Disodium 4,4'-bis[[4-anilino-6-[(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Endpoint summary

Description of key information

Not skin irritating.

Not eye irritating.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

From February 9 to 12,1993.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guidelines and performed according to GLP. Justification for Read Across is detailed in the endpoint summary and in the Category Justification Report attached to the section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1981

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: David Percival Ltd., Moston, Sandbach, Cheshire, U.K.
- Age at study initiation: twelve to sixteen weeks old.
- Weight at study initiation: 2.53 - 2.79 kg.
- Housing: individually housed in suspended metal cages.
- Diet: free access to food (RABMA Rabbit Diet, Special Diet Services Ltd., witham, Essex, U.K.).
- Water: free access to mains drinking water.
- Acclimation period: a minimum acclimatisation period of five days.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 21°C
- Humidity: 39 - 45 %.
- Air changes: approximately 15 changes per hour.
- Photoperiod: the lighting was controlled by a time switch to give continuous 12 hours light and 12 hours darkness.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

0.5 ml of the test material was introduced under a 2.5 cm x 2.5 cm gauze patch and placed in position on the shorn skin.

Number of animals:

Three

Details on study design:

TEST SITE
- Area of exposure: on the day before the test each of a group of three rabbits was clipped free of fur from the dorsal flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study. On the day of the test a suitable test site was selected on the back of each rabbit.
- Type of wrap if used: the patch was secured in position with a strip of surgical adhesive tape (BLENDERM: approximate size 2.5 cm x 4.0 cm). To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset (TUBIGRIP) and the animals were returned to their cages for the duration of the exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing: the corset and patches were removed from each animal and any residual test material removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: four hours after application.

SCORING SYSTEM
Approximately one hour following the removal of the patches, and 24,48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the following scale from Draize J.H. (1959) Association of Food and Drug Officials of the United States, Austin, Texas, "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics":
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

Oedema Formation
No oedema: O
Very slight oedema (barely perceptible): 1
Slight oedema (edges of area well-defined by definite raising): 2
Moderate oedema (raised approximately 1 millimetre): 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure): 4

Any other skin reactions, if present, were also recorded.

Irritation parameter:

erythema score

Basis:

animal: 3/3

Time point:

other: mean 24, 48 and 72 hours

Score:

< 2.3

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal: 3/3

Time point:

other: mean 24, 48 and 72 hours

Score:

< 2.3

Reversibility:

fully reversible

Irritant / corrosive response data:

The test material produced a primary irritation index of 0.3. According to the Draize classification scheme: mild irritating.
According to the CLP regulation: not irritating.

Skin reaction	Reading (hrs)	Individual scores
		147 F	156 F	161 F
Erythema/eschar formation	24	0	1	1
	48	0	0	0
	72	0	0	0
Total		0	1	1
Mean score		0	0.3	0.3
Oedema formation	24	0	0	0
	48	0	0	0
	72	0	0	0
Total		0	0	0
Mean score		0	0	0

Interpretation of results:

not irritating

Remarks:

Migrated information according to the CLP Regulation Criteria used for interpretation of results: EU

Conclusions:

Not irritating.

Executive summary:

Method

A study was performed to assess the irritancy potential of the test material to the skin of the New Zealand white rabbit. The method used followed that described in the OECD Guidelines No. 404 "Acute Dermal Irritation/Corrosion" referenced as Method B4 in Commission Directive 84/449/EEC.

Results

A single 4 -hours, semi-occluded application of the test material to the intact skin of three rabbits produced well-defined erythema and very slight oedema. All treated skin sites appeared normal at the 48-hour observation.

The test material produced a primary irritation index of 0.3 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

Conclusion

According to the CLP regulation (EC 1272/2008), test substance can be classified as non irritating, because mean value scored is less than 2.3 for erythema/eschar and for oedema in at least 2 of 3 tested animals from gradings at 24, 48 and 72 hours after patch removal.

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:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Internationally accepted guideline, well documented and scientifically acceptable. Justification for Read Across is detailed in the endpoint summary and in the Category Justification Report attached to the section 13.

Principles of method if other than guideline:

Test was conducted following the procedure used is described in the Proposed Guidelines of the United States Environmental Protection Agency (EPA) § 163.81-4 "Primary eye irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978.

GLP compliance:

no

Remarks:

Pre GLP

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: IVANOVAS, 7964 Kisslegg/Allgäu, Germany and MADOERIN AG, 4414 Füllinsdorf, Switzerland.
- Age at study initiation: adult, age not specified.
- Weight at study initiation: 2 to 3 kg.
- Housing: housed individually in metal cages.
- Diet: ad libitum, standard rabbit food - NAFAG, No. 814, Gossau SG.
- Water: ad libitum.
- Acclimation period: prior to treatment they were adapted to our laboratories for a minimum of 4 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Relative humidity: 55 ± 10 %
- Photoperiod: 10 hours light cycle day.

