Reactive Red F03-0318

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 March 2011 to 12 April 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study conducted to EU & 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 guideline

Test guideline 1

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.

Test guideline 2

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.

Principles of method if other than guideline:

The deviations are considered to have no impact on the outcome of the study and interpretation of the results.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Name: Reactive Red F03-0318

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: ~12 weeks old (adult)
Sex: Male
Body weight range at the
beginning of the life phase: 2923 – 3311 g
end of the life phase: 3514 – 3996 g
Date of receipt: 09 March 2011
Acclimatization time: 13 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: 620
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20 ± 3°C
Relative humidity: 24 - 63 %
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: untreated right eye served as control

Amount / concentration applied:

A single dose of 0.1 g of the solid test item Reactive Red F03-0318 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 using physiological saline solution.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 male

Details on study design:

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 according to a six point scale.

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

OBSERVATIONS AND SCORING

Clinical Observations and Eye Examination

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 study.

Results and discussion

In vivo

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Results 5

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

Results 6

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

Results 7

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

mean

Time point:

other: Overall at 24, 48 and 72 hours

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 3 weeks

Results 8

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

mean

Time point:

other: Overall at 24, 48 and 72 hours

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 3 weeks

Results 9

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: 3 weeks

Results 10

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

mean

Time point:

other: Overall at 24, 48 amd 72 hours

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Results 11

Irritation parameter:

chemosis 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

Results 12

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

mean

Time point:

other: Overall at 24, 48 and 72 hours

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Initial Pain Reaction (IPR) (score 3) was observed in all animals.

One hour after the application:

Conjunctival redness (score 1) and different severities of conjunctival discharge (score 2 or 3) were found in all rabbits. Two animals showed conjunctival chemosis (score 2 or 3). The conjunctiva was coloured by the test item in all animals, the cornea in one rabbit. The cornea discoloration persisted up to and including the 72 hour observation, the conjunctiva discoloration was not longer present at the 3 weeks observation.

At 24 hours after treatment:

Conjunctival redness (score 2 or 1) was found in two and one rabbit, respectively, and discharge (score 1) was observed in all animals. Two rabbits displayed conjunctival chemosis (score 1).

At 48 and 72 hours and 1 week after treatment:

Conjunctival redness (score 22 or 1) was seen in one rabbit and two rabbits, respectively.

At 2 weeks after treatment, conjunctival redness (score 1) was found in all rabbits.

At 3 weeks after treatment neither signs of eye irritation nor discoloration of the tissues of the eyes 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:

There was no mortality observed during the study.
The body weights and body weight changes were considered to be normal with no indication of treatment related effect.
The general state and behavior of animals were normal throughout the study period. There were no clinical signs observed that could be related to treatment.

Any other information on results incl. tables

INDIVIDUAL SCORES FOR OCULAR IRRITATION

 

 

Study Code:             11/008-005N                    Species:    NZW Rabbit

Dose:                        0.1 g                                 Sex:          Male

Start of Exposure:    22 March 2011                  Test Item: Reactive Red F03-0318

 

 

Abbreviations:   R    = Redness                                OD =   Opacity degree of density

                            CH = Chemosis                              OE =   Extentof opaque area

D    = Discharge                             IPR=   Initial pain reaction

 

 

Time	Animal No.	Score of irritation								IPR
		Conjunctivae			Opacity of cornea		Iris	Control eye	Other sign
		R	CH	D	OD	OE	R
1 hour	00008	1	2	3	0	0	0	0	*	3
	00009	1	0	2	0	0	0	0	*	3
	00003	1	3	3	0	0	0	0	**	3

Time of Observation: Day 0

Time	Animal No.	Score of irritation
		Conjunctivae			Opacity of cornea		Iris	Control eye	Other sign
		R	CH	D	OD	OE	R
24 hours	00008	2	1	1	0	0	0	0	*
	00009	1	0	1	0	0	0	0	*
	00003	2	1	1	0	0	0	0	**

Time of Observation: Day 1

Time	Animal No.	Score of irritation
		Conjunctivae			Opacity of cornea		Iris	Control eye	Other sign
		R	CH	D	OD	OE	R
48 hours	00008	2	0	0	0	0	0	0	*
	00009	1	0	0	0	0	0	0	*
	00003	1	0	0	0	0	0	0	**

Time of Observation: Day 2

*: The conjunctiva was coloured by the test item.

