Registration Dossier

Administrative data

Description of key information

Skin Irritation:

No signs of erythema or edema were observed on the intact and abraded skin of the 6 rabbits after 24 and 72 hours of patch removal.  

The Primary Irritation Index was calculated to be 0.0.

Based on the scores and observations, it was concluded that C.I. Direct Blue 218[CAS: 28407 -37 -6] was not irritating to skin.

Eye Irritation:

The Primary Irritation Index calculated at 1, 24, 48, 72 hours, 7 and 14 days were 11, 9.7, 14, 8, 3 and 1 respectively.

Based on the scores, C.I. Direct Blue 218 [CAS: 28407 -37 -6] can be considered to be moderately irritating to rabbit eyes.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
data is from authoritative databases and NTRL reports
Qualifier:
according to
Guideline:
other: 16 CFR 1500.41 &16 CFR 1500.3 Guidelines
Principles of method if other than guideline:
To determine the primary irritation index of C.I. Direct Blue 218 when applied to the skin of rabbits
GLP compliance:
not specified
Specific details on test material used for the study:
Name of the test chemical: Copper,[tetrahydrogen-3,3'-[(3,3'-dihydroxy-4,4'-biphenylylene)bis(azo)]bis[5-amino-4-hydroxy-2,7-naphthalenedisulfonato](4-)]di-,tetrasodium salt (7CI)
Common Name: C.I Direct Blue 218
Molecular Formula: C32H20Cu2N6Na4O16S4
Molecular Weight: 1087.84 g/mol
SMILES Notation: c12c3c(c(S(=O)(=O)[O-])cc1cc(S(=O)(=O)[O])cc2N)N=Nc1ccc(cc1O[Cu]O3)c1cc2c(N=Nc3c(cc4c(c3O[Cu]O2)c(cc(c4)S(=O)(=O)[O-])N)S(=O)(=O)[O-])cc1.[Na+].[Na+].[Na+].[Na+]
InChI: 1S/C32H24N6O16S4.2Cu.4Na/c33-19-11-17(55(43,44)45)5-15-9-25(57(49,50)51)29(31(41)27(15)19)37-35-21-3-1-13(7-23(21)39)14-2-4-22(24(40)8-14)36-38-30-26(58(52,53)54)10-16-6-18(56(46,47)48)12-20(34)28(16)32(30)42;;;;;;/h1-12,39-42H,33-34H2,(H,43,44,45)(H,46,47,48)(H,49,50,51)(H,52,53,54);;;;;;/q;2*+2;4*+1/p-8/b37-35-,38-36-;;;;;;
Substance Type: Organic
Physical State: Solid
Code Number -2146-00
Product Name - Lumisol Blue VG
Concentration:100%
Mix Number - 384
TR r-number - 78-638
Description - Dark Grey Powder





Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: approximately 2.5 kg
- Housing: housed individually in suspended wire mesh cages
- Diet (e.g. ad libitum): Purina Rabbit Chow, ad libitum
- Water (e.g. ad libitum): water, ad libitum
- Acclimation period: at least seven days equilibration
Type of coverage:
occlusive
Preparation of test site:
clipped
Remarks:
intact and abraded skin
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
0.5 g
Duration of treatment / exposure:
24 hours
Observation period:
The test sites were observed at 24 and 72 hours after removal of patches
Number of animals:
6
Details on study design:
Details on study design
TEST SITE
- Area of exposure: back of the rabbits
- % coverage: dry surgical gauze, one inch square. two layers thick.
- Type of wrap if used: adhesive tape; the trunk was wrapped wi th impervious ma terial


SCORING SYSTEM: The dermal reactions were scored by the Draize technique at 24, 72 hours and the Primary Irritation Index (PII) was calculated
Irritation parameter:
erythema score
Basis:
mean
Time point:
24 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
mean
Time point:
24 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
mean
Time point:
72 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
mean
Time point:
72 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
No signs of erythema or edema were observed on the intact and abraded skin of the 6 rabbits after 24 and 72 hours of patch removal.

