5',5'''-[ethylenebis(p-phenyleneazo)]bis[1',2'-dihydro-6'-hydroxy-4'-methyl-2'-oxo-1,3'-bipyridinium] dilactate

Endpoint summary

Description of key information

The available information indicate that the main constituent of Basic Yellow 094 is expected to not possess any skin/eye irritating potential. However, the main substance's impurity can, in the worst case, determine a classification as Eye Dam 1 (H318).

Based on the content and/or their characteristics, the remaining impurities have no impact  to the classification of the Basic Yellow 094.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 28th to 31st December, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

The test substance is the acid form of the substance under registration; justification for Read Across is detailed in the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

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: 2.23 - 2.67 g.
- Housing: animals were individually housed in suspended metal cages.
- Diet: Spillers Rabbit Diet, Dalgety Agriculture Ltd., Almondsbury, Bristol), ad libitum.
- Water: ad libitum.
- Acclimation period: minimum acclimatisation period of at least five days.

ENVIRONMENTAL CONDITIONS
- Temperature: 17 - 21 °C.
- Relative humidity: 52 - 62 %
- Air changes: approximately 15 changes per hour.
- Photoperiod: the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.5 g of the test material, moistened with 0.5 ml of distilled water.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

Three rabbits

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. Test item moinstoned with water was introduced under a 2.5 cm × 2.5 cm gauze patch and placed in position on the shorn skin.
- Type of wrap if used: the patch was secured in position with a strip of surgical adhesive tape (BLENDERM: approximate size 2.5 cm × 4.0 cm). To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset (TUBIGRIP).

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

OBSERVATION TIME POINTS
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.

SCORING SYSTEM:
The test sites were examined 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".

Evaluation of skin reactions
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 0
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

Irritation parameter:

erythema score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2.3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2.3

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

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.

Faint yellow-coloured staining caused by the test material was commonly noted during the study. This did not affect evaluation of skin responses.
Very slight erythema was noted at two treated skin sites one hour after patch removal and at the 24-hour observation and at one treated skin site at the 48-hour observation.
No adverse skin reactions were noted 72 hours after treatment.

Individual skin reactions

Skin reaction	Observation time (hrs)	Individual score
		176 (female)	181 (male)	186 (male)
Erythena	1	1STA	1STA	0STA
	24	1STA	1STA	0STA
	48	0STA	1STA	0STA
	72	0STA	0STA	0STA
Mean 24, 48, 72 hrs		0.3	0.7	0.0
Oedema	1	0	0	0
	24	0	0	0
	48	0	0	0
	72	0	0	0
Mean 24, 48, 72 hrs		0.0	0.0	0.0

STA = faint yellow-coloured staining

Interpretation of results:

other: not classified, according to the CLP Regulation (EC1272/2008)

Conclusions:

Not irritant.

Executive summary:

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 guideline 404.

A single 4-hour, semi-occluded application of the test material to the intact skin of three rabbits produced very slight erythema. No adverse skin reactions were noted 72 hours after treatment. 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. Faint yellow-coloured staining caused by the test material was commonly noted during the study. This did not affect evaluation of skin responses. Very slight erythema was noted at two treated skin sites one hour after patch removal and at the 24-hour observation and at one treated skin site at the 48-hour observation. No adverse skin reactions were noted 72 hours after treatment.

Conclusion

Mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions, thus the test item does not meet the criteria to be classified as irritating, according to the CLP Regulation (EC 1272/2008).

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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 2nd to 6th January, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

The test substance is the acid form of the substance under registration; justification for Read Across is detailed in the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1987

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: David Percival Ltd., Moston, Sandbach, Cheshire, U.K.
- Age at study initiation: at the start of the study the animals were approximately twelve to sixteen weeks old.
- Weight at study initiation: at the start of the study the animals weighed 2.90 - 3.34 kg.
- Housing: individually housed in suspended metal cages.
- Diet: SpilIers Rabbit Diet, Dalgety Agriculture Ltd., Almondsbury, Bristol, ad libitum.
- Water: drinking water, ad libitum.
- Acclimation period: minimum acclimatisation period of five days.
- Health: immediately before the start of the test, both eyes of the three provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Animals showing evidence of ocular lesions were rejected and replaced.

