Disodium 4,4'-bis[(4-anilino-6-morpholino-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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

data is from experimental reports

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Principles of method if other than guideline:

The objective of the study was to assess the irritant and/or corrosive effects of test chemical on eye, when exposed by the ocular route in rabbits.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

240-245-2

EC Name:

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

Cas Number:

16090-02-1

Molecular formula:

C40H38N12Na2O8S2

IUPAC Name:

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

Test material form:

other: fine granules

Details on test material:

- Name of test material: disodium4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-disulphonate
- Molecular formula: C40-H40-N12-O8-S2.2Na
- Molecular weight: 925
- Substance type: Organic
- Physical state: Solid

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source:Procured from RABBI ROOF Animal Facility, A. P., India.
- Age at study initiation:3.0 to 4.5 Months (Approximately).
- Weight at study initiation:Minimum: 1.534 kg and Maximum: 1.685 kg (Prior to Treatment)
- Health Status :Healthy young adult. Females were nulliparous and non pregnant.
- Housing:The animals were housed individually in stainless steel cages.
- Room Sanitation:The experimental room floor and work tops were swept and mopped with disinfectant solution every day.
- Cages and water bottle:All the cages and water bottles were changed minimum twice a week.
- Diet (e.g. ad libitum):All animals were provided conventional laboratory rabbit diet (Nutrivet Life Sciences, Pune) ad libitum.
- Water (e.g. ad libitum):Aqua guard filtered tap water was provided ad libitum.
- Acclimation period:Rabbits were acclimatised to the test conditions for a period of 7 days (Animal No.-1) and 14 days (Animal No. 2 and 3) prior to the application of the test item.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):Minimum: 19.00 °C and Maximum: 20.80 °C
- Humidity (%):Minimum: 46.10 % and Maximum: 65.80 %.
- Air changes (per hr):More than 12 changes per hour
- Photoperiod (hrs dark / hrs light):12:12

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):0.1 g
- Concentration (if solution):N/A

VEHICLE
- Amount(s) applied (volume or weight with unit):N/A
- Concentration (if solution):N/A
- Lot/batch no. (if required):N/A
- Purity:N/A

Duration of treatment / exposure:

24 hrs

Observation period (in vivo):

All the animals were observed at 1, 24, 48, 72 hours, day 7 and 14 after instillation of test item.

Number of animals or in vitro replicates:

3 female rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done):The treated eye of rabbit was washed with normal saline.
- Time after start of exposure:24 hours

SCORING SYSTEM:Draize Method

TOOL USED TO ASSESS SCORE:Ophthalmoscope and sodium fluorescein strips.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

other: for treated eye

Results 2

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

not specified

Remarks on result:

other: for treated eye

Results 3

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: for treated eye

Results 4

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: for treated eye

Results 5

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: for treated eye

Results 6

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

not specified

Remarks on result:

other: for treated eye

Results 7

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not specified

Remarks on result:

other: for treated eye

Results 8

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

not specified

Remarks on result:

other: for treated eye

Irritant / corrosive response data:

The following grading scores were observed in treated eye of tested rabbits.
Observation at 1 hour after instillation of test item revealed: Cornea- No ulceration or opacity in all 3 animals; Area of Opacity- Zero in all 3 animals; Iris: Normal in all 3 animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible in all animal; Chemosis: some swelling above normal (includes nictitating membranes) was seen in animal no. 1 and 2 whereas obvious swelling with partial eversion of lids was observed in animal no. 3.

Observation at 24 hours after instillation of test item revealed: Cornea- No ulceration or opacity was observed in all the animals; Area of Opacity- Zero in all the animals; Iris: Normal in all 3 animals; Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible in all animal; Chemosis: some swelling above normal (includes nictitating membranes) was seen in animal no.2 whereas obvious swelling with partial eversion of lids was observed in animal no. 1 and 3.

At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 60 %, 40 % and 40 % damage in animal no. 1, 2 and 3, resectively. Corneal epithelium cell damage had reversed 0% in all the trreated animals on day 7 observation period.

Observation at 48 hours and 72 hours after instillation of test item revealed: Cornea- No ulceration or opacity in all 3 animals; Area of Opacity- Zero in all 3 animals; Iris: Normal in all 3 animals; Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was observed in animal no. 1 and 3 whereas some blood vessels definitely hyperaemic was observed in animal no.2; Chemosis: some swelling above normal (including nictiating membranes) was observed in all the animals.

Observation at day 7 after instillation of test item revealed: Cornea- No ulceration or opacity was observed in all animals; Area of Opacity- zero in all animal ; Iris: normal in all the animals;conjunctivae:blood vessels norma; in all the animals;Chemosis: no swelling was observed in all animals.

The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 0.00, 0.00, 2.00, 1.33, 1.00 and 0.00, 0.00, 2.00 and 1.33, respectively.

Other effects:

No systemic toxicity was observed in treated rabbits during the experimental period.