 

Table: Individual scores for the acute dermal irritation study

Observations

Rabbit number

Mean Score

 

 

1

2

3

4

5

6

 

Erythema and eschar formation

Intact skin -24 hours

0

0

0

0

0

0

0

Abraded skin – 24 hours

0

0

0

0

0

0

0

Intact skin – 72 hours

0

0

0

0

0

0

0

Abraded skin – 72 hours

0

0

0

0

0

0

0

Edema

Intact skin – 24 hours

0

0

0

0

0

0

0

Abraded skin – 24 hours

0

0

0

0

0

0

0

Intact skin – 72 hours

0

0

0

0

0

0

0

Abraded skin - 72 hours

0

0

0

0

0

0

0

Sum of the mean scores

0

 Primary Dermal Index = sum of the mean scores/4 = 0.0

Interpretation of results:
other: not irritating
Conclusions:
No signs of erythema or edema were observed on the intact and abraded skin of the 6 rabbits after 24 and 72 hours of patch removal.
The Primary Irritation Index was calculated to be 0.0.
Based on the scores and observations, it was concluded that C.I. Direct Blue 218[CAS: 28407-37-6] was not irritating to skin.
Executive summary:

A study was performed in rabbits to determine the dermal irritation potential of C.I. Direct Blue 218. The study was conducted as per 16 CFR 1500.41 &16 CFR 1500.3 Guidelines.6 New Zealand white rabbits were used for the study. After at least seven days of equilibration, the back and sides of each rabbit were clipped free of fur with an electric clipper. Rabbits with dermal lesions or irritation were replaced. One day after clipping, the left side of the spinal column was abraded (minor incisions which did not disturb the derma or produce bleeding). 0.5 g of the test material was applied to the intact and abraded skin of the back of the rabbits. The material was covered with dry surgical gauze, one inch square, two layers thick. The patches were secured with adhesive tape; the trunk was wrapped with impervious material. After 24 hours, the patches were removed. The dermal reactions were scored by the Draize technique at 24 and 72 hours. The primary dermal index was calculated.

No signs of erythema or edema were observed on the intact and abraded skin of the 6 rabbits after 24 and 72 hours of patch removal. 

The Primary Irritation Index was calculated to be 0.0.

Based on the scores and observations, it was concluded that C.I. Direct Blue 218 [CAS: 28407-37-6] was not irritating to 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:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
data is from NTRL reports
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
To determine the primary irritation index of C.I. Direct Blue 218 when instilled to the eyes of rabbits
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Housing: housed individually in metal cages elevated above the droppings with, feed
- Diet (e.g. ad libitum): Purina Rabbit Chow, ad libitum
- Water (e.g. ad libitum): water, ad libitum
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1 ml
Duration of treatment / exposure:
The lids were held closed for 1-second for even distribution of the test substance
Observation period (in vivo):
The eyes were examined and graded for irritation and corneal damage at 1, 24, 48 and 72 hours after instillation
Number of animals or in vitro replicates:
6
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM: The eyes were examined and the grade of ocular reaction was recorded at 1, 24, 48 and 72 hours, using the scale for scoring ocular lesions as outlined by Lehman. A.J. Lehman in Appraisal of the Safety of Chemicals in Foods. Drugs and Cosmetics Assoc., Food and Drug Officials of the U.S. The evaluation of the data was done in accordance with Section 191.12 Chapter 1, Title 21, Code of Federal Regulations under the FHSA
Both eyes of each animal were examined with Fluorescein Sodium Ophthalmic solution U.S.P. 96 hours before testing and only those animals without observable eye defects or irritation were used. The eyes were not washed following instillation of the test material. The animals were immobilized in a suitable animal restrainer 1 hour subsequent to treatment.

After the initial examination at 24 hours all eyes were washed with sufficient clean tap water at body temperature to remove the residual test sample. All eyes were further examined by placing one drop of Fluorescein Sodium Ophthalmic solution U.S.P. on the cornea. After 5 minutes the excess fluorescein was flushed out with sufficient tap water. Fluorescein Sodium in 2% aqueous solution when applied topically to the ~e is
used as an ophthalmic diagnostic aid for the detection of corneal lesions, injured areas of the cornea appear greenish yellow following application of fluorescein.

