A mixture of isomers of: 1,1'-[(3,5(or 2,4 or 4,6 or 2,6)-dihydroxy-o(or m or p)-phenylene)bis(azo-meta-phenyleneazo{1-[3-(dimethylamino)propyl]-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-5,3-diyl})]dipyridinium-dichloride-dihydrochloride; 1-(1-[3-(dimethylamino)propyl]-5-{3-[x-(4-{1-[3-(dimethylamino)propyl]-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-5-pyridinio-3-pyridylazo}phenylazo)-2,4(or 2,6 or 3,5 or 4,6)-dihydroxyphenylazo]phenylazo}-1,2-dihydro-6-hydroxy-4-methyl-2-oxo-3-pyridyl)pyridinium-dichloride-dihydrochloride (where x is variable)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-05-06 to 1989-05-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1987

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of isomers of: 1,1'-[(3,5(or 2,4 or 4,6 or 2,6)-dihydroxy-o(or m or p)-phenylene)bis(azo-meta-phenyleneazo{1-[3-(dimethylamino)propyl]-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-5,3-diyl})]dipyridinium-dichloride-dihydrochloride; 1-(1-[3-(dimethylamino)propyl]-5-{3-[x-(4-{1-[3-(dimethylamino)propyl]-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-5-pyridinio-3-pyridylazo}phenylazo)-2,4(or 2,6 or 3,5 or 4,6)-dihydroxyphenylazo]phenylazo}-1,2-dihydro-6-hydroxy-4-methyl-2-oxo-3-pyridyl)pyridinium-dichloride-dihydrochloride (where x is variable)

EC Number:

404-540-1

EC Name:

A mixture of isomers of: 1,1'-[(3,5(or 2,4 or 4,6 or 2,6)-dihydroxy-o(or m or p)-phenylene)bis(azo-meta-phenyleneazo{1-[3-(dimethylamino)propyl]-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-5,3-diyl})]dipyridinium-dichloride-dihydrochloride; 1-(1-[3-(dimethylamino)propyl]-5-{3-[x-(4-{1-[3-(dimethylamino)propyl]-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-5-pyridinio-3-pyridylazo}phenylazo)-2,4(or 2,6 or 3,5 or 4,6)-dihydroxyphenylazo]phenylazo}-1,2-dihydro-6-hydroxy-4-methyl-2-oxo-3-pyridyl)pyridinium-dichloride-dihydrochloride (where x is variable)

Cas Number:

159405-95-5

Molecular formula:

C50H56Cl4N14O6

IUPAC Name:

1'-[3-(dimethylamino)propyl]-5'-(2-{3-[2-(4-{2-[3-(2-{1'-[3-(dimethylamino)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-2,6-dihydroxyphenyl)diazen-1-yl]phenyl}diazen-1-yl)-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium 1'-[3-(dimethylamino)propyl]-5'-(2-{4-[2-(4-{2-[4-(2-{1'-[3-(dimethylamino)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-2,6-dihydroxyphenyl)diazen-1-yl]phenyl}diazen-1-yl)-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium tetrahydrochloride tetrachloride

Test material form:

solid

Test animals / tissue source

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: 15 weeks
- Weight at study initiation: 2757 - 2966 gram
- Housing: individually in cages with perforated floors equipped with an automatic drinking system
- Diet (e.g. ad libitum): standard laboratory rabbit diet approx. 100 gram per day.
- Water (e.g. ad libitum): ree access to tap-water diluted with decalcified water.
- Acclimation period: at least five days under test conditions.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 30 - 70 %
- Air changes (per hr): 7.5 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Test system

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 60 mg

Duration of treatment / exposure:

The test article was instilled 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. The other eye remained untreated and served as the reference control. Immediatelely after the examination on day 2, both eyes of all three rabbits were washed out with approximately 20 ml tepid tap-water to remove the residual test article.

Observation period (in vivo):

15 d

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): immediatelely after the examination on day 2, both eyes of all three rabbits were washed out with approximately 20 ml tepid tap-water to remove the residual test article. Unfortunately, most of the test article remained sticked to the eyelids.
- Time after start of exposure: 24 h

SCORING SYSTEM:

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 palpebral and bulbar conjunctivae, 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 nictating membrane
No swelling 0
Any swelling above normal (includes nictating 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

TOOL USED TO ASSESS SCORE: fluorescein. In case standard lighting was considered inadequate for observing minor effects, eye examinations were performed using a pocket flash light.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 14 d

Results 4

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 d

Results 5

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Results 7

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

fully reversible within: 14 d

Results 8

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 72 h

Results 9

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Results 10

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Results 11

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

fully reversible within: 14 d

Results 12

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 72 h

Irritant / corrosive response data:

The most severe irritation effect was conjunctival redness. One out of three animals had an average score of 2 over 24 h, 48 h, and 72 h, while the two other animals had conjunctival score of 1.67 over the three time points. Max. individual conjunctival score at one single time point was 2. All other irritation effects were less severe. All irritation effects were fully reversible at 14 days at latest.

Other effects:

Conjunctival irritation was observed in all animals, with redness occurring from 1 hour after treatment until day 7. Conjunctival chemosis was observed from 24 hour to 48 hour in two animals and persisted until 72 hour in one animal. No signs of eye irritation were reported on day 14. Slight discharge occurred at 24 hour (3/3) and 48 hour (2/3).

Applicant's summary and conclusion

Interpretation of results:

other: not classified within the CLP Regulation (EC 1272/2008)

Conclusions:

Not eye irritant.

Executive summary:

The purpose of the study was to evaluate the eye irritation potential of test item, following OECD guideline 405.

Under the conditions of this study, the substance caused adverse effects to the conjunctivae, in terms of redness and chemosis. One out of three animals had an average score of 2 over 24 h, 48 h, and 72 h, while the two other animals had a conjunctival score of 1.67 over the three time points. Maximum individual conjunctival score at one single time point was 2. All other irritation effects were less severe. All irritation effects were fully reversible at 14 days at latest.

No corrosion nor signs of systemic intoxication were observed.

No classification as eye irritant within the CLP Regulation (EC 1272/2008) is to be applied.