Reactive Blue F08-0170

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 December 2010 - 23 December 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to EU, OECD & US EPA test standards 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 Blue F08-0170

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST 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: 12 - 13 weeks old (adult)
Sex: Male
Body weight range at the
beginning of the life phase: 3279 - 3364 g
end of the life phase: 3351 - 3422 g
Date of receipt: 01 December 2010
Acclimatization time: 19 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

Number of animal room: 618
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20 ± 3 °C
Relative humidity: 30 - 70%
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:

no

Amount / concentration applied:

A single dose of 0.1 g of the solid test item Reactive Blue F08-0170 was instilled into the conjunctival sac of the left eye.

Duration of treatment / exposure:

The treated eye of the rabbits was rinsed at 1 hour after application with physiological saline solution.

Observation period (in vivo):

The eyes were examined at 1, 24, 48 and 72 hours after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes.

Number of animals or in vitro replicates:

3 male

Details on study design:

Clinical Observations and Eye Examination

The eyes were examined at 1, 24, 48 and 72 hours 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 were recorded daily.

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

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:
corneal opacity ≥ 3 and/or
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:
corneal opacity ≥ 1 and/or
iritis ≥ 1, and/or
conjunctival redness ≥ 2 and/or
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 score

Time point:

other: overall at 24, 48 and 72 hours after the treatment

Score:

0

Max. score:

4

Reversibility:

fully reversible

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

mean score

Time point:

other: overall at 24, 48 and 72 hours after the treatment

Score:

0

Max. score:

4

Reversibility:

fully reversible

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

mean score

Time point:

other: overall at 24, 48 and 72 hours after the treatment

Score:

0

Max. score:

4

Reversibility:

fully reversible

Results 4

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

mean score

Time point:

other: overall at 24, 48 and 72 hours after the treatment

Score:

0

Max. score:

2

Reversibility:

fully reversible

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

mean score

Time point:

other: overall at 24, 48 and 72 hours after the treatment

Score:

0

Max. score:

2

Reversibility:

fully reversible

Results 6

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

mean score

Time point:

other: overall at 24, 48 and 72 hours after the treatment

Score:

0

Max. score:

2

Reversibility:

fully reversible

Results 7

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

mean score

Time point:

other: overall at 24, 48 and 72 hours after the treatment

Score:

0

Max. score:

4

Reversibility:

fully reversible

Results 8

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

mean score

Time point:

other: overall at 24, 48 and 72 hours after the treatment

Score:

0

Max. score:

4

Reversibility:

fully reversible

Results 9

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

mean score

Time point:

other: overall at 24, 48 and 72 hours after the treatment

Score:

0

Max. score:

4

Reversibility:

fully reversible

Results 10

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: 48

Max. score:

3

Reversibility:

fully reversible within: 48

Remarks on result:

other: The redness values were not described due to the discoloration caused by the test item therefore scoring and evaluation was not performed. The effects were reversible by the 48-hour endpoint.

Results 11

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

other: 48

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Remarks on result:

other: The redness values were not described due to the discoloration caused by the test item therefore scoring and evaluation was not performed. The effects were reversible by the 48-hour endpoint.

Results 12

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

other: 48

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Remarks on result:

other: The redness values were not described due to the discoloration caused by the test item therefore scoring and evaluation was not performed. The effects were reversible by the 48-hour endpoint.

Irritant / corrosive response data:

One hour after the application, chemosis (score 1) was observed in two rabbits, discharge (score 1 or 2) was seen in all rabbits, corneal opacity (score 1, area 1) was noted in one rabbit. The conjunctivae was coloured by the test item, therefore there was no possibility to score the conjunctival redness.

At 24 hours after treatment, the conjunctivae was coloured by the test item, therefore there was no possibility to score the conjunctival redness. No other signs of eye irritation were seen.

At 48 hours after treatment there were no signs of eye irritation observed.

At 72 hours after treatment, there were no signs of eye irritation observed.

Other effects:

No discharge as noted during the study.

Any other information on results incl. tables

Study Code:             10/252-005N                    Species:    NZW Rabbit

Dose:                                                                  Sex:          Male

Start of Exposure:    20 Dec 2010 – Day 0       Test Item: Reactive Blue F08-0170

 

 

Time	Animal No.	Score of irritation								IPR
		Conjunctivae			Opacity of cornea		Iris	Control eye	Other sign
		R	CH	D	OD	OE	R
1 hour	00887	*	1	2	0	0	0	0	-	3
	00612	*	1	1	0	0	0	0	-	3
	00013	*	0	1	1	1	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	00887	*	0	0	0	0	0	0	-
	00612	*	0	0	0	0	0	0	-
	00013	*	0	0	0	0	0	0	-

Time of Observation: Day 1

 Abbreviations:   R    = Redness                                OD =   Opacity degree of density

                            CH = Chemosis                              OE =   Extension of opaque area

D    = Discharge                              IPR=   Initial pain reaction

-     = No data

 

*: The redness values were not described due to the discoloration caused by the test item therefore scoring and evaluation was not performed.

Time	Animal No.	Score of irritation
		Conjunctivae			Opacity of cornea		Iris	Control eye	Other sign
		R	CH	D	OD	OE	R
48 hours	00887	0	0	0	0	0	0	0	-
	00612	0	0	0	0	0	0	0	-
	00013	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	00887	0	0	0	0	0	0	0	-
	00612	0	0	0	0	0	0	0	-
	00013	0	0	0	0	0	0	0	-

Time of Observation: Day 3

 

Abbreviations:   R    = Redness                                OD =   Opacity degree of density

                            CH = Chemosis                              OE =   Extension of opaque area

D    = Discharge                              IPR=   Initial pain reaction

-     =  No data

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item Reactive Blue F08-0170, applied to the rabbits’ eye mucosa, caused significant conjunctival irritant effects at one hour after application. The effects were fully reversible within 24 hours.

According to Regulation (EC) No 1272/2008, Reactive Blue F08-0170 does not require classification as an eye irritant.

Executive summary:

An acute eye irritation study of the test item Reactive Blue F08-0170 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. A volume 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 and 72 hours 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 after test item administration.

 

One hour after the application, chemosis (score 1) was observed in two rabbits, discharge (score 1 or 2) was seen in all rabbits, corneal opacity (score 1, area 1) was noted in one rabbit. The conjunctivae was coloured by the test item, therefore there was no possibility to score the conjunctival redness.

 

At 24 hours after treatment, the conjunctivae was coloured by the test item, therefore there was no possibility to score the conjunctival redness. No other signs of eye irritation were seen.

 

At 48 hours after treatment, there were no signs of eye irritation observed.

 

At 72 hours after treatment, there were no signs of eye irritation observed.

 

As no local or systemic clinical signs were observed 72 hours after test item administration, the study was terminated after the 72-hour 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
00887	0.00	0.00	0.00*	0.00	0.00
00612	0.00	0.00	0.00*	0.00	0.00
00013	0.00	0.00	0.00*	0.00	0.00

*: Conjunctival redness could not be assessed at 24 hours due to the discoloration caused by the test item. The means scoring is based on the results at 48 and 72 hours.

The test item Reactive Blue F08-0170, applied to the rabbits’ eye mucosa, caused significant conjunctival irritant effects at one hour after application. The effects were fully reversible within 24 hours. Discoloration was reversible within 48 hours.

 

According to Regulation (EC) No 1272/2008, Reactive Blue F08-0170 does not require classification as an eye irritant.