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 ml.
- Application: test substance was inserted into the conjunctival sac of the left eye of the rabbits and the lids were gently closed for a few seconds.

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

Three males and three females.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: in 3 of the 6 rabbits approximately 30 seconds after treatment the treated eye was flushed with 10 ml of physiological saline.

TOOL USED TO ASSESS SCORE
The eye irritation was appraised with a slit-lamp on day 1, 2, 3, 4 and 7.

SCORING SYSTEM

Cornea
A Opacity-degree of density (area most dense taken for reading)
No opacity: 0
Scattered or diffuse area, details of iris clearly visible: 1
Easily discernible translucent areas, details of iris slightly obscured: 2
Opalescent areas, no details of iris visible, size of pupil barely discernible: 3
Opaque, iris invisible: 4
B Area of cornea involved
One quarter (or less) but not zero: 1
Greater than one quarter, but less than half: 2
Greater than half, but less than three quarters: 3
Greater than three quarters, up to whole area: 4
A x B x 5 Total maximum = 80

Iris
A Values
Normal: 0
Folds above normal, congestion, swelling, circumcorneal injection (any or all of these or combination of any thereof) iris still reacting to light (sluggish reaction is positive): 1
No reaction to light, hemorrhage, gross destruction (any or all of these): 2
A x 5 Total maximum = 10

Conjunctivae
A Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)
Vessels normal: 0
Vessels definitely injected above normal: 1
More diffuse, deeper crimson red, individual vessels not easily discernible: 2
Diffuse beefy red: 3
B Chemosis
No swelling: 0
Any swelling above normal (includes nictitating membrane): 1
Obvious swelling with partial eversion of lids: 2
Swelling with lids about half closed: 3
Swelling with lids about half closed to completely closed: 4
C Discharge
No discharge: 0
Any amount different from normal (does not include small amounts observed in inner canthus of normal animals): 1
Discharge with moistening of the lids and hairs just adjacent to lids: 2
Discharge with moistening of the lids and hairs, and considerable area around the eye: 3
(A + B + C) x 2 Total maximum = 20

Irritation parameter:

cornea opacity score

Basis:

animal: 6/6

Time point:

other: mean 24, 48 and 72 hours

Score:

< 1

Reversibility:

fully reversible

Remarks on result:

other: rinsed and unrinsed eyes

Irritation parameter:

iris score

Basis:

animal: 6/6

Time point:

other: mean 24, 48 and 72 hours

Score:

< 1

Reversibility:

fully reversible

Remarks on result:

other: rinsed and unrinsed eyes

Irritation parameter:

conjunctivae score

Basis:

animal: 6/6

Time point:

other: mean 24, 48 and 72 hours

Score:

< 2

Reversibility:

fully reversible

Remarks on result:

other: rinsed and unrinsed eyes

Irritation parameter:

chemosis score

Basis:

animal: 6/6

Time point:

other: mean 24, 48 and 7 hours

Score:

< 2

Reversibility:

fully reversible

Remarks on result:

other: rinsed and unrinsed eyes

Irritant / corrosive response data:

The substance was found to cause no irritation when applied to the rabbit eye mucosa.

Rabbit eye irritation scores

Animal	Eye	24 hrs	48 hrs	72 hrs	4 days	7 days	Mean 24, 48, 72 hrs
Cornea
274	eye not rinsed	0	0	0	0	0	0.00
275	eye not rinsed	0	0	0	0	0	0.00
276	eye not rinsed	0	0	0	0	0	0.00
277	rinsed after 30 sec.	0	0	0	0	0	0.00
278	rinsed after 30 sec.	0	0	0	0	0	0.00
279	rinsed after 30 sec.	0	0	0	0	0	0.00
Iritis
274	eye not rinsed	0	0	0	0	0	0.00
275	eye not rinsed	0	0	0	0	0	0.00
276	eye not rinsed	0	0	0	0	0	0.00
277	rinsed after 30 sec.	0	0	0	0	0	0.00
278	rinsed after 30 sec.	0	0	0	0	0	0.00
279	rinsed after 30 sec.	0	0	0	0	0	0.00
Conjunctival-redness
274	eye not rinsed	0	0	0	0	0	0.00
275	eye not rinsed	0	0	0	0	0	0.00
276	eye not rinsed	0	0	0	0	0	0.00
277	rinsed after 30 sec.	0	0	0	0	0	0.00
278	rinsed after 30 sec.	0	0	0	0	0	0.00
279	rinsed after 30 sec.	0	0	0	0	0	0.00
Conjunctival chemosis
274	eye not rinsed	0	0	0	0	0	0.00
275	eye not rinsed	0	0	0	0	0	0.00
276	eye not rinsed	0	0	0	0	0	0.00
277	rinsed after 30 sec.	0	0	0	0	0	0.00
278	rinsed after 30 sec.	0	0	0	0	0	0.00
279	rinsed after 30 sec.	0	0	0	0	0	0.00