Mortality
No mortality was observed during the observation period.

Body weight
Increase in body weight of all the animals weighed at termination as comp[ared to day 0 body weight.

Any other information on results incl. tables

Table – : Individual Animal Eye Irritation Scores

 

Treated Dose: 0.1 g (pulverized form)                                                                    Sex: Female

 

Animal number	1
Application side	Right
Eye Reactions	*	Hour(s)				Day
Cornea	0	1	24	48	72	7
Area of Opacity	0	0	0	0	0	0
Iris	0	0	0	0	0	0
Conjunctiva	0	2	2	2	2	0
Chemosis	0	1	2	1	1	0
Corneal Damage (%)	0	/	60	50	30	0

 

 

Animal number	2
Application side	Right
Eye Reactions	*	Hour(s)				Day
Cornea	0	1	24	48	72	7
Area of Opacity	0	0	0	0	0	0
Iris	0	0	0	0	0	0
Conjunctiva	0	2	2	1	1	0
Chemosis	0	1	1	1	1	0
Corneal Damage (%)	0	/	40	30	20	0

 

Animal number	3
Application side	Right
Eye Reactions	*	Hour(s)				Day
Cornea	0	1	24	48	72	7
Area of Opacity	0	0	0	0	0	0
Iris	0	0	0	0	0	0
Conjunctiva	0	2	2	2	2	0
Chemosis	0	2	2	1	1	0
Corneal Damage (%)	0	/	40	30	20	0

 

Key : * = Pre-treatment eye examination

 

Table:1 Continued…..

Eye Irritation Scores – Mean Value after 24,48,72 hours ( Treated Eye) 

 

Animal No.     Eye Reaction	1	2	3
Corneal capacity	0.00	0.00	0.00
Iris	0.00	0.00	0.00
Conjunctiva	2.00.	1.33	2.00
Chemosis	1.33	1.00	1.33

 

Eye Irritation Scores – Mean Value after 24,48,72 hours ( Untreated Eye) 

 

Animal No.     Eye Reaction	1	2	3
Corneal capacity	0.00	0.00	0.00
Iris	0.00	0.00	0.00
Conjunctiva	0.00.	0.00	0.00
Chemosis	0.00	0.00	0.00

 

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the experimental conditions tested, all the three animals were fully irreversible within an observation period of 7 days.
Hence under the experimental test conditions,test chemical is “Mildly Irritating to Eyes” of New Zealand White Female rabbit eyes.Thus the test chemical is being classified as an eye irritant in 'Category 2' as per the CLP regulation.

Executive summary:

Acute Eye Irritation/Corrosion Study of test chemical in Rabbits.This study was performed as per OECD guideline no. 405.Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 0.1 g (pulverized) of test item was instilled in the other (treated) eye of each rabbit. The eye was observed at 1, 24, 48, 72 hour and day 7 for all the three animals post test item instillation. Ophthalmoscope was used for scoring of eye lesions.

In the initial test, 0.1 g (pulverized) of test item was instilled into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. As animal no. 1 showed no severe ocular lesions; hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 0.1 g (pulverized) of test item was instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control.All the animals were observed till day 7 post test item instillation.Untreated eye of all the three rabbits were normal throughout the experimental period.The following grading scores were observed in treated eye of tested rabbits.Observation at 1 hour after instillation of test item revealed: Cornea- No ulceration or opacity was observed in all the animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was observed in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in animal no. 1 and 2 whereas obvious swelling with partial eversion of lids was observed in animal no. 3.Observation at 24 hours after instillation of test item revealed: Cornea- No ulceration or opacity was observed in all the animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was observed in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in animal no. 2 whereas obvious swelling with partial eversion of lids was observed in animal no. 1 and 3.At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 60%, 40% and 40% damage in animal no. 1, 2 and 3 respectively. Corneal epithelium cell damage had reversed 0% in all the treated animals on day 7 observation period Observation at 48 and 72 hours after instillation of test item revealed: Cornea- No ulceration or Opacity was observed in all the animals: Area of Opacity- Zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was observed in animal no. 1 and 3 whereas some blood vessels definitely hyperaemic (injected) was observed in animal no. 2; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all the animals. Observation on day 7 after instillation of test item revealed: Cornea- No ulceration or Opacity was observed in all the animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Blood vessels normal in all the animals; Chemosis: No swelling (Normal) was observed in all the animals.The individual mean score of treated eyes for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 0.00, 0.00, 2.00, 1.33; 0.00. 0.00, 1.33, 1.00, and 0.00, 0.00, 2.00, 1.33, respectively.

Under the experimental conditions tested, all the three animals were fully irreversible within an observation period of 7 days.

Hence under the experimental test conditions, test chemical is “Mildly Irritating to Eyes” of New Zealand White Female rabbit eyes. Thus the test chemical is being classified as an eye irritant in 'Category 2' as per the CLP regulation.