Reaction mass of Trisodium 4-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-{[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazenyl}benzenesulfonate and Trisodium 4-({4-chloro-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-{[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazenyl}benzenesulfonate

Endpoint summary

Description of key information

FAT 40000 is to be considered as a non-irritant to the skin and eye of rabbits.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 02 July 1992; Experiment start date - 07 July 1992; Experiment completion date - 10 July 1992; Study completion date - 05 October 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Code number: FAT 40000/G
Batch number: 67/92
Purity: 79.5%
Appearance: solid
Solubility: miscible
Storage: room temperature
Expiration date: 01 June 1993.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH Postfach 1755 0·7950 Biberach a.d. Riss
- Age at study initiation: 15 weeks
- Weight at study initiation: male : 2.8 kg females: 3.2 kg
- Housing: Individually in stainless steel cages (size: 86 x 54 x 33 cm) equipped with feed hoppers and drinking water bottles.
- Diet (e.g. ad libitum): Pelleted standard Kliba 341, Batch 69/92 rabbit maintenance diet ("Kliba", Klingentalmuehle AG, CH-4303 Kaiseraugst), ad libitum.
- Water (e.g. ad libitum): Community tap water from FOllinsdorf, ad libitum
- Acclimation period: Five days under test conditions after veterinary examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C
- Humidity (%): 40 ± 70%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark, music during the light period.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.5 g of substance was applied.

Duration of treatment / exposure:

4 hours

Number of animals:

1 male
2 females

Details on study design:

Approximately 24 hours before treatment, the dorsal fur was clipped with an electric clipper, exposing an area of approximately 100 square centimeters (10 cm x 10 cm). The skin of the animals was examined once, 1 day prior to test ( .article administration. Only those animals with no signs of skin injury or irritation were used in the test. On test day 1, 0.5 g of the test article was applied to approx. 6 sq.cm of the intact skin of the clipped area. It was covered with a 3.0 cm x 3.0 cm patch of surgical gauze. The gauze was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with an elastic bandage. The duration of treatment was 4 hours. Then the dressing was removed and the skin was flushed with lukewarm-tap water.

EVALUATION OF SKIN REACTION
Erythema and eschar formation
No erythema........................................................................................................................ 0
Very slight erythema (barely perceptible)................................................................................ 1
Well defined erythema.......................................................................................................... 2
Moderate to severe erythema................................................................................................. 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth).............................. 4

Edema formation
No edema............................................................................................................................. 0
Very slight edema (barely perceptible)..................................................................................... 1
Sli~ht edema (edges of area well defined by definite raising) ......................................................2
Moderate edema (raised approximately 1 mm).......................................................................... 3
Severe edema (raised more than 1 mm and extending beyond area of exposure)..........................4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

FAT 40'000/G showed a primary irritation score of 0.0 (max. 8.0) when applied to healthy intact rabbit skin. Local signs (mean values from 1 hour to 72 hours) consisted of grade 0.25 erythema and grade 0.0 edema.

Other effects:

No systemic symptoms were observed in the animals during the test and observation period, and no mortality occurred.

COLORATION: In the area of application yellow staining of the treated skin by pigment or coloring of the test article was observed in two animals from 1 hour after removal of the dressing to the study termination and in one animal from 1 hour to 24 hours after removal of the dressing.

CORROSION: No destructions or irreversible alterations of the treated skin were observed. No corrosive effect was evident on the skin.

BODY WEIGHTS: The body weight gain of all rabbits was within the normal range of variability.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is to be considered as a non-irritant to the skin of rabbits.

Executive summary:

A study was performed according to OECD test guideline 404 and EEC Directive B.4 to determine the primary-irritation index, which serves as a measure of the acute irritation, provoked by the test item, on the skin of the rabbits.

0.5 g of the test article was applied to approx. 6 sq.cm of the intact skin of the clipped area. It was covered with a 3.0 cm x 3.0 cm patch of surgical gauze. The gauze was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with an elastic bandage. The duration of treatment was 4 hours. Then the dressing was removed and the skin was flushed with lukewarm-tap water. The animals were checked daily for systemic symptoms and mortality. The skin reactions were evaluated 1, 24, 48, and 72 hours after removing the gauze patches according to the OECD scoring system. The application site was yellow stained up to 24 hours after the application; therefore the evaluation of a possible erythema formation was impeded.

The mean 24/48/72h scores edema and erythema score was zero. Based on this findings, the test item was considered to be "non-irritant" to the rabbit skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 16 July 1992; Experiment completion date - 28 July 1992; Study completion date - 30 September 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Code number: FAT 40000/G
Batch number: 67/92
Purity: 79.5%
Appearance: solid
Solubility: miscible
Storage: room temperature
Expiration date: 01 June 1993.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Postfach 1755, D-7950 Biberach an der Riss
- Age at study initiation:15 weeks
- Weight at study initiation: male : 2.9 kg females: 2.4 - 2.8 kg
- Housing: Individually in stainless steel cages (size: 86 x 54 x 33 cm) equipped with feed hoppers and drinking water bottles.
- Diet (e.g. ad libitum): Pelleted standard Kliba 341, Batch 69/92 rabbit maintenance diet ("Kliba", Klingentalmuehle AG, CH-4303 Kaiseraugst), ad libitum. Results of analysis for contaminants are included in this report.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are included in this report.
- Acclimation period: Five days under laboratory conditions after veterinary examination. Only animals without any visual signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C
- Humidity (%): 40-70 %
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light (approx. 100 Lux)/12 hours dark, music during the light period.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 g of the test article

Observation period (in vivo):

1, 24, 48 and 72 hours and 7 days after administration.

