Reactive Orange F08-0314

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2011 to 24 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD, EU and US 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:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Name: Reactive Orange F08-0314

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

EXPERIMENTAL ANIMALS

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: 2933 – 3023 g, end of the life phase: 3689 – 3700 g
Date of receipt: 26 October 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: 607
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20 ± 3°C
Relative humidity: 28 - 65 %
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. Minor variations from the target humidity (min. 28%) range were observed during the study. These deviations were considered to have no impact on the animal health, as certified by the Clinical Veterinarian, or on the outcome of the study and interpretation of the results.

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 CiToxLAB Hungary Ltd.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: contralateral eye served as the control

Amount / concentration applied:

A single dose of 0.1 g of the solid test item REACTIVE ORANGE F08-0314 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

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.

For ethical reasons, since data on pain response with this test item is available, the animals were analgised using by Butorphanole (0.3-0.4 mg/ kg bw im) administered approximately 15 - 20 minutes before treatment. This procedure follows the principals of the draft OECD 405 guideline.
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.

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

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 i.v. 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).

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:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Results 8

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

(mean)

Time point:

other: Overall at 24, 48 and 72 hours

Score:

0

Max. score:

3

Reversibility:

other: Not applicable

Results 9

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

(mean)

Time point:

other: Overall at 24, 48 and 72 hours

Score:

0

Max. score:

3

Reversibility:

other: Not applicable

Results 10

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:

other: Not applicable

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

Max. score:

4

Reversibility:

other: Not applicable

Results 13

Irritation parameter:

other: Discharge

Basis:

animal #1

Remarks:

(mean)

Time point:

other: Overall at 24, 48 and 72 hours

Score:

0

Max. score:

3

Reversibility:

other: Not applicable

Results 14

Irritation parameter:

other: Discharge

Basis:

animal #2

Remarks:

(mean)

Time point:

other: Overall at 24, 48 and 72 hours

Score:

0

Max. score:

3

Reversibility:

other: Not applicable

Results 15

Irritation parameter:

other: Discharge

Basis:

animal #3

Remarks:

(mean)

Time point:

other: Overall at 24, 48 and 72 hours

Score:

0

Max. score:

3

Reversibility:

other: Not applicable

Irritant / corrosive response data:

The eyes were examined at 1, 24, 48, 72 hours and 1, 2 and 3 weeks after the application.

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

Initial Pain Reaction (IPR) (score 2 or 3) was observed in all animals after test item administration (data from study 10/296-005N).

One hour after the application:

Conjunctival redness (score 1) was found in all rabbits, conjunctival chemosis (score 1) were observed in one animal and all rabbits showed conjunctival discharge (score 3 or 2).

At 24 hours after treatment:
Conjunctival redness (score 1) was found in 1 rabbit.

At 48 and 72 hours after treatment:

No signs of eye irritation or other clinical signs were observed.

Other effects:

The surface of the cornea was coloured by the test item. The discoloration of the cornea was fully reversible within 1 week in two animals, and within 2 weeks in one animal.

The conjunctivae 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.

Any other information on results incl. tables

There was no mortality observed during the study.

The body weight and body weight change were considered to be normal with no indication of treatment related effect.

There were no clinical signs observed that could be related to treatment.

TABLE 1: INDIVIDUAL SCORES FOR OCULAR IRRITATION: 

 

Study Code:             10/296-005NR                Species:    NZW Rabbit

Dose:                        0.1 g                               Sex:          Male

Start of Exposure:    03 November 2011          Test Item: REACTIVE ORANGE F08-0314

 

Time	Animal No.	Score of irritation
		Conjunctivae			Opacity of cornea		Iris	Control eye	Other sign
		R	CH	D	OD	OE	R
1 hour	00832	1	1	3	0	0	0	0	*
	00838	1	0	3	0	0	0	0	*
	00843	1	0	2	0	0	0	0	*

 

