hexasodium 5-[(4-{3-[(8-benzamido-1-hydroxy-3,6-disulfonato-2-naphthyl)diazenyl]-4-sulfonatoanilino}-6-[(2-sulfonatoethyl)amino]-1,3,5-triazin-2-yl)amino]isophthalate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 24 January 2007 and 08 February 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Magenta T-43

IUPAC Name:

Magenta T-43

Test material form:

solid: particulate/powder

Details on test material:

Sponsor's identification: JPD Magenta T-43
Description: dark red solid
Storage conditions: room temperature in the dark
Batch number: H186-5

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animals and Animal Husbandry

Three New Zealand White rabbits were supplied by an accredited supplier. At the start of the study the animals were in the weight range of 2.0 to 3.5 kg and were twelve to twenty weeks old. After an acclimatisation period of at least five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended cages. Free access to mains drinking water and food (Certified Rabbit Diet) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

A volume of 0.1 ml of the test material, which was found to weigh approximately 95 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye.

Duration of treatment / exposure:

The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test material.

Observation period (in vivo):

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours.
An additional observation was made in one treated eye on Day 7 to assess the reversibility of the ocular effects

Number of animals or in vitro replicates:

3 male rabbits

Details on study design:

Preparation of Test Material:
For the purpose of the study the test material was used as supplied.
The absorption of the test material was not determined.

Measurement of pH:
The pH of the test material was determined prior to commencement of the study. The results were tabulated.

Procedure:
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

Initially, a single rabbit was treated. A volume of 0.1 ml of the test material, which was found to weigh approximately 95 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test material, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test material, an assessment of the initial pain reaction was made.

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

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation 'Draize Scale for Scoring Ocular Irritation.'

Due to a technical error the 72-hour observation was missed in one treated eye but an observation was performed on Day 5. This deviation was considered not to affect the purpose or integrity of the study as reactions were still present in this treated eye at Day 5. Full recovery followed by redevelopment of reactions between the 48-hour observation and the Day 5 observation was considered unlikely.

Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

An additional observation was made in one treated eye on Day 7 to assess the reversibility of the ocular effects

Interpretation of Results:
The numerical values corresponding to each animal, tissue and observation time were recorded.
The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:

Score for conjunctivae = (A + B + C) x 2
Score for iris = D x 5
Score for cornea = (E x F) x 5

Using the numerical data obtained a modified version of the system described by Kay J H and Calandra J C (1962), J. Soc. Cosmet. Chern. 13, 281-289 was used to classify the ocular irritancy potential of the test material. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test material.

If evidence of irreversible ocular damage is noted, the test material will be classified as corrosive to the eye.

The results were also evaluated according to Regulation (EC) No. 1272/2008 (CLP).

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Time point:

other: mean of scores at 24, 48 h and 5 day

Score:

1.67

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: Due to a technical error the 72-hour observation was missed in one treated eye but an observation was performed on Day 5.

Remarks:

This deviation was considered not to affect the purpose or integrity of the study as reactions were still present in this treated eye at Day 5. Full recovery followed by redevelopment of reactions between the 48-hour observation and the Day 5 observation was considered unlikely.

Results 2

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 h

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 h

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: mean of scores at 24, 48 h and 5 day

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: Due to a technical error the 72-hour observation was missed in one treated eye but an observation was performed on Day 5.

Remarks:

This deviation was considered not to affect the purpose or integrity of the study as reactions were still present in this treated eye at Day 5. Full recovery followed by redevelopment of reactions between the 48-hour observation and the Day 5 observation was considered unlikely.

Results 5

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 h

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 h

Remarks on result:

no indication of irritation

Results 7

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: mean of scores at 24, 48 h and 5 day

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: Due to a technical error the 72-hour observation was missed in one treated eye but an observation was performed on Day 5.

Remarks:

This deviation was considered not to affect the purpose or integrity of the study as reactions were still present in this treated eye at Day 5. Full recovery followed by redevelopment of reactions between the 48-hour observation and the Day 5 observation was considered unlikely.

Results 8

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

no indication of irritation

Results 9

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

no indication of irritation

Results 10

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: mean of scores at 24, 48 h and 5 day

Score:

0.33

Max. score:

2

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: Due to a technical error the 72-hour observation was missed in one treated eye but an observation was performed on Day 5.

Remarks:

This deviation was considered not to affect the purpose or integrity of the study as reactions were still present in this treated eye at Day 5. Full recovery followed by redevelopment of reactions between the 48-hour observation and the Day 5 observation was considered unlikely.

Results 11

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 24 h

Remarks on result:

no indication of irritation

Results 12

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 24 h

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Reversibility of any observed effect: Changes were fully reversible within 7 days.

