Disodium 2,2'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[3-bromo-5-butyltoluene-4-sulphonate]

Endpoint summary

Description of key information

Skin Irritation (in vivo), Driscoll (1993)

Under the conditions of the study the test material was not irritating.

Eye Irritation (in vivo), Driscoll (1993)

Under the conditions of the study, the test material was irritating to the eye.

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:

18 August 1993 to 25 August 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: twelve to sixteen weeks old
- Weight at study initiation: 2.32 - 2.47 kg
- Housing: The animals were individually housed in suspended metal cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: minimum of five days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 24 °C
- Humidity: 56 - 66 %
- Air changes: approximately 15 changes per hour
- Photoperiod: the lighting was controlled by a time switch to give continuous 12 hours light and 12 hours darkness

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.5g of the test material, moistened with 0.5 mL of distilled water

Duration of treatment / exposure:

Four hours

Observation period:

72 hours

Number of animals:

Three

Details on study design:

TEST SITE
- Area of exposure: dorsal flank area
- On the day before the test each of a group of three rabbits was clipped free of fur from the dorsal flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.
- On the day of the test a suitable test site was selected on the back of each rabbit. A quantity of 0.5 g of the test material, moistened with 0.5 mL of distilled water, was introduced under a 2.5 cm x 2.5 cm gauze patch and placed in position on the shorn skin. The patch was secured in position with a strip of surgical adhesive tape (BLENDERM: approximate size 2.5 cm x 4.0 cm). To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset (TUBIGRIP) and the animals were returned to their cages for the duration of the exposure period.

REMOVAL OF TEST SUBSTANCE
- Four hours after application the corset and patches were removed from each animal and any residual test material removed by gentle swabbing with cotton wool soaked in distilled water followed by diethyl ether.

OBSERVATION TIME POINTS
- Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to scale from Draize J.H. (1959) Association of Food and Drug Officials of the United States, Austin, Texas, "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics".

SCORING SYSTEM:
EVALUATION OF SKIN REACTIONS
- Erythema and Eschar Formation Value:
No erythema = 0
Very slight erythema (barely perceptible) = 1
We 11-defi ned erythema = 2
Moderate to severe erythema = 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) = 4

- Oedema Formation Value:
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 millimetre) = 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) = 4

- Any other skin reactions, if present, were also recorded.
- An additional observation was made on day 7 to assess the reversibility of skin reactions.

INTERPRETATION OF RESULTS
- The scores for erythema and oedema at the 24 and 72-hour readings were totalled for the three test rabbits (12 values) and this total was divided by 6 to give the primary irritation index of the test material. The test material was classified according to the following scheme:
Primary Irritation Index
0 = Non-irritant
> 0 – 2 = Mild irritant
> 2 – 5 = Moderate irritant
> 5 – 8 = Severe irritant
- If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study Director, which may include ulceration and clear necrosis or signs of scar tissue, the test material is classified as corrosive to rabbit skin. Classification according to Draize may, therefore, not be applicable.
- The results were interpreted according to the EEC Commission Directive of 91/325/EEC which adapts Council Directive 67/548/EEC on the regulations relating to the classification, packaging and labelling of dangerous substances as follows:
i) Interpretation according to Annex VI Part II (B): The test material will be classified as irritant or corrosive to the skin according to the following criteria:
a) Corrosion: The test material will be considered to be corrosive and will require the appropriate "C" symbol if it produces full thickness destruction of the skin tissue on at least one animal. If this criterion is not satisfied, the test material will be classified as non-corrosive.
b) Irritation: The test material will be considered to be irritant and will require the appropriate "Xi" symbol if it causes inflammation of the skin, which persists for at least 24 hours, and if either erythema/eschar formation or oedema formation equivalent to a mean value of two or more, calculated for each animal separately is observed in two or more animals. The 24, 48 and 72-hour readings for each animal will be used to calculate the mean values. If these criteria are not satisfied the test material will be classified as non-irritant.
ii) Interpretation according to Annex VI, Part II (D): In addition, the following risk (R) phrases will be assigned to the test material, if appropriate, according to the following criteria:
Corrosive:
R 35 CAUSES SEVERE BURNS: If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs as a result of up to three minutes exposure.
R 34 CAUSES BURNS: If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs as a result of up to four hours exposure.
Irritant
R 38 IRRITATING TO SKIN: If, when applied to healthy intact skin for up to four hours, significant inflammation is caused which is present 24 hours or more after the end of the exposure period. Inflammation is significant if the mean score for either erythema/eschar formation or oedema formation, observed in two or more animals, is equivalent to a value of two or more. The 24, 48 and 72-hour readings for each animal will be used to calculate the mean scores.

