Tetrasodium 4,4'-bis[[4-morpholino-6-(p-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 May 7 2008 to 16 May 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted April 24, 2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 4,4'-bis[[4-morpholino-6-(p-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

EC Number:

249-323-0

EC Name:

Tetrasodium 4,4'-bis[[4-morpholino-6-(p-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Cas Number:

28950-61-0

Molecular formula:

C40H40-xN12NaxO14S4

IUPAC Name:

tetrasodium 2,2'-ethene-1,2-diylbis[5-({4-morpholin-4-yl-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate]

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 0582900
- Expiration date of the lot/batch: 25-FEB-2013
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (range of 20 ± 5 °C), light protected.
- Stability under storage conditions: Stable under storage conditions.

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands BV.
- Age at study initiation: 16 weeks (male) and 14-15 weeks (females).
- Weight at study initiation: 2515 g - 3155 g.
- Allocation: Male No. 64 and Female Nos. 65, 66.
- Accommodation: individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (RCC Ltd, Füllinsdorf) and haysticks 4642 (batch no. 36/07, Provimi Kliba AG) were provided for gnawing.
- Diet: ad libitum, pelleted standard Provimi Kliba 3418 rabbit maintenance diet (batch no. 03/08) provided by Provimi Kliba AG, CH-4303 Kaiseraugst.
- Water: ad libitum, community tap water from Füllinsdorf.
- Acclimation period: under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: continuously monitored environment with ranges for room temperature 17-23 °C.
- Humidity: between 30-70 % (values above 70 % during cleaning process possible).
- Air changes: air-conditioned with 10-15 air changes per hour.
- Photoperiod: automatically controlled light cycle of 12 hours light and 12 hours dark.
- Other: music during the daytime light period.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 g (per animal) of test item was weighed and applied undiluted as it was delivered by the sponsor.
- pH solution: the pH of a 1 % (w/w) solution was measured and was found to be 7-8.

Duration of treatment / exposure:

FAT 66042/A TE 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 test item.

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48, 72 hours after administration.

Number of animals or in vitro replicates:

Three (one male and two females)

Details on study design:

TREATMENT
The eyes of the animals were examined one day prior to test item 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 test item 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 test item.

Necropsy:
No necropsy was performed on the animals sacrificed at termination of observation. The animals were killed by intravenous injection of Pentobarbitone into the ear vein at a dose of at least 1 ml/kg body weight (162 mg sodium pentobarbitone/kg body weight) and discarded.

SCORING SYSTEM
The eye reactions were assessed according to the numerical scoring system listed in the Commission Directive 2004173/EC, April 29, 2004 (see page 24). Scleral reddening and ocular discharge were also assessed.

TOOL USED TO ASSESS SCORE: Varta Cliptrix diagnostic-lamp (Roth AG, CH-4153 Reinachi Switzerland); the eyes of each animal were examined approximately 1, 24, 48, 72 hours after administration.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

iris score

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 h

Results 4

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No abnormal findings were observed in the cornea or iris of any animal at any of the measurement intervals. Slight to moderate reddening of the conjunctivae was noted in all animals 1 hour after treatment and persisted as slight up to the 48-hour reading. A slight to moderate reddening of the sclerae was present in all animals 1 hour after treatment and persisted as slight up to the 24-hour observation. A Slight swelling (chemosis) of the conjunctivae was observed 1 hour after instillation. Slight to moderate ocular discharge was recorded in all three animals 1 hour after treatment. No abnormal findings were observed in the treated eye of any animal 24, 48 or 72 hours after treatment, the end of the observation period for all animals.

Coloration: no staining produced by the test item of the treated eye was observed.
Test Item Remnants: yellow test item remnants were evident in the eye or conjunctival sac of the male animal 1 hour after treatment.
Corrosion: no corrosion of the cornea was observed at any of the reading times.

Other effects:

No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.

Any other information on results incl. tables

Eye irritation scores

Animal N.	Sex	Parameter	1 hr	24 hrs	48 hrs	72 hrs	Mean*
64	M	Corneal Opacity	0	0	0	0	0.00
		Iritis	0	0	0	0	0.00
		Conjunctival redness	2	1	1	0	0.67
		Conjunctival chemosis	1	0	0	0	0.00
65	F	Corneal Opacity	0	0	0	0	0.00
		Iritis	0	0	0	0	0.00
		Conjunctival redness	1	1	1	0	0.67
		Conjunctival chemosis	1	0	0	0	0.00
66	F	Corneal Opacity	0	0	0	0	0.00
		Iritis	0	0	0	0	0.00
		Conjunctival redness	1	1	1	0	0.67
		Conjunctival chemosis	1	0	0	0	0.00

* mean calculated across 24, 48 and 72 hours after instillation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 66042/A is considered as not irritating to rabbit eye.

Executive summary:

The primary eye irritation potential of test item was investigated according to OECD test guideline no. 405. The test item 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 hours after test item instillation. The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.67 for reddening for all animals and 0.00 for chemosis for all animals. The instillation of test item into the eye resulted in mild to moderate, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and chemosis. These effects were reversible and were no longer evident 24, 48 or 72 hours after treatment. No abnormal findings were observed in the cornea or iris of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. Yellow test item remnants were evident in the eye or conjunctival sac of the male animal 1 hour after treatment. No staining of the treated eyes by the test item was observed and no clinical signs were observed. Based on the study results, FAT 66042/A is considered as not irritating to rabbit eye.