Reaction products of 4,4'-methylenebis(2,6-dimethylaniline) and 4-aminobenzenesulfonic acid with 2,4,6-trichloro-1,3,5-triazine, diazotised and subsequently coupled with N-(2-methoxyphenyl)-3-oxobutanamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 March 2006 to 27 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 4-{4-[4-(4-Amino-3,5-dimethyl-benzyl)-2,6-dimethyl-phenylamino]-6-chloro-[1,3,5]triazin-2-ylamino}-benzenesulfonic acid, sodium nitrite, hydrochloric acid and acetoacet-o-anisidin

Cas Number:

1165939-52-5

IUPAC Name:

Reaction product of 4-{4-[4-(4-Amino-3,5-dimethyl-benzyl)-2,6-dimethyl-phenylamino]-6-chloro-[1,3,5]triazin-2-ylamino}-benzenesulfonic acid, sodium nitrite, hydrochloric acid and acetoacet-o-anisidin

Test material form:

solid: particulate/powder

Remarks:

Yellow solid

Details on test material:

- Expiry date: 20 Dec 2007
- Storage conditions: At room temperature (ca 20°C)

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: 12 – 13 weeks (male) 12 – 14 weeks (female)
- Housing: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 – 23 °C
- Humidity (%): 30 – 70 %
- Air changes (per hr): 10 – 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 h light and 12 h darkness.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 g (per animal) of the test material was weighed and applied undiluted as it was delivered.
The test material was applied at 0.1 g/animal the dose specified in the test guidelines for a solid test material.
The pH of at 1 % (w/w) solution of the test material was measured for a previous study and was found to be pH 5.

Duration of treatment / exposure:

0.1 g of the test material per animal

Observation period (in vivo):

72 h

Number of animals or in vitro replicates:

Three

Details on study design:

The eyes of the animals were examined one day prior to the test material administration.
Animals with overt signs of ocular injury or irritation which may have interfered with the interpretation of the results were not used in the test.
On the day of treatment 0.1 g of the test material 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 material. The right eye remained untreated and served as the reference control.

REMOVAL OF TEST SUBSTANCE
- Washing: The treated eyes were not rinsed after instillation.

OBSERVATIONS
- Viability / mortality: Daily from acclimatisation of the animals to the termination of the test.
- Clinical signs: Daily from acclimatisation of the animals to the termination of the test.
- Body weights: At the start of the acclimatisation, on the day of application and at termination of observation.


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

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

Conjunctivae
Redness (refers to most severe reading of palpebral and bulbar conjunctivae when compared with control eye).
0: Blood vessels normal.
1: Some blood vessels definitely hyperemic (injected).
2: Diffuse, crimson colour, individual vessels not easily discernible.
3: Diffuse beefy red.
Chemosis: Lids and / or nictitating membranes.
0: No swelling.
1: Any swelling above normal (including nictitating membranes).
2: Obvious swelling with partial eversion of lids.
3: Swelling with lids about half-closed.
4: Swelling with lids more than half-closed.

Additionally, ocular discharge, reddening of the sclerae and staining of conjunctivae, sclerae and cornea by the test material was assessed according to the following scheme:
Ocular discharge:
0: No discharge
1: Slight: Any amount different to normal
2: Moderate: Discharge with moistening of the lids and hair just adjacent to the lids
3: Marked: Discharge with moistening of the lids and hairs, and a considerable area around the eye (running)
Sclerae reddness
0: Normal: blood vessels normal
1: Slight reddening: Some blood vessels definitely hyperemic (injected)
2: Moderate reddening: diffuse, crimson colour, individual vessels not easily discernible
3: Marked reddening: diffuse beefy red
Staining of conjunctivae, sclerae and cornea by test material
0: Not observed
1: Slight staining
2: Marked staining

- Scleral reddening and ocular discharge were also assessed.
- The scores of each animal at the following reading times (24, 48 and 72 h) were used in calculating the respective mean values (with the exception of the sclerae) for each type of lesion.