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

46 Female

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

50 Female

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

54 Female

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

46 Female

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

50 Female

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

54 Female

Time point:

24/48/72 h

Score:

0.7

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritant / corrosive response data:

- Faint blue-coloured staining caused by the test material was noted at all treated skin sites after patch removal and persisted up to the 72- hour observation. This did not affect evaluation of skin responses.
- Very slight erythema was noted at one treated skin site at the one, 24, 48 and 72-hour observations. Desquamation was noted at two treated skin sites at the 7-day observation.
- Very slight oedema was noted at one treated skin site at the 24 and 48-hour observations.

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study the test material was not irritating.

Executive summary:

The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 404 and EU Method B4, under GLP conditions.

On the day before the test each of a group of three female New Zealand White rabbits was clipped free of fur from the dorsal flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study. On the day of the test a suitable test site was selected on the back of each rabbit. A quantity of 0.5g of the test material, moistened with 0.5 mL of distilled water, was introduced under a 2.5 cm x 2.5 cm gauze patch and placed in position on the shorn skin. Four hours after application the corset and patches were removed from each animal. Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation.

Faint blue-coloured staining caused by the test material was noted at all treated skin sites after patch removal and persisted up to the 72- hour observation. This did not affect evaluation of skin responses. Very slight erythema was noted at one treated skin site at the one, 24, 48 and 72-hour observations. Desquamation was noted at two treated skin sites at the 7-day observation. Very slight oedema was noted at one treated skin site at the 24 and 48- hour observations.

The test material produced a primary irritation index of 0.5 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

Under the conditions of this study the test material was not irritating.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

06 September 1993 and 20 September 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately twelve to sixteen weeks old
- Weight at study initiation: 2.38 - 2.80 kg
- Housing: The animals were individually housed in suspended metal cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: minimum of five days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 – 23 °C
- Humidity: 54 – 74 %
- Air changes: approximately 15 changes per hour
- Photoperiod: lighting was controlled by a time switch to give 12 hours light and 12 hours darkness

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 mL (99 mg)

Duration of treatment / exposure:

The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test material, and then released.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

Three

Details on study design:

PROCEDURE
- One rabbit was initially treated. A volume of 0.1 mL of the test material, which was found to weigh approximately 99 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test material, and then released.
- The left eye remained untreated and was used for control purposes.
- Immediately after administration of the test material, an assessment of the initial pain reaction was made.
- After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

SCORING SYSTEM:
- Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment (from Draize J.H. 1959, Association of Food and Drug Officials of the United States, Austin, Texas, "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics").
- Any other ocular effects were also noted. Examination of the eye was facilitated by use of the light source from a standard ophthalmoscope.
- Additional observations were made on days five, seven and fourteen to assess the reversibility of the ocular effects.

DRAIZE SCALE FOR SCORING OCULAR IRRITATION
CONJUNCTIVAE
(A) Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)
Vessels normal = 0
Vessels definitely injected above normal = 1
More diffuse, deeper crimson red, individual vessels not easily discernible = 2
Diffuse beefy red = 3
(B) Chemosis
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 half closed to completely closed = 4
(C) 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 a considerable area around the eye = 3
THE TOTAL SCORE = (A + B + C) x 2 MAXIMUM TOTAL = 20

2. IRIS
(D) Values
Normal = 0
Folds above normal, congestion, swelling, circumcorneal injection (any or all of these or combination of any thereof) iris still reacting to light (sluggish reaction is positive) = 1
No reaction to light, haemorrhage, gross destruction (any or all of these) = 2
THE TOTAL SCORE = D x 5 MAXIMUM TOTAL = 10

3. CORNEA
(E) Degree of Opacity (most dense area used)
No opacity = 0
Scattered or diffuse areas, details of iris clearly visible = 1
Easily discernible translucent areas, details of iris slightly obscured = 2
Opalescent areas, no details of iris visible, size of pupil barely discernible = 3
Opaque, iris invisible = 4
(F) Area of Cornea involved
One quarter (or less) but not zero = 1
Greater then 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
THE TOTAL SCORE = (E x F) x 5 MAXIMUM TOTAL - 80