ENVIRONMENTAL CONDITIONS
- Temperature: 17 - 19 °C
- Humidity: 48 - 60 %
- Air changes: approximately 15 changes per hour.
- Photoperiod: the lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

The test material was used as supplied.
0.1 ml of the test material, which was found to weigh approximately 66 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 arway from the eyeball. The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test material, and then released.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

Three rabbits

Details on study design:

PROCEDURE
One rabbit was initially treated. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

TOOL USED TO ASSESS SCORE: examination of the eye was facilitated by use the light source from a standard ophthalmoscope.

SCORING SYSTEM
Immediately after administration of the test material, an assessment of the initial pain reaction was made. Assessment of ocular damage/irritation rras made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation proposed by 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".
Any other adverse ocular effects were also noted.

Grades of ocular lesions
A. CORNEA
Opacity: Degree of density (area most dense taken for reading).
No ulceration or opacity 0
Scattered or diffuse areas of opacity (other than slight dulling of normal lustre) details of iris clearly visible 1
Easily discernible translucent area, details of iris slightly obscured 2
Opalescent area, no details of iris visible, size of pupil barely discernible 3
Opaque cornea, iris not discernible through the opacity 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

The total score = (A x B) x 5
Maximum total = 80

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

The total score = C x 5
Maximum total = 10

CONJUNCTIVAE
D. Redness: (refers to palpebral and bulbar conjunctivae excluding cornea and iris)
Blood vessels normal 0
Some blood vessels definitely hyperaemic (injected) 1
Diffuse, crimson colour, individual vessels not easily discernible 2
Diffuse beefy red 3

E. Chemosis
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

F. Discharge
No discharge 0
Any anount different from nornal (does not include small anounts observed in inner canthus of nornal animals) 1
Di scharge with moistening of the lids and hairs just adjacent to lids 2
Di scharge with moistening of the lids and hairs a considerable area around the eye 3

The total score = (D + E + F) x 2
Maximum total = 20

Maximum total score possible = 110

Irritation parameter:

cornea opacity score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 1

Reversibility:

fully reversible within: 72 hrs

Irritation parameter:

iris score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 1

Reversibility:

fully reversible within: 72 hrs

Irritation parameter:

conjunctivae score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 72 hrs

Irritation parameter:

chemosis score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Reversibility:

fully reversible within: 72 hrs

Irritant / corrosive response data:

The test material produced a maximum group mean score of 16.3 and was classified as a mild irritant to the rabbit eye, according to a modified Kay and Calandra classification system.
A dulling of the normal lustre of the corneal surface was noted in one treated eye one hour after treatment with diffuse corneal opacity at the 24 and 48-hour observations. No other adverse corneal effects were noted.
Iridial inflammation was noted in all treated eyes one hour after treatment and in one treated eye at the 24 and 48-hour observations. No other adverse iridial effects were noted.
Moderate conjunctival irritation was noted in all treated eyes one hour after treatment and in one treated eye at the 24-hour observation. Minimal conjunctival irritation was noted in two treated eyes at the 24-hour observation and in one treated eye at the 48-hour observation. Residual test material was noted around the treated eye of all animals during the study.
All treated eyes appeared normal 72 hours after treatment.

Individual mean 24, 48 and 72 hours score

Skin reaction	244 female				239 female				265 female
	1 hr	24 hrs	48 hrs	72 hrs	1 hr	24 hrs	48 hrs	72 hrs	1 hr	24 hrs	48 hrs	72 hrs
Cornea opacity	d	1	1	0	0	0	0	0	0	0	0	0
Mean 24, 48, 72 hrs	0.7				0.0				0.0
Iris	1	1	1	0	1	0	0	0	1	0	0	0
Mean 24, 48, 72 hrs	0.7				0.0				0.0
Redness	2	2	1	0	2	1	0	0	2	1	0	0
Mean 24, 48, 72 hrs	1.0				0.3				0.0
Chemosis	2	2	1	0	1	0	0	0	1	0	0	0
Mean 24, 48, 72 hrs	1.0				0.3				0.0