Other effects:

Due to a technical error, the 72 hour observation of one animal was not conducted until Day 5. This deviation was considered not to affect the purpose or integrity of the study as reactions were still present in this treated eye at Day 5. Full recovery followed by redevelopment of reactions between the 48-hour observation and the Day 5 observation was considered unlikely.

The mean scores for animal 1 were based upon the values from the 24 hour, 48 hour and 5 day observations.

Any other information on results incl. tables

Results

 

Individual and group mean scores for ocular irritation were tabulated. Red-coloured staining of the fur was noted around all treated eyes throughout the study.

 

Scattered or diffuse corneal opacity was noted in all treated eyes at the 24-hour observation and in one treated eye at the 48-hour and 5-day observations. Staining of the cornea was noted in all treated eyes one hour after treatment and at the 24 and 48-hour observations and in one treated eye at the 5-day observation.

 

Iridial inflammation was noted in all treated eyes one hour after treatment and in one treated eye at the 24-hour observation.

 

Moderate conjunctival irritation was noted in all treated eyes one hour after treatment with minimal to moderate conjunctival irritation notedinall treated eyes at the 24 and 48-hour observations and minimal conjunctival irritation noted in one treated eye at the 5-day observation.

 

Two treated eyes appeared normal at the 72-hour observation and the remaining treated eye appeared normal at the 7-day observation.

 

Individual Score and Individual Total Scores for Ocular Irritation

 

Rabbit No. & Sex	65649 Male					65755 Male				65756 Male
	IPR = 2					IPR = 2				IPR = 2
Time After Treatment	1 Hour	24 Hours	48 Hours	5 Days	7 Days	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA E= degree of opacity F = Area of Cornea Involved	0S          1S            1S          1S          0           0S           1S           0S           0           0S          1S           0S           0
	0            2              1             1            0           0              1            0             0            0             1             0            0
Score (E x F) x 5	0	10	5	5	0	0	5	0	0	0	5	0	0
IRIS D	1	1	0	0	0	1	0	0	0	1	0	0	0
Score (D x 5)	5	5	0	0	0	5	0	0	0	5	0	0	0
CONJUNCTIVAE A= Redness B = Chemosis C = Discharge
	2	2	2	1	0	2	1	1	0	2	1	1	0
	2	2	1	1	0	2	1	1	0	2	1	1	0
	3Sf	2Sf	1Sf	0Sf	0Sf	2Sf	1Sf	0Sf	0Sf	2Sf	1Sf	0Sf	0Sf
Score (A + B + C) x 2	14	12	8	4	0	12	6	4	0	12	6	4	0
Total Score	19	27	13	9	0	17	11	4	0	17	11	4	0

 

IPR = Initial pain reaction

S = Staining of the cornea

Sf = Red coloured staining of the fur around the treated eye

 

Individual Total Scores and Group Mean Scores for Ocular Irritation 

 

Rabbit No. and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours	7 Days
65649 Male	19	27	13	9*	0
65755 Male	17	11	4	0	-
65756 Male	17	11	4	0	-
Group Total	53	49	21	+	0
Group Mean Total	17.7	16.3	7.0	+	0.0

 

*= Observation performed on Day 5 due to a technical error

+ = As the 72-hour observation was not performed on one treated eye, it was impossible to calculate the true Group Total and Group Mean Score at the 72-hour observation, however, as reactions were still present in this treated eye at the Day 5 observation, it was considered unlikely that the Group Mean Score at the 72-hour observation would 0.0

-         = Observation not required

 

Applicant's summary and conclusion

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

The test material produced a maximum group mean score of 17.7 and was classified as a moderate irritant (class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item is not classified as an eye irritant according to the Classification, Labelling and Packaging Regulation (EC) 1272/2008 (CLP).

Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test material to the eye of the New Zealand White rabbit. The method was designed to meet the requirements of the following:

• OECD Guidelines for the Testing of Chemicals No. 405 "Acute Eye Irritation/Corrosion" (adopted 24 April 2002)

• Method B5 Acute Toxicity (Eye Irritation) of Commission Directive 2004/73/EC

Result

A single application of the test material to the non-irrigated eye of three rabbits produced scattered or diffuse corneal opacity, iridial inflammation and moderate conjunctival irritation. Two treated eyes appeared normal at the 72-hour observation and the remaining treated eye appeared normal at the 7-day observation.

Conclusion

The test material produced a maximum group mean score of 17.7 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item is not classified as an eye irritant according to the Classification, Labelling and Packaging Regulation (EC) 1272/2008 (CLP).