Number of animals or in vitro replicates:

1 male
2 females

Details on study design:

SCORING SYSTEM:

CORNEAL IRRITATION
Opacity: degree of density (densest area used for assessment)
No ulceration or opacity..................................................................................................................................0
Scattered or diffuse areas of opacity (other than slight dulling of normal luster), details of iris clearly visible............1
Easily discernible translucent area, details of iris slightly obscured.....................................................................2
Nacrous area, no details of iris visible, size of pupil barely discernible.................................................................3
Opaque cornea, iris not discernible through the opacity.....................................................................................4

IRIDIC IRRITATION
Normal..............................................................................................................................0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperemia, or injection, any of these or combination thereof, iris still reacting to light (sluggish reaction is positive)........................................................................1
No reaction to light, hemorrhage, gross destruction (any or all of these)...................................2

CONJUNCTIVAL IRRITATION
Redness (refers to palpebral and bulbar conjunctivae, cornea and iris). Blood vessels normal.....................0
Some blood vessels definitely hyperemic (injected)............................................................................... 1
Diffuse, crimson color, individual vessels not easily discernible............................................................... 2
Diffuse beefy red.................................................................................................................................3

Chemosis: lids and/or nictitating membranes
No swelling................................................................................................. 0
Any swelling above normal (includes nictitating membranes)........................... 1
Obvious swelling with partial eversion of lids................................................... 2
Swelling with lids about half closed............................................................... 3
Swelling with lids more than half closed..........................................................4

TOOL USED TO ASSESS SCORE: hand-slit lamp

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0.88

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within:

Remarks:

48 h

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

FAT 40'000/G showed a primary irritation score of: 1.67, when applied to the conjunctival sac of the rabbit eye. Only one animal was found to show opacity in the first hour of the test period. No corrosion was observed in all the other animals.

Other effects:

No acute systemic clinical symptoms were observed in the animals during the test and observation period, and no mortality occurred.

The lidhairs were yellowish dicolored in all animals at 1 hour after test article installation.

Female no. 61 lost weight (up to 8 %) during the aclimatization period. The body weight of the other animals was within the normal range of variability.

Observation table

OBESERVATION PERIOD	ANIMAL	SEX	CORNEA OPACITY	IRIS	CONJUNCTIVAE REDNESS	CONJUNCTIVAE CHEMOSIS
1 HR	59	M	1	0	1	1
	60	F	0	0	1	1
	61	F	0	0	2	2
24 HR	59	M	0	0	1	1
	60	F	0	0	1	1
	61	F	0	0	1	1
48 HR	59	M	0	0	1	0
	60	F	0	0	1	0
	61	F	0	0	1	0
72 HR	59	M	0	0	1	0
	60	F	0	0	1	0
	61	F	0	0	0	0
7 DAYS	59	M	0	0	0	0
	60	F	0	0	0	0
	61	F	0	0	0	0

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is considered to be "non-irritant" to the eyes.

Executive summary:

A GLP study was performed according to OECD test guideline 405 to determine the eye irritation potential of test substance.

The test substance was instilled in the eyes of 3 rabbits and observations for irritation were recorded at the time points of 1 hour, 24 hours, 48 hours 72 hours. On test day 1, 0.1 g of the test article (undiluted) was placed in the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test article. The right eye remained untreated and served as the reference control. The observation was made at 1h, 24h, 48h, 96h and 7 days timepoint.

The mean corneal opacity, iris, conjuctivae and chemosis score was found to be 0, 0, 0.88, 0.3 respectively. Based on the findings of this study, the test item is considered to be a "non-irritant" to the eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation/corrosion

A study was performed to determine the acute irritation, provoked by the compound, FAT 40000, on the skin of the rabbits. 0.5 g of the test article was applied to approx. 6 cm² of the intact skin of the clipped area. It was covered with a 3.0 cm x 3.0 cm patch of surgical gauze. The gauze was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with an elastic bandage. The duration of treatment was 4 hours. Then the dressing was removed and the skin was flushed with lukewarm-tap water. The animals were checked daily for systemic symptoms and mortality. The skin reactions were evaluated 1, 24, 48, and 72 hours after removing the gauze patches according to the OECD scoring system. The application site was yellow stained up to 24 hours after the application; therefore the evaluation of a possible erythema formation was impeded. Because no erythema reactions were observed at 4 8 to 72 hrs and no edema reactions at 24 to 72 hrs after removing the bandages, the test was ended after the 72 hours evaluation. According to the EC classification of the results obtained, FAT 40000/G can be classified as non-irritant in albino rabbits.

In addition, several other studies have been conducted involving different batches of the test item all proving non-irritant test results of the key study selected. Based on these data batch and production process specific effects on skin irritation can be ruled out.

Eye irritation/corrosion

In a study performed to determine the eye irritation potential of FAT 40000/G, test substance was instilled in the eyes of 3 rabbits and observations for irritation were recorded at the time points of 1 hour, 24 hours, 48 hours 72 hours. On test day 1, 0.1 g of the test article (undiluted) was placed in the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test article. The right eye remained untreated and served as the reference control. FAT 40000/G is considered to be a non-irritant to the eyes.

In addition, several other studies have been conducted involving different batches of the test item all proving non-irritant test results of the key study selected. Based on these data batch and production process specific effects on eye irritation can be ruled out.

Justification for classification or non-classification

Based on the above stated assessment, FAT 40000 is to be considered as a non-irritant to the skin and eye of rabbits, hence no classification is warranted according to CLP (Regulation (EC) No 1272/2008).