Salts of 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthenium chloride and 4-{[1-hydroxy-6-({[5-hydroxy-6-(phenyldiazo)-7-sulfonaphthalen-2-yl]carbamoyl}amino)-3-sulfonaphthalen-2-yl]diazo}benzoic acid (2:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2008-07-14 to 2008-08-04

Reliability:

1 (reliable without restriction)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

not applicable

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Species and strain: New Zealand White rabbit
Source: TETRABBIT Kft. 2173 Kartal, Császár út 135, HUNGARY
Hygienic level during the study: Good conventional
Justification of strain: The albino New Zealand White rabbit is the preferred animal in acute eye irritation studies.
Number of animals: 3 animals
Sex: Male
Age of animals at arrival: Young adult rabbit, 12 weeks old
Body weight at the beginning of the study: 3101-3257 g
Body weight at the end of the study: 3602-3752 g
Acclimatisation time: 11 days
Only animals in acceptable health condition were used for the test as certified by the veterinarian.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

A total volume of 0.1 g of the test item was used for the study in pure state, in a single dose. The eye of test animal was washed out with physiological saline one hour after the test item application. The pH of the test item was measured before the study initiation and was found to be 6.0.

Duration of treatment / exposure:

one hour

Observation period (in vivo):

The eyes were examined at 60 minutes, 24, 48, 72 hours then one, two and three weeks after the treatment. The duration of the observation period was sufficient for the statement of reversibility or irreversibility of changes. All of the observations were performed with using a hand slit lamp. All of the observations were performed with using a hand slit lamp. There was no indication of using fluorescein dying as the signs were properly observable without using this method. At the end of the observation period euthanasia of the animal was carried out by intra-muscular injections of CP-Ketamin 10% and CP-Xylazin 2% followed by iv. Euthasol® 40% anaesthesia.

Number of animals or in vitro replicates:

3 males

Details on study design:

not applicable

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

animal #1

Remarks:

8579

Time point:

other: 24, 48, 72 h

Score:

1

Max. score:

4

Reversibility:

not reversible

Remarks:

within 21 days

Results 2

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

animal #2

Remarks:

8586

Time point:

other: 24, 48, 72 h

Score:

1

Max. score:

4

Reversibility:

not reversible

Remarks:

within 21 days

Results 3

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

animal #3

Remarks:

8585

Time point:

other: 24, 48, 72 h

Score:

1

Max. score:

4

Reversibility:

not reversible

Remarks:

within 21 days

Results 4

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

mean

Time point:

other: 24, 48, 72 h

Score:

1

Max. score:

4

Reversibility:

not reversible

Remarks:

within 21 days

Results 5

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

8579

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

2

Reversibility:

not specified

Results 6

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

8586

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

2

Reversibility:

not specified

Results 7

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

8585

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

2

Reversibility:

not specified

Results 8

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

2

Reversibility:

not specified

Results 9

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Remarks:

8579

Time point:

other: 24,48,72 h

Score:

1

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Results 10

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #2

Remarks:

8586

Time point:

other: 24, 48, 72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Results 11

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #3

Remarks:

8585

Time point:

other: 24, 48, 72 h

Score:

1.33

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Results 12

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

mean

Time point:

other: 24, 48, 72 h

Score:

1.44

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Results 13

Irritation parameter:

cornea opacity score

Remarks:

(chemosis)

Basis:

animal #1

Remarks:

8579

Time point:

other: 24, 48, 72 h

Score:

1.67

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Results 14

Irritation parameter:

conjunctivae score

Remarks:

(chemosis)

Basis:

animal #2

Remarks:

8586

Time point:

other: 24, 48, 72 h

Score:

2.33

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Results 15

Irritation parameter:

conjunctivae score

Remarks:

(chemosis)

Basis:

animal #3

Remarks:

8585

Time point:

other: 24, 48, 72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Results 16

Irritation parameter:

conjunctivae score

Remarks:

(chemosis)