Individual scores and individual total scores for ocular irritation

Skin reaction	244 female				239 female				265 female
	1 hr	24 hrs	48 hrs	72 hrs	1 hr	24 hrs	48 hrs	72 hrs	1 hr	24 hrs	48 hrs	72 hrs
A. Cornea opacity	d	1	1	0	0	0	0	0	0	0	0	0
B. Area involved	4	2	1	0	0	0	0	0	0	0	0	0
Score (A x B) x 5	0	10	5	0	0	0	0	0	0	0	0	0
C. Iris	1	1	1	0	1	0	0	0	1	0	0	0
Score C x 5	5	5	5	0	5	0	0	0	5	0	0	0
D. Redness	2	2	1	0	2	1	0	0	2	1	0	0
E. Chemosis	2	2	1	0	1	0	0	0	1	0	0	0
F. Discharge	3Re	2Re	1Re	0Re	2Re	0Re	0Re	0Re	2Re	0Re	0Re	0Re
Score (D + E + F) x 2	14	12	6	0	10	2	0	0	10	2	0	0
Total score	19	27	16	0	15	2	0	0	15	2	0	0

d: dulling of the normal lustre of the corneal surface

Re: residual test material around the eye

Interpretation of results:

other: not classified, according to the CLP Regulation (EC1272/2008)

Conclusions:

Not irritant.

Executive summary:

A study was performed to assess the irritancy potential of the test material to the eye of the New Zealand White rabbit. The method used followed that described in the OECD guideline 405. A single application of the test material to the non-irrigated eye of three rabbits produced iridial inflammation and moderate coniunctival irritation. Diffuse corneal opacity was confined to one treated eye. All treated eyes appeared normal 72 hours after treatment. The test material produced a maximum group mean score of 16.3 and was classified as a mild irritant to the rabbit eye according to a modified Kay and Calandra classification system. A dulling of the normal lustre of the corneal surface was noted in one treated eye one hour after treatment with diffuse corneal opacity at the 24 and 48-hour observations. No other adverse corneal effects were noted. Iridial inflammation was noted in all treated eyes one hour after treatment and in one treated eye at the 24 and 48-hour observations. No other adverse iridial effects were noted. Moderate conjunctival irritation was noted in all treated eyes one hour after treatment and in one treated eye at the 24-hour observation. Minimal conjunctival irritation was noted in two treated eyes at the 24-hour observation and in one treated eye at the 48-hour observation. Residual test material was noted around the treated eye of all animals during the study. All treated eyes appeared normal 72 hours after treatment.

Conclusion

The mean values from gradings at 24, 48 and 72 hours were lower than 1 for corneal opacity, lower than 1 for iritis, lower than 2 for both conjunctival redness and oedema, in all the three tested animals. Therefore, the substance does not meet the criteria to be classified as eye irritating, according to the CLP Regulation (EC 1272/2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

SKIN IRRITATION

There are no information about the skin irritation potential of Basic Yellow 094, thus the available information on the structural analogues Similar Substance 01 and Similar Substance 02 have been taken into consideration. Details about the read-across approach are reported into the IUCLID section 13.

A study was performed to assess the irritancy potential of Similar Substance 01 to the skin of the New Zealand White rabbit. The method used followed that described in the OECD guideline 404. A single 4-hour, semi-occluded application of the test material to the intact skin of three rabbits produced very slight erythema. No adverse skin reactions were noted 72 hours after treatment. 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. Faint yellow-coloured staining caused by the test material was commonly noted during the study. This did not affect evaluation of skin responses. Very slight erythema was noted at two treated skin sites one hour after patch removal and at the 24-hour observation and at one treated skin site at the 48-hour observation. No adverse skin reactions were noted 72 hours after treatment.

The mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions; all the reactions resulted to be fully reversible within 72 hours. Thus the test item does not meet the criteria to be classified as irritating, according to the CLP Regulation (EC 1272/2008).

In addition, a study on the irritancy potential of the Similar Substance 02 to the skin of rabbits is also available. The method used followed that described in the OECD guideline 404. Under the conditions of the experiment, the substance was found to cause a primary irritation score of 0, when applied to intact skin. In the area of application, a clearly visible brown-orange discolouration of the skin was observed which could be related to effects proper to the test article. No corrosive effect had occurred on the skin at any measuring interval.