MAXIMUM TOTAL SCORE POSSIBLE = 110

INTERPRETATION OF RESULTS
- The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of opacity). For each tissue the score was calculated as follows:
Score for conjunctivae = (A+ B + C) x 2
Score for iris = D X 5
Score for cornea = (E X F) X 5
- Using the numerical data obtained a modified version of the system described by Kay J.H. and Calandra J.C., J. Soc. Cosmet. Chem., 1962 13 281-289 was used to classify the ocular irritancy potential of the test material. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test material. The results were also interpreted according to Commission Directive 91/325/EEC which adapts Council Directive 67/548/EEC on the regulations relating to the classification, packaging and labelling of dangerous substances, as follows:
- Interpretation according to Annex VI Part II {B) Eve Irritation
Criteria: The test material will be classified as irritant and will require the appropriate "Xi" symbol if ocular lesions occur within 72 hours after exposure, persist for at least 24 hours and correspond to one or more of the following mean values in two or more animals:
- corneal opacity: 2 or more
- iridial lesion: 1 or more
- redness of conjunctivae: 2.5 or more
- chemosis of conjunctivae: 2 or more
The 24, 48 and 72-hour readings for each animal will be used to calculate the mean values.
If these criteria are not satisfied the test material will be classified as non-irritant.
- Interpretation according to Annex VI Part II (D)
In addition the following risk (R) phrases will be assigned to the test material, if appropriate, according to the criteria indicated below:
R 36 IRRITATING TO EYES: If, when applied to the eye of three rabbits, significant ocular lesions are caused which are present 24 hours or more after the instillation of the test material in two or more animals. Ocular lesions are significant if the mean of the 24, 48 and 72-hour readings comply with any of the following criteria:
- corneal opacity: equal to or greater than 2 but less than 3
- iridial lesion: equal to or greater than 1
- conjunctival redness: equal to or greater than 2.5
- conjunctival chemosis: equal to or greater than 2

R 41 RISK OF SERIOUS DAMAGE TO EYES: If, when applied to the eye of three rabbits, severe ocular lesions are caused in two or more animals which are present 24 hours or more after instillation of the test material. Ocular lesions are severe if the mean of the 24, 48 and 72- hour readings comply with either of the following criteria:
- corneal opacity: equal to or greater than 3
- iridial lesion: equal to 2

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

115 Female

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

3 Female

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

101 Female

Time point:

24/48/72 h

Score:

1.7

Max. score:

2

Reversibility:

not fully reversible within: 5 days

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

115 Female

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

101 Female

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: 5 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

115 Female

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

3 Female

Time point:

24/48/72 h

Score:

1.7

Max. score:

2

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

101 Female

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 5 days

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

115 Female

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

3 Female

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

101 Female

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 5 days

Irritant / corrosive response data:

- Residual test material was noted around the treated eye of all animals throughout the study.
- Blue-coloured staining was noted in all treated eyes during the study.
- Blue-coloured staining prevented accurate evaluation of the cornea in one treated eye one hour after treatment. A dulling of the normal lustre of the corneal surface was noted in two treated eyes one hour after treatment. Areas of diffuse corneal opacity were noted in all treated eyes at the 24-hour observation with areas of diffuse to translucent corneal opacity at the 48 and 72-hour observations. Areas of opalescent corneal opacity were noted in one treated eye at the 5-day observation. Areas of diffuse or opalescent corneal opacity were noted in two treated eyes at the 7-day observation. Opalescent corneal opacity over approximately 50 % of the cornea with diffuse to translucent corneal opacity in the remaining area was noted in one treated eye at the 14-day observation. Vascularisation of the cornea was noted in one treated eye at the 5-day observation, in two treated eyes at the 7-day observation and in one treated eye at the 14-day observation.
- Blue-coloured staining prevented accurate evaluation of the iris in one treated eye one hour after treatment. Iridial inflammation was noted in two treated eyes one hour after treatment, in all treated eyes at the 24, 48, 72-hour and 5 or 7-day observations and in one treated eye at the 14-day observation.
- Blue-coloured staining prevented accurate evaluation of conjunctival redness in all treated eyes one hour after treatment. Slight to severe conjunctival chemosis and discharge were noted in all treated eyes at this time. Moderate conjunctival irritation was noted in all treated eyes at the 24, 48, 72-hour and 5 or 7-day observations. Moderate conjunctival irritation was noted in one treated eye at the 14-day observation. Ectropion was noted in one treated eye at the 5-day observation and in another treated eye at the 7-day observation.
- The animals were showing signs of pain and discomfort and were therefore killed for humane reasons immediately after the 5, 7 or 14-day observations in accordance with current UK Home Office guidelines.

