 0 0.00
Conjunctivae chemosis 2 1 0 0 0.33
Interpretation of results:
other: not classified, according to the CLP Regulation (EC) No 1272/2008
Conclusions:
The substance is not an eye irritant to rabbits.
Executive summary:

The irritancy potential of the test material to the eye of New Zeland white rabbit was assessed, according to the OECD Guideline 405. The substance was applied to the right eye of three rabbits (two males and one female) and the animals were examined for signs of irritation/damage (in iris, cornea and conjuctivae) at 1, 24, 48 and 72 hours following treatment.

Black staining of the cornea, iris, conjuctivae, nictitating membrane, sclera and of the eyelashes by the test material was noted in all animals until termination of observation. No iridal or cornea irritation effects were observed. Erythema was not visible in any of the animals through the study. Conjunctivae discharge and chemosis was observed in all animals after 1 hour and persisted after 24 hours for two out of three animals. No corossion and no mortality occured. The mean score for conjunctival chemosis was 0.33 for 2 rabbits and 0 for 1 rabbit.

Conclusion

The mean values from gradings at 24, 48 and 72 hours were lower than 1 for corneal opacity, lower than 1 for irtitis, lower than 2 for both conjunctival redness and oedema, in all animals, thus the substance can be considered as non irritating to eye, according to the CLP Regulation (EC) No 1272/2008.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: readings was difficult and in almost all the cases was not feasible, due to substance staining
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
24/02/1987
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: authorised supplier.
- Weight at study initiation: 2.5 - 2.7 kg.
- Housing: individually in stainless steel cage, brand Tecniplast.
- Diet: daily dose of 150 g of special diet for test rabbits.
- Water: ad libitum, supplied automatically, treated and filtered.
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 55 ± 25 %
- Air changes: 15 air changes per hr, prefiltered at 5 μm.
- Photoperiod: 12 hrs dark / 12 hrs light.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
Amount applied: 0.1 g
Observation period (in vivo):
21 days
Number of animals or in vitro replicates:
3 males
Details on study design:
APPLICATION OF TEST MATERIAL
The test substance was instilled, undiluted, in a single application. The animal was immobilized and the product was instilled into the conjunctival sac of only the one eye, made accessible by pulling out the lower lid. The untreated eye was used as a control.

TOOL USED TO ASSESS SCORE: a manual lamp was used to assess the eye lessions. After 24 hours fluorescein was also used to evaluate the corena reaction(s).

SCORING SYSTEM
The general state of the animal and the state of the eyes are observed. Special attention was paid to the appearance of alterations in the treated eye: the cornea, the iris, the conjunctiva, as well as any other lesions detected. The observations were made after 1, 24, 48 and 72 hours and on the 7th, 14th and 21st day following application.

Corneal Irritation
Opacity: degree of density (densest area used for assessment)
No ulceration or opacity............................................................................................................................................ 0
Scattered or diffuse areas of opacity (other than slight dulling of normal luster), details of iris clearly visible....... 1
Easily discernible translucent area, details of iris slightly obscured.........................................................................2
Nacrous area, no details of iris visible, size of pupil barely discernible....................................................................3
Opaque cornea, iris not discernible through the opacity.......................................................................................... 4

Iridic Irritation
Normal............................................................................................................................................................................0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperemia, or injection, any of these or
combination thereof, iris still reacting to light (sluggish reaction is positive)................................................................. 1
No reaction to light, hemorrhage, gross destruction (any or all of these)........................... ........................................ 2

Conjuctival Irritation
Redness (refers to palpebral and bulbar conjunctivae, cornea and iris). Blood vessels normal................................ 0
Some blood vessels definitely hyperemic (injected).................................................................................................... 1
Diffuse, crimson color, individual vessels not easily discernible................................................................................. 2
Diffuse beefy red......................................................................................................................................................... 3

Chemosis: lids and/or nictitating membranes
No swelling.................................................................................................................................................................... 0
Any swelling above normal (includes nictitating membranes)....................................................................................... 1
Obvious swelling with partial eversion of lids............................................................................................................... 2
Swelling wi th lids about half closed.............................................................................................................................. 3
Swelling with lids more than half closed........................................................................................................................ 4
Irritation parameter:
cornea opacity score
Basis:
animal: 3/3
Time point:
24/48/72 h
Reversibility:
fully reversible within: 14 days
Remarks on result:
not determinable
Remarks:
due to colouration
Irritation parameter:
iris score
Basis:
animal: 3/3
Time point:
24/48/72 h
Reversibility:
fully reversible within: 7 days
Remarks on result:
not determinable
Remarks:
due to colouration
Irritation parameter:
conjunctivae score
Basis:
animal: 3/3
Time point:
24/48/72 h
Reversibility:
fully reversible within: 21 days
Remarks on result:
not determinable
Remarks:
due to colouration
Irritation parameter:
chemosis score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
2 - 2.67
Reversibility:
fully reversible within: 21 days
Irritant / corrosive response data:
Rabbit 1: at reading at 1, 24 and 48 hours staining is obseved and possible eye lesions cannot be assessed, except for edema; at 72 hours and 7 days the staining only affected the nictitante membrane, disappearing in the reading at the 14th day.
The opacity observed at 72nd hour reading and at 7th day afftected for 1/4 the eye, disappearing in the reading at the 14th day.

