3,10-diamino-6,13-dichloro-2-((6-(((4-(1,1-dimethylethyl)phenyl)sulfonyl)amino)-2-naphthalenyl)sulfonyl)-4,11-triphenodioxazinedisulfonic acid, lithium potassium sodium salt

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 13, 2001 to January 10, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

July 31, 1992.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted February 24, 1987.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

440-770-9

EC Name:

-

Cas Number:

371921-63-0

Molecular formula:

C38 H29 Cl2 N5 O12 S4 .x K .x Li .x Na

IUPAC Name:

3,10-diamino-2-{[6-(4-tert-butylbenzenesulfonamido)naphthalen-2-yl]sulfonyl}-6,13-dichloro-5,12-dioxa-7,14-diazapentacene-4,11-disulfonic acid lithium hydride potassium hydride sodium hydride

Test material form:

solid

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Elevage Scientifique des Dombes F-01400 Chatillon sur Chalaronne I France
- Age at study initiation: 11-12 weeks (male #79); 10-11 weeks (females #80 & 81)
- Weight at study initiation: 2069g (male #79); 2273g (female #80); 2252g (female #81)
- Housing: Individually in stainless steel cages equipped with feed hoppers, drinking water bowls, with wood (RCC Ltd, Fullinsdorf) and haysticks for gnawing.
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch no. 91 /01) provided by Provimi Kliba AG, CH-4303 Kaiseraugst. Results of analysis are archived at RCC Ltd, ltingen.
Haysticks (QS no. 24/01) provided by Eberle Nafag AG, CH-9200 Gossau.
- Water (e.g. ad libitum): Community tap water from Fullinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at RCC Ltd, ltingen.
- Acclimation period: 5 days (November 2001, 13 to 18). Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1g/animal
- Concentration (if solution): undiluted

Duration of treatment / exposure:

21 days as the treated eyes were not rinsed after instillation

Observation period (in vivo):

21 days after application

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none
- Time after start of exposure: not relevant

SCORING SYSTEM: same grading of ocular lesions as the one shown in OECD TG 405

TOOL USED TO ASSESS SCORE: Eye examinations were made with a Varta Cliptrix diagnostic-lamp (A. Riegger, Basel I Switzerland)

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal: #79, 80 & 81

Time point:

other: 24h, 48h, 72h, 7d, 10d, 14d, 17d & 21d

Score:

0

Reversibility:

other: not relevant as scores remain nil

Results 2

Irritation parameter:

iris score

Basis:

animal: #79, 80 & 81

Time point:

other: 24h, 48h, 72h, 7d, 10d, 14d, 17d & 21d

Score:

0

Reversibility:

other: not relevant as scores remain nil

Results 3

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal: #79 & 80

Time point:

24/48/72 h

Score:

2

Reversibility:

fully reversible within: 10d

Results 4

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal: #79 & 80

Time point:

7 d

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 10d

Results 5

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal: #81

Time point:

24/48 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7d

Results 6

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal: #81

Time point:

72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7d

Results 7

Irritation parameter:

chemosis score

Basis:

animal: #79

Time point:

24/48 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 72h

Results 8

Irritation parameter:

chemosis score

Basis:

animal: #80

Time point:

24/48 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7d

Results 9

Irritation parameter:

chemosis score

Basis:

animal: #80

Time point:

72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7d

Results 10

Irritation parameter:

chemosis score

Basis:

animal: #81

Time point:

24 h

Score:

3

Max. score:

3

Reversibility:

fully reversible within: 7d

Results 11

Irritation parameter:

chemosis score

Basis:

animal: #81

Time point:

48 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7d

Results 12

Irritation parameter:

chemosis score

Basis:

animal: #81

Time point:

72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7d

Irritant / corrosive response data:

