4-[(4,6-bis{[3-(diethylamino)propyl]amino}-heteromonocycle-2-yl)amino]-N-[2-(4,5,6,7-tetrachloro-1,3-dioxo-2,3-dihydro-1H-inden-2-yl)quinolin-8-yl]benzenesulfonamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 September to 18 October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

No.476 YELLOW

IUPAC Name:

No.476 YELLOW

Details on test material:

- Substance type: Yellow to orange powder
- Lot/batch No.: 00082

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex and Harlan, Belton, Leics, England.
- Age at study initiation: Animals used within the study were at least 6 weeks old*
- Weight at study initiation: body weights were at least 1.0 kg.*
- Housing: labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
- Diet (e.g. ad libitum): Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least three times a week.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimatization period: at least 5 days before start of treatment under laboratory conditions.

* Animal specifications (sex, age and body weight) are specified in the attached table.

A health inspection was performed prior to commencement of treatment, to ensure that the animals were in a good state of health. Special attention was paid to the eyes, which were free from any abnormality.

Results of analysis for diet (nutrients and contaminants), hay and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.2 – 22.3ºC
- Humidity (%): 40 - 85%
Temporary deviations from the maximum level of relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

IN LIFE DATES: From: 27 September to 18 October 2010

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: One eye of each animal remained untreated and served as the reference control.

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied: 41.2 mg (range 40.7 – 41.6 mg)

Duration of treatment / exposure:

Single instillation on Day 1.

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 days after instillation of the test substance.

Number of animals or in vitro replicates:

3 males

Details on study design:

STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner approximately 2 weeks later, after considering the degree of eye irritation observed in the first animal.

TREATMENT
Animals were treated by instillation of, on average, 41.2 mg (range 40.7 – 41.6 mg) of the test substance (a volume of approximately 0.1 mL) as the maximum required amount in the conjunctival sac of one of the eyes 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 substance. The other eye remained untreated and served as the reference control.

Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.

After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

REMOVAL OF TEST SUBSTANCE
-Washing (if done): No

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and after the final observation.
- Necropsy: No necropsy was performed according to protocol.
- Irritation:
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 days after instillation of the test substance.
The irritation scores and a description of all other (local) effects were recorded.The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:

CORNEAL IRRITATION
Opacity: degree of density (area most dense taken for reading)
0: No ulceration or opacity (may include slight dulling of normal luster)
1: Scattered or diffuse areas of opacity, details of iris clearly visible
2: Easily discernible translucent area, details of iris slightly obscured
3: Nacreous area, no details of iris visible, size of pupil barely discernible
4: Opaque cornea, iris not discernible through the opacity

Area of cornea involved:
0: No ulceration or opacity
1: One quarter or less but not zero
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 or combination thereof, iris still reacting to light (sluggish reaction is positive)
2: No reaction to light, hemorrhage, gross destruction (any or all of these)

CONJUNCTIVAL IRRITATION
Redness (refers to palpebrae and sclera, excluding cornea and iris):
0: Blood vessels normal
1: Some blood vessels definitely hyperaemic (injected)
2: Diffuse, crimson color, individual vessels not easily discernible
3: Diffuse beefy red

Chemosis (refers to lids and/or nictitating membranes):
0: No swelling
1: Any swelling above normal (includes 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

Discharge:
0: No discharge (may include small amounts observed in inner canthus of normal animals)
1: Any amount different from normal and/or lacrimation
2: Discharge with moistening of the lids and hairs just adjacent to lids
3: Discharge with moistening of the lids and hairs (considerable area around the eye)

Where standard lighting was considered inadequate for observing minor effects, eye examinations were performed using an ophthalmic examination lamp.
In cases of equivocal results when comparing the treated and untreated eyes, the illustrated guide from the Consumer Product Safety Commission, Washington, D.C. 20207 was used for additional control purposes.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

animal #1

Remarks:

(mean)

Time point:

other: 24, 48 and 72 hrs.

Score:

0

Max. score:

4

Results 2

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

animal #2

Remarks:

(mean)

Time point:

other: 24, 48 and 72 hrs.

Score:

0

Max. score:

4

Results 3

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

animal #3

Remarks:

(mean)

Time point:

other: 24, 48 and 72 hrs.

Score:

0

Max. score:

4

Results 4

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

(mean)

Time point:

other: 24, 48 and 72 hrs.

Score:

0

Max. score:

2

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

(mean)

Time point:

other: 24, 48 and 72 hrs.

Score:

0

Max. score:

2

Results 6

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

(mean)

Time point:

other: 24, 48 and 72 hrs.

Score:

0

Max. score:

2

Results 7

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Remarks:

(mean)

Time point:

other: 24, 48 and 72 hrs.

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Results 8

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #2

Remarks:

(mean)

Time point:

other: 24, 48 and 72 hrs.

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Results 9

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #3

Remarks:

(mean)

Time point:

other: 24, 48 and 72 hrs.

Score:

1.3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Results 10

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

(mean)

Time point:

other: 24, 48 and 72 hrs.

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Results 11

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

(mean)

Time point:

other: 24, 48 and 72 hrs.

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Results 12

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

(mean)

Time point:

other: 24, 48 and 72 hrs.

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Instillation of approximately 42 mg of the test substance (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation completely resolved within 72 hours in one animal and within 7 days in the other two animals.

No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.


Individual and mean eye irritation scores are specified in the attached table.

Any other information on results incl. tables

Remnants of the test substance were present in the eye of all animals on Day 1.

Yellow/orange staining of the fur on the head and paws, caused by the test substance, was noted during the observation period.

 

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Applicant's summary and conclusion

Interpretation of results:

slightly irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: GHS (2007), EC (1272/2008)

Conclusions:

In an in vivo eye irritation study in rabbits, the test substance induced slight irritation, which was fully reversible within 2-7 days.
Based on these results the test substance does not have to be classified and has no obligatory labelling requirement for eye irritation according to the:
- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007),
- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.