Interpretation of results:

other: EU criteria: Causes serious eye damage

Conclusions:

Under the conditions of this study, the test material was irritating to the eye.

Executive summary:

The eye irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 405 and EU Method B5, under GLP conditions.

One rabbit was initially treated. A volume of 0.1 mL of the test material, which was found to weigh approximately 99 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test material, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test material, an assessment of the initial pain reaction was made. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment.

Blue-coloured staining prevented accurate evaluation of the cornea in one treated eye one hour after treatment. A dulling of the normal lustre of the corneal surface was noted in two treated eyes one hour after treatment. Areas of diffuse corneal opacity were noted in all treated eyes at the 24-hour observation with areas of diffuse to translucent corneal opacity at the 48 and 72-hour observations. Areas of opalescent corneal opacity were noted in one treated eye at the 5-day observation. Areas of diffuse or opalescent corneal opacity were noted in two treated eyes at the 7-day observation. Opalescent corneal opacity over approximately 50 % of the cornea with diffuse to translucent corneal opacity in the remaining area was noted in one treated eye at the 14-day observation. Vascularisation of the cornea was noted in one treated eye at the 5-day observation, in two treated eyes at the 7-day observation and in one treated eye at the 14-day observation.

The animals were showing signs of pain and discomfort and were therefore killed for humane reasons immediately after the 5, 7 or 14-day observations in accordance with current UK Home Office guidelines.

Under the conditions of this study, the test material was irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation (in vivo), Driscoll (1993)

The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 404 and EU Method B4, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

On the day before the test each of a group of three female New Zealand White rabbits was clipped free of fur from the dorsal flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study. On the day of the test a suitable test site was selected on the back of each rabbit. A quantity of 0.5g of the test material, moistened with 0.5 mL of distilled water, was introduced under a 2.5 cm x 2.5 cm gauze patch and placed in position on the shorn skin. Four hours after application the corset and patches were removed from each animal. Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation.

Faint blue-coloured staining caused by the test material was noted at all treated skin sites after patch removal and persisted up to the 72- hour observation. This did not affect evaluation of skin responses. Very slight erythema was noted at one treated skin site at the one, 24, 48 and 72-hour observations. Desquamation was noted at two treated skin sites at the 7-day observation. Very slight oedema was noted at one treated skin site at the 24 and 48- hour observations.

The test material produced a primary irritation index of 0.5 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

Under the conditions of the study the test material was not irritating.

Eye Irritation (in vivo), Driscoll (1993)

The eye irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 405 and EU Method B5, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

One rabbit was initially treated. A volume of 0.1 mL of the test material, which was found to weigh approximately 99 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test material, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test material, an assessment of the initial pain reaction was made. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment.

Blue-coloured staining prevented accurate evaluation of the cornea in one treated eye one hour after treatment. A dulling of the normal lustre of the corneal surface was noted in two treated eyes one hour after treatment. Areas of diffuse corneal opacity were noted in all treated eyes at the 24-hour observation with areas of diffuse to translucent corneal opacity at the 48 and 72-hour observations. Areas of opalescent corneal opacity were noted in one treated eye at the 5-day observation. Areas of diffuse or opalescent corneal opacity were noted in two treated eyes at the 7-day observation. Opalescent corneal opacity over approximately 50 % of the cornea with diffuse to translucent corneal opacity in the remaining area was noted in one treated eye at the 14-day observation. Vascularisation of the cornea was noted in one treated eye at the 5-day observation, in two treated eyes at the 7-day observation and in one treated eye at the 14-day observation.

The animals were showing signs of pain and discomfort and were therefore killed for humane reasons immediately after the 5, 7 or 14-day observations in accordance with current U.K. Home Office guidelines.

Under the conditions of the study, the test material was irritating to the eye.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin irritation.

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does require classification as irritating to the eye (irreversible damage) and is assigned the hazard phrase H318: causes serious eye damage.