Rabbit 2: at reading at 1, 24 and 48 hours staining is obseved and possible eye lesions cannot be assessed, except for edema; from 72nd hour the staining only affected the nictitante membrane, persisting until the reading at 21st day.
The opacity observed at 72nd hour reading and at 7th day afftected for 1/4 the eye, disappearing in the reading at the 7th day.
The reactions observed were recovered until within 21 days.

Rabbit 3: at reading at 1, 24 and 48 hours staining is obseved and possible eye lesions cannot be assessed, except for edema; from 72nd hour the staining only affected the nictitante membrane, persisting until the reading at 21st day.
The opacity observed at 72nd hour reading and at 7th day afftected for 1/4 the eye, disappearing in the reading at the 7th day.
The reactions observed were recovered until within 21 days.

The eye irritation scores for each animal are presented below.

Animal 1 Reaction 1 hr 24 hrs 48 hrs 72 hrs 7 d 14 d 21 d Mean 24/48/72 nhrs
1 Cornea opacity - - - 1 1 0 0 -
Iris - - - 1 0 0 0 -
Conjunctival redness - - - 2 2 1 0 -
Conjunctival oedema 3 4 2 2 1 0 0 2.67
2 Cornea opacity - - - 1 0 0 0 -
Iris - - - 1 0 0 0 -
Conjunctival redness - - - 2 2 1 0 -
Conjunctival oedema 3 3 3 2 2 1 0 2.67
2 Cornea opacity - - - 1 0 0 0

-

Iris

-

-

-

1

0

0

0

-

Conjunctival redness

-

-

-

2

2

1

0

-

Conjunctival oedema

3

3

3

1

1

0

0

2.33

* staining is obseved and possible eye lesions cannot be assessed

All the lessions observed where reversible in 21 days.

Interpretation of results:
study cannot be used for classification
Conclusions:
The substance is considered to be an eye irritant to rabbits.
Executive summary:

The substance was assessed for eye irritation potential to rabbits, according to the OECD guideline 405. One of the eyes of three New Zealand White rabbits was treated with 0.1 g of test substance, non-diluted. The eyes were scored for irritation in conjuctivae, cornea and iris at 1, 24, 48 and 72 hours and at 7, 14 and 21 days following treatment.

The observations of the effects in cornea, iris and conjunctivae erythema at 1, 24 and 48 hours was not possible due to the black staining produced by the substance; at 72 hours and 7 days the staining only affected the nictitante membrane, disappearing in the reading at the 14th and 21st day. Formation of reversible conjunctivae chemosis was noted in all animals.

The mean conjunctivae chemosis score was greater than 2 in all animals. Effects resulted to be fully recovered within the 21st day.

Conclusion

The substance is considered to be an eye irritant to rabbits.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

SKIN IRRITATION

The irritancy potential of the test material to the skin of New Zealand white rabbit was assessed according to the OECD Guideline 404. The intact skin of the rabbits was exposed for 4 hours to the substance (0.5 g moistened with bi-distilled water), in a semi-occlusive dressing. No acute clinical symptoms were observed and no mortality occurred. A black coloration on the back was observed in all animals from 1 to 72 hours, but no signs of irritations were observed. Test item showed a primary irritation score of 0.00 when applied to intact rabbit skin. Local signs (mean values from 1 hour to 72 hours) consisted of grade 0.00 erythema and grade 0.00 edema.

The study outcomes are confirmed by the results of a second experiment, during which three rabbits were treated with the substance (0.5 g, unchanged). The study was conducted according to the OECD guideline 404. 30 minutes after the patches were removed, very slight erythema (grade 1) was observed in two of the animals. In the readings carried out 24, 48 and 72 hours after administration, no alterations were observed on the skin of any of the animals. Mean scores for erythema and edema, 24 , 48 and 72 hours after treatment were 0.00 for the three animals. Following the last reading, the study was ended.