TOOL USED TO ASSESS SCORE: Eye examinations were made with a Varta Cliptrix diagnostic lamp (RothAG, CH-4153, Reinach, Switzerland). The eyes of each animal were examined approximately 1, 24, 48 and 72 h after administration.

PATHOLOGY
- No necropsy was performed on animals treated at termination of observation.
- All rabbits were sacrificed by intravenous injection of Vetanarcol into the ear vein at a dose of at least 1 mL/kg body weight (equivalent to 162 mg sodium pentobarbitone / kg bodyweight) and discarded.

STATISTICAL ANALYSIS
No statistical analysis was performed.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Results 7

Irritation parameter:

conjunctivae score

Remarks:

Reddening

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Results 8

Irritation parameter:

conjunctivae score

Remarks:

Reddening

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Results 9

Irritation parameter:

conjunctivae score

Remarks:

Reddening

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Results 10

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Results 11

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Results 12

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

The mean score was calculated across 3 scoring times (24, 48 and 72 h after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately.
No abnormal findings were observed in the iris of any animal at any of the measurement intervals.
Very slight corneal opacity affecting the whole area at the 1 h reading and affecting one quarter (or less) but not zero was noted in one animal.
Moderate reddening of the conjunctivae was noted in all animals at the 1-h reading and persisted as slight reddening until the 24 h reading in two animals, and until the 48 h reading in one animal.
Slight swelling (chemosis) of the conjunctivae to obvious swelling with partial eversion of the lids was observed in all animals at the 1 h reading.
Slight to moderate reddening of the sclerae was present in all animals at the 1 h reading and persisted as slight reddening in two animals at the 24 h reading.
Slight ocular discharge was noted in all animals at the 1 h reading.
No abnormal findings were observed in the treated eye of any animal 72 h after treatment, the end of the observation period for all animals.
No corrosion of the cornea was observed at any of the reading times.

Other effects:

- Lesions and clinical observations: No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
Other observations:
- Colouration: Yellow remnants of the test material were observed in the eye or conjunctival sac of all animals one hour after treatment.
- Bodyweights: One animal showed a slight loss of body weight (0.9 %) at the end of the observation period. The bodyweights of the remaining rabbits were considered to be within the normal range of variability.

Any other information on results incl. tables

Eye Irritation Scores – Individual Values

Animal No.	Sex	Evaluation interval*	Corneal opacity	Area of corneal opacity	Iris	Conjunctivae		Sclera
						Redness	Chemosis
1	M	1 h	1	4	0	2	2	2
2	F		0	0	0	2	1	1
3	F		0	0	0	2	1	1
1	M	24 h	1	1	0	1	0	1
2	F		0	0	0	1	0	0
3	F		0	0	0	1	0	1
1	M	48 h	0	0	0	1	0	0
2	F		0	0	0	0	0	0
3	F		0	0	0	0	0	0
1	M	72 h	0	0	0	0	0	0
2	F		0	0	0	0	0	0
3	F		0	0	0	0	0	0

* Examinations were performed at the specified times after installation of the test material.

Applicant's summary and conclusion

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

Under the conditions of the study the test material did not induce significant or irreversible damage to the rabbit eye.

Executive summary:

The primary irritation potential of the test material was investigated according to OECD Test Guideline 405 and EU Method B.5 and in compliance with GLP.

The test material was applied by instillation of 0.1 g into the left eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 h after test material instillation.

The mean score was calculated across three scoring times (24, 48 and 72 h after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual means scores for iris were 0.0 for all three animals. The individual means scores for corneal opacity were 0.33, 0.0 and 0.0 respectively. The individual means scores for the conjunctivae were 0.67, 0.33 and 0.33 for reddening and 0.0 for chemosis for all three animals.

The instillation of the test material into the eye resulted in mild, early-onset and transient ocular changes such as corneal opacity, reddening of the sclerae, discharge and chemosis. These effects were reversible and were no longer evident 72 h after treatment, the end of the observation period for all animals. No abnormal findings were observed in the iris of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. Yellow remnants of the test material were observed in the eye or conjunctival sac of all animals on hour after treatment. No clinical signs were observed.

Under the conditions of the study the test material did not induce significant or irreversible damage to the rabbit eye.