Application of the test item to healthy rabbit conjunctivae resulted in a primary irritation score of 3.33.
The eye reactions (mean values from 24 to 72 hours) consisted of grade 0.00 corneal opacity, grade 0.00 iris lesions, grade 1.89 redness of the conjunctivae and grade 1.44 chemosis of the conjunctivae.
Assessment of the cornea and iris was not possible in two animals 1 hour after treatment due to blue staining produced by the test item. No abnormal findings were noted in the cornea or iris of any animal at any of the other measurement intervals.
Reddening of the conjunctivae could not be fully evaluated in two animals 1 hour after treatment due to blue staining. Moderate reddening was however observed in the other animal at this examination. Slight to moderate reddening was evident in all animals from
24 to 72 hours after treatment and slight reddening persisted in two animals up to the 7-day reading.
Swelling, with partial eversion of lids, or with lids about half closed, was noted in all animals 1 hour after treatment and continued to be observed in two animals up to the 48-hour reading. The swelling gradually decreased in severity and was no longer evident in any animal 7 days after treatment.
A slight to moderate watery discharge was observed in all animals 1 hour after treatment and persisted in two animals up to the 24-hour reading.
Assessment of the sclerae was not possible at a number of readings due to either staining or swelling of the conjunctivae. Slight to moderate reddening was however subsequently observed in all animals but diminished to clear within 10 days of treatment.

Other effects:

- Mortality, lesions and clinical observations:
No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.

- Coloration
Blue remnants of the test item were observed in the eye or conjunctiva! sac of all animals 1 hour after treatment and continued to be observed in two animals at the 24- and 48-hour readings.
A light blue staining was observed in the treated eye of one animal 1 hour after treatment and light to marked staining was noted in another animal from 1 hour to 7 days after treatment. A light to marked blue staining was similarly observed in the remaining animal from 1 hour after treatment, however, the staining in this animal was present throughout the entire 21 day observation period.

Any other information on results incl. tables

The body weights (in grams) of all rabbits were considered to be within the normal range of variability:

 

Animal #	Sex	First Day of Acclimatization	Day of Treatment	Last Day of Observation
79	male	2069	2277	2815
80	female	2273	2620	3540
81	female	2252	2423	3276

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The test item produces at least in 2 of 3 tested animals, a positive response of conjunctival redness = 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.
However, a light to moderate blue staining was however noted in the treated eye of all animals and continued to be observed in one animal throughout the entire 21 day observation period.
Based on criteria defined in Regulation (EC) No 1272/2008, the substance Blue GS 5664.80 must be classified as Serious eye damage (Category 1) due to the persistance of Blue staining.

Executive summary:

The primary eye irritation potential of BLUE GS 5664.80 was investigated by instillation of 0.1 g into one eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours, as well as 7, 10, 14, 17 and 21 days after test item application. The scores of each animal at the following reading times (24, 48 and 72 hours) were used in calculating the respective mean values for each type of lesion.

The primary irritation score was calculated by totaling the mean cumulative scores at 24, 48 and 72 hours and then dividing the resulting total by the number of data points. The primary irritation score was 3.33 (max. 13). The eye reactions (mean values from 24 to 72 hours) consisted of grade 0.00 corneal opacity, grade 0.00 iris lesions, grade 1.89 redness of the conjunctivae and grade 1.44 chemosis of the conjunctivae.

The instillation of BLUE GS 5664.80 into the eye resulted in moderate, early-onset and transient signs of irritation. These changes included watery discharge, swelling and reddening of the conjunctivae and sclerae. These effects were reversible and were no longer evident 1 O days after treatment. No abnormal findings were observed in the cornea or iris of any animal at any reading. No corrosion was observed at any of the measuring intervals.

A light to moderate blue staining was however noted in the treated eye of all animals and continued to be observed in one animal throughout the entire 21 day observation period.

Based upon the referred classification criteria (EEC Commission Directive 93/21/EEC of April 27, 1993), with the exception of the staining which was still present in one animal at termination, BLUE GS 5664.80 is considered to be "not irritating" to the rabbit eye.