**: The conjunctiva and the cornea were coloured by the test item.

 

Time	Animal No.	Score of irritation
		Conjunctivae			Opacity of cornea		Iris	Control eye	Other sign
		R	CH	D	OD	OE	R
72 hours	00008	2	0	0	0	0	0	0	*
	00009	1	0	0	0	0	0	0	*
	00003	1	0	0	0	0	0	0	**

 

Time of Observation: Day 3

 

Time	Animal No.	Score of irritation
		Conjunctivae			Opacity of cornea		Iris	Control eye	Other sign
		R	CH	D	OD	OE	R
1 week	00008	2	0	0	0	0	0	0	*
	00009	1	0	0	0	0	0	0	*
	00003	1	0	0	0	0	0	0	*

 

Time of Observation: 1 week

 

*: The conjunctiva was coloured by the test item.

**: The conjunctiva and the cornea were coloured by the test item.

Time	Animal No.	Score of irritation
		Conjunctivae			Opacity of cornea		Iris	Control eye	Other sign
		R	CH	D	OD	OE	R
2 weeks	00008	1	0	0	0	0	0	0	*
	00009	1	0	0	0	0	0	0	*
	00003	1	0	0	0	0	0	0	*

 Time of Observation: 2 weeks

*: The conjunctiva was coloured by the test item.

 

Time	Animal No.	Score of irritation
		Conjunctivae			Opacity of cornea		Iris	Control eye	Other sign
		R	CH	D	OD	OE	R
3 weeks	00008	0	0	0	0	0	0	0	-
	00009	0	0	0	0	0	0	0	-
	00003	0	0	0	0	0	0	0	-

 Time of Observation: 3 weeks

 

MEAN VALUES OF EYE IRRITATION (24, 48, 72 hour reading)

Animal Number	Sex	Cornea Opacity	Iris	Conjunctivae
				Redness	Chemosis	Discharge
00008	male	0.00	0.00	2.00	0.33	0.33
00009	male	0.00	0.00	1.00	0.00	0.33
00003	male	0.00	0.00	1.33	0.33	0.33

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item Reactive Red F03-0318, applied to rabbit eye mucosa, caused significant conjunctival irritant effects at one hour after application. The effects were fully reversible within 3 weeks.

According to Regulation (EC) No 1272/2008, REACTIVE RED F03-0318 does not require classification as an eye irritant.

Executive summary:

An acute eye irritation study of the test item Reactive Red F03-0318was 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 3) was observed in all animals immediately after test item administration.

 

One hour after the application:

 

Conjunctival redness (score 1) and different severities of conjunctival discharge (score 2 or 3) were found in all rabbits. Two animals showed conjunctival chemosis (score 2 or 3). The conjunctiva was coloured by the test item in all animals, the cornea in one rabbit. The cornea discoloration persisted up to and including the 72 hour observation, the conjunctiva discoloration was not longer present at the 3 weeks observation.

At 24 hours after treatment:

 

Conjunctival redness (score 2 or 1) was found in two and one rabbit, respectively, and discharge (score 1) was observed in all animals. Two rabbits displayed conjunctival chemosis (score 1).

At 48 and 72 hours and 1 week after treatment:

 

Conjunctival redness (score 2 or 1) was seen in one rabbit and two rabbits, respectively.

 

At 2 weeks after treatment, conjunctival redness (score 1) was found in all rabbits.

 

At 3 weeks after treatment, neither signs of eye irritation nor discoloration of the tissues of the eyes were observed.

 

The study was terminated after the 3 weeks 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
00008	0.00	0.00	2.00	0.33	0.33
00009	0.00	0.00	1.00	0.00	0.33
00003	0.00	0.00	1.33	0.33	0.33

The test item REACTIVE RED F03-0318, applied to rabbit eye mucosa, caused significant conjunctival irritant effects at one hour application. The effects were fully reversible within 3 weeks.

According to Regulation (EC) No 1272/2008, REACTIVE RED F03-0318 does not require classification as an eye irritant.