Basis:

mean

Time point:

other: 24, 48, 72 h

Score:

2.33

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Results 17

Irritation parameter:

conjunctivae score

Remarks:

(discharge)

Basis:

animal #1

Remarks:

8579

Time point:

other: 24, 48, 72 h

Score:

2.67

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Results 18

Irritation parameter:

conjunctivae score

Remarks:

(discharge)

Basis:

animal #2

Remarks:

8586

Time point:

other: 24, 48, 72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Results 19

Irritation parameter:

conjunctivae score

Remarks:

(discharge)

Basis:

animal #3

Remarks:

8585

Time point:

other: 24, 48, 72 h

Score:

2.67

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Results 20

Irritation parameter:

conjunctivae score

Remarks:

(discharge)

Basis:

mean

Time point:

other: 24, 48, 72 h

Score:

2.5

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Irritant / corrosive response data:

During the study, no primary irritation symptoms occurred in the control eye of the animals.
24 hours after treatment:
In case of animal No. 8579 redness (grade 1), chemosis (grade 1) and discharge (grade 3) were observed. In case of animal No. 8586 and animal No.8585 chemosis (grade 1) and discharge (grade 2) were observed. All animals had initial pain reaction (grade 1).
48 hours after treatment:
Animal No. 8579 had conjunctival redness (grade 1), chemosis (grade 2), discharge (grade 2) and cornea opacity (grade 1) with an area of 4. The signs observed on animal No. 8586 were conjunctival redness (grade 2), chemosis (grade 3), discharge (grade 1) and cornea opacity (grade 1) with an area of 4. In case of animal No. 8585 conjunctival redness (grade 1), chemosis (grade 3), discharge (grade 3) and cornea opacity (grade 1) with an area of 4 were observed.
72 hours after the treatment:
On animal No. 8579 conjunctival redness (grade 1), chemosis (grade 1), discharge (grade 3) and cornea opacity (grade 1) with an area of 4 were observed. Animal No. 8586 had conjunctival redness (grade 2), chemosis (grade 3), discharge (grade 2) and cornea opacity (grade 1) with an area of greater than three quarters of the cornea. In case of animal No. 8585 conjunctival redness (grade 1), chemosis (grade 3), discharge (grade 2) and cornea opacity (grade 1) with an area of greater than three quarters of the cornea were observed
At one week after the treatment:
In case of animal No. 8579 cornea opacity (grade 3) with an area of 4 was observed. On animal No. 8586 conjunctival redness (grade 1), discharge (grade 3) and cornea opacity (grade 3) with an area of greater than three quarters of the cornea were observed. On animal No. 8585 chemosis (grade 1), discharge (grade 3) and cornea opacity (grade 3) with an area of greater than three quarters of the cornea were noted. Due to the severe cornea opacity, the correct observation of the iris was not possible.
At two weeks after the treatment:
In case of animal No. 8579 cornea opacity (grade 2) with an area of greater than three quarters of the cornea was observed. Both animal No. 8586 and 8585 had cornea opacity (grade 3) with an area of greater than three quarters of the cornea. Correct observation of the cornea was impossible due to the severe corneal opacity.
At three weeks after the treatment:
On animals No. 8579, 8586 and 8585 cornea opacity (grade 2) with an area of 4 was observed. Details of the iris was not observable in case of all animals.

Other effects:

During the study the control eyes of the animals were symptom-free.
General state and the behaviour of animal were normal throughout the study period.
Treating with Test Item F 213 Red had no impact on the body weight change of the animals showed under the duration of the study.