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	00832	1	0	0	0	0	0	0	*
	00838	0	0	0	0	0	0	0	*
	00843	0	0	0	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	00832	0	0	0	0	0	0	0	*
	00838	0	0	0	0	0	0	0	*
	00843	0	0	0	0	0	0	0	*

Time of Observation: Day 2

Time	Animal No.	Score of irritation
		Conjunctivae			Opacity of cornea		Iris	Control eye	Other sign
		R	CH	D	OD	OE	R
72 hours	00832	0	0	0	0	0	0	0	*
	00838	0	0	0	0	0	0	0	*
	00843	0	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	00832	0	0	0	0	0	0	0	*
	00838	0	0	0	0	0	0	0	**
	00843	0	0	0	0	0	0	0	**

Time of Observation: Day 7

 

Time	Animal No.	Score of irritation
		Conjunctivae			Opacity of cornea		Iris	Control eye	Other sign
		R	CH	D	OD	OE	R
2 weeks	00832	0	0	0	0	0	0	0	**
	00838	0	0	0	0	0	0	0	**
	00843	0	0	0	0	0	0	0	**

Time of Observation: Day 14

 

Time	Animal No.	Score of irritation
		Conjunctivae			Opacity of cornea		Iris	Control eye	Other sign
		R	CH	D	OD	OE	R
3 weeks	00832	0	0	0	0	0	0	0	**
	00838	0	0	0	0	0	0	0	**
	00843	0	0	0	0	0	0	0	**

Time of Observation: Day 21

 

Abbreviations:   R    = Redness                                OD =   Opacity degree of density

                           CH = Chemosis                               OE =   Extent of opaque area

D    = Discharge                              

*: The surface of the cornea and the conjunctivae were coloured by the test item.

**: The conjunctivae were coloured by the test item.

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 ORANGE F08-0314, applied to rabbit eye mucosa, caused conjunctival irritant effects at one hour which were reduced at 24 hours after application. There were no corneal irritation effects observed at any timepoint, all conjunctival irritation effects were completely reversed after the 48 hours observation period. Discoloration of the cornea was fully reversible within 2 weeks. Some discoloration of the conjuctivae remained at the observation period of 3 weeks, but this is not considered as a classifiable effect under CLP or GHS guidelines.
REACTIVE ORANGE F08-0314 is “not classified” according to Regulation (EC) No 1272/2008.

Executive summary:

An acute eye irritation study of the test item REACTIVE ORANGE F08-0314 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 of 0.1 g of 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 behaviour 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 animals.

Initial Pain Reaction (IPR) (score 2 or 3) was observed in all animals after test item administration (data from study 10/296-005N).

One hour after the application:

Conjunctival redness (score 1) was found in all rabbits, conjunctival chemosis (score 1) were observed in one animal and all rabbits showed conjunctival discharge (score 3 or 2).

At 24 hours after treatment:

Conjunctival redness (score 1) was found in 1 rabbit.

At 48 and 72 hours after treatment:

No signs of eye irritation or other clinical signs were observed.

At 1, 2 and 3 weeks after treatment:

No signs of eye irritation or other clinical signs were observed.

The surface of the cornea was coloured by the test item. The discoloration of the cornea was fully reversible within 1 week in two animals, and within 2 weeks in one animal.

The conjunctivae 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
00832	0.00	0.00	0.33	0.00	0.00
00838	0.00	0.00	0.00	0.00	0.00
00843	0.00	0.00	0.00	0.00	0.00

 

The test item, REACTIVE ORANGE F08-0314, applied to rabbit eye mucosa, caused conjunctival irritant effects at one hour which were reduced at 24 hours after application. There were no corneal irritation effects observed at any timepoint, all conjunctival irritation effects were completely reversed after the 48 hours observation period. Discoloration of the cornea was fully reversible within 2 weeks. Some discoloration of the conjuctivae remained at the observation period of 3 weeks, but this is not considered as a classifiable effect under CLP or GHS guidelines.

REACTIVE ORANGE F08-0314 is “not classified” according to Regulation (EC) No 1272/2008.