disodium 7-(4,6-difluoropyrimidin-2-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate; reaction mass of: disodium 7-(2,4-difluoropyrimidin-6-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Delivery of the animals : October, 30, 1996 - Euthanasia of the animals : November 25, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted February 24, 1987

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 31, 1992

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

426-840-1

EC Name:

-

IUPAC Name:

tetrasodium 7-[(2,6-difluoropyrimidin-4-yl)amino]-4-hydroxy-3-[2-(4-methoxy-2-sulfophenyl)diazen-1-yl]naphthalene-2-sulfonate 7-[(4,6-difluoropyrimidin-2-yl)amino]-4-hydroxy-3-[2-(4-methoxy-2-sulfophenyl)diazen-1-yl]naphthalene-2-sulfonate

Test material form:

solid

Details on test material:

Identity: Scarlet RN 1165
Appearance : Solid powder, dark-red

Specific details on test material used for the study:

PROTOCOL DEVIATION: the test article classification was omitted at the request of the sponsor.

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Remarks:

Chbb:NZW (SPF)

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH; Birkendorferstrasse 65; D - 88397 Biberach / Riss
- Age at treatment: 15 weeks
- Weight at study initiation:
body weight at start of acclimatization: male 2.5 kg, females 2.8 kg
body weight at start of treatment: male 2.5 kg, females 2.8-2.9 kg
- Housing: Individually in stainless steel cages with an automatic cleaning system, equipped with feed hoppers, drinking water bowls and wood for gnawing.
- Diet (e.g. ad libitum): Pelleted standard Kliba 341 rabbit maintenance diet (batch 70/96 and 71/96) ad libitum from Kliba Muhlen AG, CH-4303 Kaiseraugst. Results of analysis for contaminants are included in this report.
- Water (e.g. ad libitum): Community tap water from Itingen, ad libitum. Results of bacteriological, chemical and contaminant analyses are included in this report.
- Acclimation period: Four days under laboratory conditions after health examination. Only healthy animals were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): between 40-70 % (values above 70% during cleaning process possible)
- Air changes (per hr): Air-conditioned with 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light/12 hours dark, music during the light period

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

the test article was applied as delivered by the sponsor

Amount / concentration applied:

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

Duration of treatment / exposure:

21 days as the treated eyes were not rinsed after instillation days

Observation period (in vivo):

Scoring of irritation effects was performed approximatively 1, 24, 48 and 72 hours as well as 7, 14 and 21 days after test article application

Number of animals or in vitro replicates:

3: 1 male (#16) and 2 females (#17 and #18)

Details on study design:

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

SCORING SYSTEM: see page 14 and 15 of the study report

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: #16, #17 & #18

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: Not relevant as scores remain nil

Results 2

Irritation parameter:

iris score

Basis:

animal: #16, #17, #18

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: Not relevant as scores remain nil

Results 3

Irritation parameter:

conjunctivae score

Basis:

animal: #16, #17 & #18

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 48h

Results 4

Irritation parameter:

conjunctivae score

Basis:

animal: #16, #17 & #18

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible within: 48h

Results 5

Irritation parameter:

other: Conjunctivae chemosis

Basis:

animal: #16 & #18

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: Not relevant as scores remain nil

Results 6

Irritation parameter:

chemosis score

Basis:

animal: #17

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 48h

Results 7

Irritation parameter:

chemosis score

Basis:

animal: #17

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

fully reversible within: 48h

Results 8

Irritation parameter:

other: Staining of conjunctivae and sclera

Basis:

animal: #16, #17 & #18

Time point:

other: 24/48/72h & 7/14/21d

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

IRRITATION
SCARLET RN 1165 showed a primary irritation score of 0.44, when applied to the conjunctival sac of the rabbit eye.
Slight to moderate swelling and slight swelling of the conjunctivae was seen in all animals. These findings were reversible in all animals after 48 hours.

CORROSION
No corrosion of the cornea was observed at any of the reading times.

Other effects:

VIABILITY/MORTALITY AND CLINICAL SIGNS
No clinical signs of systemic toxicity were observed in the animals during the test and observation period, and no mortality occurred.

COLORATION
Irreversible light red staining of the conjunctivae and sclera by the test article was observed, although reversible light red staining of the iris and cornea of all animals was also seen.

BODY WEIGHTS
The body weight of the animals was within the normal range of variability (for results, please refer to the point "Any other information on results incl. tables" hereafter).

Any other information on results incl. tables

EYE IRRITATION SCORES - INDIVIDUAL VALUES

Animal	Sex	Cornea opacity	Iris	Conjunctivae		Cumul. score	Mean cumul. score
				Redn.	Chem.
1 hour
16	M	0	0	-	2*	2	1.33
17	F	0	0	-	1	1
18	F	0	0	-	1	1
24 hours
16	M	0	0	1	0	1	1.33
17	F	0	0	1	1	2
18	F	0	0	1	0	1
48 hours
16	M	0	0	0	0	0	0.00
17	F	0	0	0	0	0
18	F	0	0	0	0	0
72 hours
16	M	0	0	0	0	0	0.00
17	F	0	0	0	0	0
18	F	0	0	0	0	0
7 days
16	M	0	0	0	0	0	0.00
17	F	0	0	0	0	0
18	F	0	0	0	0	0
14 days
16	M	0	0	0	0	0	0.00
17	F	0	0	0	0	0
18	F	0	0	0	0	0
21 days
16	M	0	0	0	0	0	0.00
17	F	0	0	0	0	0
18	F	0	0	0	0	0

BODY WEIGHTS

Animal #	Sex	First Day of Acclimatization	Day of Treatment	Last Day of Observation
16	male	2456	2507	3161
17	female	2802	1761	3670
18	female	2813	2945	3769

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Based on criteria defined in Regulation (EC) No 1272/2008, SCARLET RN 1165 is classified as Serious eye damage (category 1). Eye irritation test shows persistence of red light staining at the end of the 21 days recovery period which is according to the CLP guideline a reason for Serious eye damage Cat 1 (discoloration of the cornea by a dye substance).

Executive summary:

The primary irritation potential of the test article was investigated by instillation of 0.1 g into one eye of each of three young adult New Zealand rabbits. The treated eyes were not rinsed after application. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours as well as 7, 14 and 21 days after test article 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 totalling the individual cumulative scores at 24, 48 and 72 hours and then dividing the resulting total by the number of figures. The primary irritation score was 0.44 (max. 13).

Swelling and reddening of the conjunctivae was seen in all animals. These findings were reversible after 48 hours.

Irreversible light red staining of the conjunctivae and sclera by the test article was observed, although reversible light .red staining of the iris and cornea of all animals was also seen.

No corrosion was observed at any of the measuring intervals.

The mean values of the scores for each type of lesion, calculated for each animal separately, are the following:

Animal No.	Mean values 24-72 hours
	Cornea Opacity	Iris	Conjunctivae
			Redness	Chemosis
16, male	0.00	0.00	0.33	0.00
17, female	0.00	0.00	0.33	0.33
18, female	0.00	0.00	0.33	0.00