Any other information on results incl. tables

During the study, no primary irritation symptoms occurred in the control eye of the animals. One hour after treatment: In case of animal No. 8579 redness (grade 1), chemosis (grade 1) and discharge (grade 3) were observed. In case of animal No. 8586 and animal No.8585 chemosis (grade 1) and discharge (grade 2) were observed. All animals had initial pain reaction (grade 1). At 24 hours after treatment: Animal No. 8579 had conjunctival redness (grade 1), chemosis (grade 2), discharge (grade 3) and cornea opacity (grade 1) with an area of 4. On animal No. 8586 conjunctival redness (grade 2), chemosis (grade 1), discharge (grade 3) and cornea opacity (grade 1) with an area of 4 were observed. In the case of animal No. 8585 conjunctival redness (grade 2), chemosis (grade 3), discharge (grade 3) and cornea opacity (grade 1) with an area of 4 were noted. At 48 hours after the treatment: Animal No. 8579 had conjunctival redness (grade 1), chemosis (grade 2), discharge (grade 2) and cornea opacity (grade 1) with an area of 4. The signs observed on animal No. 8586 were conjunctival redness (grade 2), chemosis (grade 3), discharge (grade 1) and cornea opacity (grade 1) with an area of 4. In case of animal No. 8585 conjunctival redness (grade 1), chemosis (grade 3), discharge (grade 3) and cornea opacity (grade 1) with an area of 4 were observed. At 72 hours after the treatment: On animal No. 8579 conjunctival redness (grade 1), chemosis (grade 1), discharge (grade 3) and cornea opacity (grade 1) with an area of 4 were observed. Animal No. 8586 had conjunctival redness (grade 2), chemosis (grade 3), discharge (grade 2) and cornea opacity (grade 1) with an area of greater than three quarters of the cornea. In case of animal No. 8585 conjunctival redness (grade 1), chemosis (grade 3), discharge (grade 2) and cornea opacity (grade 1) with an area of greater than three quarters of the cornea were observed At one week after the treatment: In case of animal No. 8579 cornea opacity (grade 3) with an area of 4 was observed. On animal No. 8586 conjunctival redness (grade 1), discharge (grade 3) and cornea opacity (grade 3) with an area of greater than three quarters of the cornea were observed. On animal No. 8585 chemosis (grade 1), discharge (grade 3) and cornea opacity (grade 3) with an area of greater than three quarters of the cornea were noted. Due to the severe cornea opacity, the correct observation of the iris was not possible. At two weeks after the treatment: In case of animal No. 8579 cornea opacity (grade 2) with an area of greater than three quarters of the cornea was observed. Both animal No. 8586 and 8585 had cornea opacity (grade 3) with an area of greater than three quarters of the cornea. Correct observation of the cornea was impossible due to the severe corneal opacity.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Since the cornea opacity caused by the test item was irreversible within 21 days observation period, F 213 Red is classified as ”R41 - Risk of serious damage to eyes”.

Executive summary:

The acute eye irritation study of the test item F 213 Red was performed in three albino New Zealand White rabbits. The irritation effect of the test item was evaluated according to the Draize method (OECD No.: 405, 2002). The test item was placed into the conjunctival sac of the left eye of the animals. The untreated right eye served as control. 0.1 g of the test item was used for the study in pure state, as a single dose. The eyes of the test animals were washed out with physiological saline at observation one hour after the treatment. One hour after the single application of test item F 213 Red into one eye of the rabbits, chemosis and discharge of conjunctivae were observed in all rabbits, whereas redness was only noted in one animal. Initial pain reaction was observed in all test animals. 24, 48 and 72 hours after the treatment conjunctival redness, chemosis, discharge and slight cornea opacity with an area of greater, than three quarters of the cornea were found in all animals. One week after the treatment conjunctival redness and discharge were found in one rabbit. In one other animal, conjunctival chemosis and discharge were noted. In all rabbits cornea opacity was observed, no details of iris were visible in an area of greater than three quarters of the cornea. Two weeks after the treatment cornea opacity with an area of greater than three quarters of the cornea was still observed in all animals. Three weeks after the treatment cornea opacity was observed in case of all rabbits. The area of the cornea involved did not change compared to the one week and two week observation. Treating with test item F 213 Red had no impact on the body weight change of the animals showed under the duration of the study. During the study the control eyes of the animals were symptom-free. General state and the behaviour of animal were normal throughout the study period.