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

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

277-429-7

EC Name:

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

Cas Number:

73398-29-5

Molecular formula:

C36H34Br2N2O8S2.2Na

IUPAC Name:

disodium 2-bromo-3-({4-[(2-bromo-4-butyl-6-methyl-3-sulfonatophenyl)amino]-9,10-dioxo-9,10-dihydroanthracen-1-yl}amino)-6-butyl-4-methylbenzene-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: dark blue powder
- Storage conditions: room temperature in the dark

Test animals / tissue source

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

Test system

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

Results and discussion

In vivo

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Results 5

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

Results 6

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

Results 7

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

Results 8

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

Results 9

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

Results 10

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

Results 11

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

Results 12

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.

Applicant's summary and conclusion

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.