Interpretation of results:

not irritating

Remarks:

Migrated information according to the CLP Regulation Criteria used for interpretation of results: EU

Conclusions:

Not irritating.

Executive summary:

Method

Test was conducted following the procedure used is described in the Proposed Guidelines of the United States Environmental Protection Agency (EPA) § 163.81-4 "Primary eye irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978.

Results

The substance was found to cause no irritation when applied to the rabbit eye mucosa.

Conclusion

According to the CLP Regulation (EC 1272/2008), the substance can be classified as not irritating, because when applied to the eye of an animal does not produce 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Several studies were performed on similar substances according to the procedure described in the Proposed Guidelines of the United States Environmental Agency (EPA) § 163.81-5 "Primary dermal irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978 (Pre-GLP) and § 163.81-4 "Primary eye irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978. The procedures are well described and the results can be converted into the CLP criteria.

The considered similar substances are CAS 16324-27-9, CAS 4193-55-9 and the CAS 70942-01-7.

The substance under registration (CAS 17958-73-5) shares with CAS 16324-27-9 the same organic functional group and substitution on the triazino moyety with monohydroxyethylamino. Therefore, they are similar both according to the chemical structure (Tanimoto similarities > 90%) than according to metabolic pathway (OECD Toolbox profiling). The two substances differ in the sulphonation degree: CAS 17958-73-5 is the disulphonato derivative, while CAS 16324-27-9 is tetrasulphonated. The difference in solubility is of almost one order of magnitude (30.2 g/l for CAS 17958-73-5 vs 140 g/l for CAS 16324-27-9), but this fact has no impact from a toxicological point of view, because both the substances are in any case highly soluble, stable and completely dissociated in water.

Further studies conducted on the analogous substances CAS 4193-55-9 and CAS 70942 -01 -7 are reported. These substances are the analogous disulphonated dihydroxyethylamino substituted. Despite the CAS 4193-55-9 is salificated with sodium salt and the CAS 70942-01-7 is salificated with dosium/potassium salts, in both the susbtances the suphonation degree is the same as the CAS 17958-73-5.

The structural difference interests the substitution on the triazino ring with dihydroxyethyl (CAS 4193-55-9 and CAS 70942-01-7) instead than monohydroxyethyl (CAS 17958-73-5). From a metabolic point of view the substances can be considered very similar because the monohydroxyl derivative is a metabolite of the dihydroxy derivative. In this respect the water solubilities of CAS 17958-73-5 and CAS 4193-55-9, CAS 709472-01-7 are more similar (30.2 vs 48.2 g/l vs 13.45 g/l, respectively).

Within the whole category, eleven over fourteen registered substances were tested and none of the existing tests arisen any concern for skin and eye irritation.

All substances of the category were modelled with OECD Toolbox and the provisional results about skin and eye irritation have been calculated for all members. No alerts have been reported (see category Justification Report) for inclusion for any substance.

The substance under registration can be considered not irritant for skin and eye.

Justification for selection of skin irritation / corrosion endpoint:

Several studies with the same reliability on similar substances agree on the result. The most recent has been taken as a reference.

Justification for selection of eye irritation endpoint:

Several studies with the same reliability on similar substances agree on the result. The most recent has been taken as a reference.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.2 Skin corrosion/irritation section, skin Irritation means the production of reversible damage to the skin following the application of a test substance for up to 4 hours.

In all the studies available, the mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in at least 2 of 3 animals for both erythema/eschar and oedema reactions.

 

According to the Regulation 1272/2008 (CLP) substances that have the potential to induce reversible eye irritation are classified in Category 2 (irritating to eyes) 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.

The mean values from gradings at 24, 48 and 72 hours for both rinsed and unrinsed eyes were 0 in all animals for all the reactions scored, in all the test performed; therefore, the substance resulted not irritating.

 

In conclusion, the available experimental data are adequate for classification and labelling and the substance is not classified for the eye/skin irritation, according to the CLP Regulation (EC 1272/2008).