EYE IRRITATION

There are no information about the eye irritation potential of Basic Yellow 094, thus the available information on the structural analogues Similar Substance 01 and similar Substance 02 have been taken into consideration. Details about the read-across approach are reported into the IUCLID section 13.

A study was performed to assess the irritancy potential of Similar Substance 01 to the eye of the New Zealand White rabbit. The method used followed that described in the OECD guideline 405. A single application of the test material to the non-irrigated eye of three rabbits produced iridial inflammation and moderate coniunctival irritation. Diffuse corneal opacity was confined to one treated eye. All treated eyes appeared normal 72 hours after treatment. The test material produced a maximum group mean score of 16.3 and was classified as a mild irritant to the rabbit eye according to a modified Kay and Calandra classification system. A dulling of the normal lustre of the corneal surface was noted in one treated eye one hour after treatment with diffuse corneal opacity at the 24 and 48-hour observations. No other adverse corneal effects were noted. Iridial inflammation was noted in all treated eyes one hour after treatment and in one treated eye at the 24 and 48-hour observations. No other adverse iridial effects were noted. Moderate conjunctival irritation was noted in all treated eyes one hour after treatment and in one treated eye at the 24-hour observation. Minimal conjunctival irritation was noted in two treated eyes at the 24-hour observation and in one treated eye at the 48-hour observation. Residual test material was noted around the treated eye of all animals during the study. All treated eyes appeared normal 72 hours after treatment.

The mean values from gradings at 24, 48 and 72 hours were lower than 1 for corneal opacity, lower than 1 for iritis, lower than 2 for both conjunctival redness and oedema, in all the three tested animals; as mentioned, all treated eyes appeared normal 72 hours after treatment. Therefore, the substance does not meet the criteria to be classified as eye irritating, according to the CLP Regulation (EC 1272/2008).

In addition, a study was performed to assess the irritancy potential of Similar Substance 02 to rabbit's eyes. The method used followed that described in the OECD guideline 405. Test item showed a primary irritation score of 0.3 when applied to the rabbit eye mucosa. In the area of application a slight yellow discolouration of the cornea and conjunctivae was observed which could be related to the colour proper to the test article. No corrosion of the cornea was observed at any of the measuring intervals.

DISCUSSION AND CONCLUSION

The available information indicate that the main constituent of Basic Yellow 094 is expected to not possess any skin/eye irritating potential; this conclusion is supported by data on both the acid form and data on the analogue Similar Substance 02, which is salified with lactic acid, as well as Basic Yellow 094.

However, the composition of the Basic Yellow 094 lots currently manufactured have more lactic acid, in addition to that involved into the salification of the base, since the substance is produced directly in a highly concentrated lactic acid solution. The final free lactic acid content can range from 0 to less than 10 % (w/w).

Lactic acid is currently classified as: Skin Irrit 2 (H315); Eye Dam 1 (H318). Specific concentration limits are: Skin Irrit. 2 conc. ≥ 10 %; Eye Dam 1 conc ≥ 3 %; Eye Irrit 2 conc. ≥ 1 - 3 %.

Therefore, the contribute of lactic acid to the classification of the substance should be taken into consideration. Based on the lactic acid content, i.e. below 10 %, Basic Yellow 094 should be classified as able to cause serious eye damage (Eye Dam 1, H318). As for skin irritation, based on the typical concentration range of lactic acid, the classification threshold is not reached.

No other impurities may influence the classification of Basic Yellow 094.

Justification for classification or non-classification

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

On the basis of the available information, the main constituent is expected to not possess any skin irritating potential; based on their content and/or their characteristics, the remaining impurities have no impact  to the classification of Basic Yellow 094.

According to the CLP Regulation (EC 1272/2008), serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance, which is not fully reversible within 21 days of application.

On the basis of the available information, the main constituent is expected to not possess any eye irritating potential. However, the possible main impurity, i.e. lactic acid, can determine a classification as Eye Dam 1 (H318). Based on their content and/or their characteristics, the remaining impurities have no impact  to the classification of Basic Yellow 094.

In conclusion, the substance does not meet the criteria to be classified as skin irritating; however, a classification as able to cause serious eye damage (i.e. Eye Dam 1, H318) is proposed, according to the CLP Regulation (EC 1272/2008).