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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 March 2011 to 31 March 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU and OECD test guidance in compliance with GLP and reported with a valid GLP certificate.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
The relative humidity (min 24%) was out of the target range (30-70%) during the study.
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
The relative humidity (min 24%) was out of the target range (30-70%) during the study.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Details on test material:
Name: Reactive Red F08-0146

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 rabbits
Source: S&K-LAP Kft., 2173 Kartal, Császár út 135, Hungary
Justification of strain: The New Zealand White rabbit is one of the standard strains used for acute irritation toxicity studies.
Animal health: Only animals in acceptable health condition were used for the test. Both eyes of each animal provisionally selected for testing were examined approximately one hour before starting the study. Animals showing eye irritation, ocular defects or pre-existing corneal injury were not used.
Number of animals: 3 animals
Age of animals at treatment:~11 weeks old (adult)
Sex: Male
Body weight range at the beginning of the life phase: 2899 – 3214 g
end of the life phase: 3474 – 3924 g
Date of receipt: 02 March 2011
Acclimatization time: 8 days
Animal identification: The individual identification was by engraved ear tag. The cages were marked with individual identity cards with information about study code, sex, dose group, cage number and individual animal number.

HUSBANDRY
Animal health: Only healthy animals were used for the test. The veterinarian certified health status.
Number of animal room: 619
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20 ± 3°C
Relative humidity: 24 - 59 %
Housing/Enrichment: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages
Ventilation: 15-20 air exchanges/hour

The environmental parameters were recorded twice daily during the study.

FOOD AND FEEDING

Animal received PURINA Base – Lap gr. diet for rabbits produced by AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi road, Hungary, ad libitum.

WATER SUPPLY AND QUALITY CONTROL OF WATER

The animals received municipal tap water, as for human consumption, ad libitum, from an automatic system.

The quality control analysis is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). The quality control results are retained in the archives of LAB Research Ltd.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: The contralateral eye served as the control.
Amount / concentration applied:
A single dose of 0.1 g of the solid test item REACTIVE RED F08-0146 was administered to each animal.
Duration of treatment / exposure:
The eyes of the test animals were washed out at 1 hour after application of test item.
Observation period (in vivo):
The eyes were examined at 1, 24, 48, 72 hours and 1, 2 and 3 weeks after treatment.
Number of animals or in vitro replicates:
3 male animals
Details on study design:
Application of the Test Item

Three male animals in acceptable health condition were selected for the test. Care was taken to select only those animals that had a normal eye condition and any with ocular lesions were rejected.

An initial test was performed using one animal. The test item was instilled into the conjunctival sac of the left eye. The eyelids were held closed for several seconds to prevent the loss of the test item. The contralateral eye served as the control. Immediately after the administration of the test item, an assessment of the initial pain reaction was made.

After consideration of the ocular responses produced in the first animal, two additional animals were treated.

OBSERVATIONS AND SCORING

Clinical Observations

The eyes were examined at 1, 24, 48, 72 hours and 1, 2 and 3 weeks after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes. Any clinical signs of toxicity or signs of ill- health during the study were recorded.

At the end of the observation period, each animal was sacrificed by intramuscular injections of CP-Ketamin 10% and CP-Xylazin 2% followed by iv. Euthasol® 40% anaesthesia. Death was verified by checking pupil and corneal reflex and the absence of respiration.

Scoring and Assessment of Local Reaction

The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (24 April 2002).

Classification of the Test Items

Individual reactions of the animal were recorded at each observation time. The nature, severity and duration of all lesions observed were described.

Results were presented and interpreted according to Regulation (EC) No 1272/2008, as follows:

Irreversible effects on the eye/serious damage to eyes (Category 1)

Substances that have the potential to seriously damage the eyes are classified in Category 1 (irreversible effects on the eye). These observations include animals with grade 4 cornea lesions and other severe reactions (e.g., destruction of cornea) observed at any time during the test, as well as persistent corneal opacity, discoloration of the cornea by a dye substance, adhesion, pannus, and interference with the function of the iris or other effects that impair sight.

Category for irreversible eye effects
If, when applied to the eye of an animal, a substance produces:
— at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days;
and/or
— at least in 2 of 3 tested animals, a positive response of:
o corneal opacity ≥ 3 and/or
o iritis > 1.5
calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material.

Reversible effects on the eye/irritating to eyes (Category 2)

Substances that have the potential to induce reversible eye irritation are classified in Category 2 (irritating to eyes).

Category for reversible eye effects
If, when applied to the eye of an animal, a substance produces:
— at least in 2 of 3 tested animals, a positive response of:
o corneal opacity ≥ 1 and/or
o iritis ≥ 1, and/or
o conjunctival redness ≥ 2 and/or
o conjunctival oedema (chemosis) ≥ 2

— calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days

Measurement of Body Weight

Individual body weight was recorded at the beginning and end of the experiment.




Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
other: Not applicable
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
other: Not applicable
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
other: Not applicable
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
not specified
Remarks:
Not applicable
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 1 week
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Irritation parameter:
conjunctivae score
Basis:
animal #1
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 3 weeks
Irritation parameter:
conjunctivae score
Basis:
animal #2
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 3 weeks
Irritation parameter:
conjunctivae score
Basis:
animal #3
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 1 week
Irritation parameter:
other: Discharge
Basis:
animal #1
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
other: Discharge
Basis:
animal #2
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
other: Discharge
Basis:
animal #3
Remarks:
mean
Time point:
other: Overall at 24, 48 and 72 hours
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
Initial Pain Reaction (IPR) (score 2) was observed in all animals after test item application.

One hour after the application:
Conjunctival redness (score 2) was found in two rabbits and redness (score 1) was seen in one animal. Different severities of conjunctival chemosis (score 1, 2 or 3) were observed in all animals and all the rabbits showed conjunctival discharge (score 3). Corneal opacity (score 1, area 2) was noted in one animal.

At 24 hours after treatment:
Conjunctival redness (score 2 or 1) was found in two or one rabbits, respectively and discharge (score 1) was observed in all animals. One rabbit displayed conjunctival chemosis (score 1).

At 48 hours after treatment:
Conjunctival redness (score 2) was seen in all rabbits.

At 72 hours after treatment:
Conjunctival redness (score 1) was observed in two animals and redness (score 2) was noted in one rabbit.

At 1 and 2 weeks after treatment:
Conjunctival redness (score 1) was observed in 2 rabbits.

At 3 weeks after treatment:
No signs of eye irritation or other clinical signs were observed.

The study was terminated after the 3 weeks observation.

During the study, no signs of eye irritation were observed in the control eye of all animals
Other effects:
The surface of the cornea and the conjunctiva were coloured by the test item during the study up to and including the 3-week observation.
There was no mortality observed during the study.
There were no clinical signs observed that could be related to treatment.
The body weight and body weight change were considered to be normal with no indication of treatment related effect.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item REACTIVE RED F08-0146, applied to rabbit eye mucosa, caused significant conjunctival and corneal irritant effects at one hour which were reduced at 24 hours after application. All irritation effects were completely reversed after the 3-week observation period; however, discoloration of the cornea has not fully reversed within the observation period of 3 weeks.
Due to to not fully reversible staining effect on the cornea, REACTIVE RED F08-0146 requires classification as an eye irritant (Irreversible effects on the eye/serious damage to eyes (Category 1)) according to Regulation (EC) No 1272/2008.
Executive summary:

An acute eye irritation study of the test item REACTIVE RED F08-0146 was performed in New Zealand White rabbits. The irritation effects of the test item were evaluated according to the Draize method (OECD No.: 405, 2002).

The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. An amount ofof the test item was administered as a single dose.

 

Individual body weight was recorded at the beginning and end of the study. Morbidity and clinical signs of toxicity were checked daily. The eyes were examined at 1, 24, 48, 72 hours and 1, 2 and 3 weeks after the application.

 

No adverse effects on body weight development were noted during the study period. The general state and behavior of animals were normal throughout the study period.

 

Eye examination:

 

During the study, no signs of eye irritation were observed in the control eye of all animals.

 

 

Initial Pain Reaction(IPR) (score 2) was observed in all animals after test item administration.

 

 

One hour after the application: Conjunctival redness (score 2) was found in two rabbits and redness (score 1) was seen in one animal. Different severities of conjunctival chemosis (score 1, 2 or 3) were observed in all animals and all rabbits showed conjunctival discharge (score 3). Corneal opacity (score 1, area 2) was noted in one animal.

At 24 hours after treatment: Conjunctival redness (score 2 or 1) was found in two or one rabbits, respectively and discharge (score 1) was observed in all animals. One rabbit displayed conjunctival chemosis (score 1).

At 48 hours after treatment: Conjunctival redness (score 2) was seen in all rabbits.

 

At 72 hours after treatment: Conjunctival redness (score 1) was observed in two animals and redness (score 2) was noted in one rabbit.

 

At 1 and 2 weeks after treatment: Conjunctival redness (score 1) was observed in 2 rabbits.

 

At 3 weeks after treatment: No signs of eye irritation or other clinical signs were observed.

 

The surface of the cornea and the conjunctiva were coloured by the test item during the study up to and including the 3-week observation. The study was terminated after the 3-week observation.

 

The animal’s individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

Animal Number

Cornea Opacity

Iris

Conjunctivae

Redness

Chemosis

Discharge

00726

0.00

0.00

1.33

0.00

0.33

00786

0.00

0.00

2.00

0.33

0.33

00779

0.00

0.00

1.33

0.00

0.33

The test item REACTIVE RED F08-0146, applied to rabbit eye mucosa, caused significant conjunctival and corneal irritant effects at one hour which were reduced at 24 hours after application. All irritation effects were completely reversed after the 3-week observation period; however, discoloration of the cornea had not fully reversed within the observation period of 3 weeks.

Due to not fully reversible staining effect on the cornea, REACTIVE RED F08-0146 requires classification as an eye irritant (Irreversible effects on the eye/serious damage to eyes (Category 1)) according to Regulation (EC) No 1272/2008.