A third experiment is available, which gave different results. The skin of the rabbits was exposed for 4 hours to the substance (0.5 g in physiological saline); after exposure period the skin was cleaned. All animals presented edema at 1 and 24 hours, while erythema was observed throughout the first observation period (72 hrs). The erythema was not reversible in two out of three animals even after 14 days, while edema was fully reversible after 48 hours following patch removal.

Although the test conditions employed in this experiment seems to be comparable to those characterizing the key study and although the composition of the tested substances is strictly comparable, the results obtained are significantly different and identify a different skin irritation potential.

Based on the available information, an explanation is not clearly evident. The substance has an intense colour, thus readings in irritation tests may be difficult, due to substance staining.

In order to clarify the skin irritation potential of Direct Black 168, the available data on the structural analogous Similar Substance 01 have been taken into account. Similar Substance 01 shares with Direct Black 168 the same scaffold chemical structure base; the differences are considered to be not able to impact the study outcomes, thus the read across can be considered as reliable and appropriated to evaluate the endpoint. Details on read-across approach are given in the IUCLID section 13.

The irritancy potential of the Similar Substance 01 to the skin of rabbits was assessed in accordance with the method and procedures outlined into the OECD Guideline 404. No evidence of skin irritation was noted at 48 or 72-hr observations. Black staining caused by the test material was noted at all treated skin sites during the study. The mean score for erythema was 0 for 2 rabbits and 0.3 for 1 rabbit. The mean score for edema for all the rabbits was 0.

EYE IRRITATION

The irritancy potential of the test material to the eye of New Zealand white rabbit was assessed, according to the OECD Guideline 405. Black staining of the cornea, iris, conjunctivae, nictitating membrane, sclera and of the eyelashes by the test material was noted in all animals until termination of observation. No iridal or cornea irritation effects were observed. Erythema was not visible in any of the animals through the study. Conjunctivae discharge and chemosis was observed in all animals after 1 hour and persisted after 24 hours for two out of three animals. No corrosion and no mortality occurred.

A second experiment was conducted under similar conditions, according to the OECD guideline 405. The right eye of three male rabbits was treated with the substance (non diluted) and after 24 hours the eyes were washed. In the course of the first 24 hours after administration, the substance induced edematic lesions. Also observed were injected vessels in the bulbar conjunctivae of all animals and discharge ranging from mild to moistening of the lids and surrounding hair, affecting a considerable area around the eye. All the animals showed opacity of the cornea. Due to the colouring produced by the test substance in the palpebral conjunctivae and iris of some of the animals, diffused crimson vessels were observed in only one animal. 2, 3 and 4 days following administration, observed ocular injuries had begun to subside. In the reading carried out 7 days post-administration, one animal showed vessels clearly more injected than normal in the palpebral conjunctivae. Also, another animal showed nearly half closed, swollen lids and opacity of the cornea which occupied less than one quarter of the total area.

In a third experiment, the substance was assessed according to the OECD guideline 405. The observations of the effects in cornea, iris and conjunctivae erythema at 1, 24 and 48 hours was not possible due to the black staining produced by the substance; at 72 hours and 7 days the staining only affected the nictitante membrane. Formation of reversible conjuctivae chemosis was noted in all animals. The mean conjunctivae chemosis score was greater than 2 in all animals. Effects on the cornea, iris or conjunctiva resulted to be fully recovered within the 21st day; only staining of the nictitante membrane persisted until the reading at 21st day.

The differences between the conclusion reached in the first test respect those trace in the other two experiments may be attribute to a different approach about how to consider the staining. In fact, although black staining of the cornea, iris, conjunctivae, nictitating membrane, sclera and of the eyelashes by the test material was noted in all animals until termination of observation (i.e. 72 hours), it was concluded that the substance was not irritating.

Based on the information given in the other two experiments, the substance seems to be able to cause eye irritation. All the effects on the cornea, iris or conjunctiva resulted to be fully recovered within the 21st day; only staining of the nictitante membrane persisted until the reading at 21st day. However, considering that humans have only a rudimentary nictitante membrane, a classification as Eye Irrit 2 (H319) seems to be appropriate.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.2 Skin corrosion/irritation section, skin irritation means the production of reversible damage to the skin following the application of a test substance for up to 4 hours.

In the key study, the mean values from grading at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions.

According to the CLP Regulation (EC) No 1272/2008, serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance, which is not fully reversible within 21 days of application.

Based on the review of all the experiments available, the substance seems to be able to cause eye irritation; however, effects can be recovered within 21 days.

In conclusion, the substance does not meet the criteria to be classified for the skin irritation; however, it should be classified as eye irritating, category 2 (H319), according to the CLP Regulation (EC) No 1272/2008.