GRADING OF OCULAR LESIONS

Cornea
A - Opacity: Degree of density (readings should be taken from most dense area) *
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
Nacrous area; no details of iris visible; size of pupil barely discernible = 3
Opaque cornea; iris not discernible through the opacity = 4
Maximum possible: 4
* The area of corneal opacity should be noted
B - Area of cornea involved
Zero = 0
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
Score = A*B*5 ; Total Maximum = 80
Iris (A)
Normal = 0
Markedly deepened rugae, congestion, swelling, moderate circum corneal hyperaemia; or injection; iris reactive to light (a sluggish reaction is considered to be an effect) = 1
Hemorrhage, gross destruction, or no reaction to light (any or all of these) = 2
Maximum possible: 2
Score= A*5 ; Total Maximum = 20
Conjunctivae
A - Redness(refers to palpebral and bulbar conjunctivae; excluding cornea and iris)
Blood vessels normal = 0
Some blood vessels definitely hyperaemic (injected) = 1
Diffuse, crimson color; individual vessels not easily discernible= 2
Diffuse beefy red = 3
Maximum possible: 3
B - Chemosis
Swelling(refers to lids and/or nictating membranes)
No swelling (Normal)= 0
Some 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
Maximum possible: 4
C. DISCHARGE
Any amount different from normal (does not not include small observed in inner pannus of animals = 1
Discharge with moistening of lids and hairs just adjacent to the lids = 2
Discharge with moistening of the lids and hairs and considerable area around the eye = 3
Score- (A+B+C)*2 – Total Maximum = 20
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24 h
Score:
13.5
Reversibility:
not specified
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
moderate eye damage at 24 hours in the form of slight to moderate erythema of the palpebral and bulbar conjunctivae, slight to marked chemosis of the lids, slight to moderate accumulation of watery to watery-mucoid discharge and moderate corneal opacity (4 of 6 test eyes) involving approximately 1/4 of-the corneal surface. Some improvement was noted by 72 hours, but all retained some erythema and 3/6 test eyes showed corneal opacity.
A maximum average score of 13.5 was recorded at 24 hours.

Table: Individual scores for the acute eye irritation study

Rabbit number

Structure

Duration

1 hour*

24 hours

48 hours

72 hours

1

Cornea

0

10

10

0

Iris

0

0

0

0

Conjunctivae

0

12

8

6

2

Cornea

0

10

5

0

Iris

0

0

0

0

Conjunctivae

8

8

6

6

3

Cornea

0

0

0

0

Iris

0

0

0

0

Conjunctivae

8

8

8

6

4

Cornea

0

0

0

0

Iris

0

0

0

0

Conjunctivae

8

10

8

6

5

Cornea

0

0

0

0

Iris

0

0

0

0

Conjunctivae

12

8

6

4

6

Cornea

0

5

0

0

Iris

0

0

0

0

Conjunctivae

8

10

8

6

Average

8.7

13.5

13.1

8.2

*1 hour erythema readings were obscured by deep bluish stain of conjunctival sac and nictitating was present. In 5/6 test eyes at 1 hour, a corneal haze was present, this does not show in the scoring

 

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Undiluted C.I Direct Blue 218 when instilled into the conjunctival sac of 6 New Zealand White rabbits produced moderate eye damage at 24 hours in the form of slight to moderate erythema of the palpebral and bulbar conjunctivae, slight to marked chemosis of the lids, slight to moderate accumulation of watery to watery-mucoid discharge and moderate corneal opacity (4 of 6 test eyes) involving approximately 1/4 of-the corneal surface. Some improvement was noted by 72 hours, but all retained some erythema and 3/6 test eyes showed corneal opacity.
A maximum average score of 13.5 was recorded at 24 hours.
Based on the scores, C.I. Direct Blue 218 can be considered to be moderately irritating to rabbit eyes.
Executive summary:

A study was performed in rabbits to determine the ocular irritation potential of C.I. Direct Blue 218.

6 New Zealand white rabbits were used for the study.Both eyes of each animal were examined with Fluorescein Sodium Ophthalmic solution U.S.P. 96 hours before testing and only those animals without observable eye defects or irritation were used. 0.1 ml undiluted C.I Direct Blue 218 was instilled into the conjunctival sac of 6 New Zealand White rabbits.The lids were held closed for 1-second for even distribution of the test substance.The other eye served as an untreated control.The eyes were not washed following instillation of the test material. The animals were immobilized in a suitable animal restrainer 1 hour subsequent to treatment.After the initial examination at 24 hours all eyes were washed with sufficient clean tap water at body temperature to remove the residual test sample. All eyes were further examined by placing one drop of Fluorescein Sodium Ophthalmic solution U.S.P. on the cornea. After 5 minutes the excess fluorescein was flushed out with sufficient tap water. Fluorescein Sodium in 2% aqueous solution when applied topically to the eye is used as an ophthalmic diagnostic aid for the detection of corneal lesions, injured areas of the cornea appear greenish yellow following application of fluorescein. The eyes were examined and the grade of ocular reaction was recorded at 1, 24, 48 and 72 hours, using the scale for scoring ocular lesions as outlined by Lehman. The evaluation of the data was done in accordance with Section 191.12 Chapter 1, Title 21, Code of Federal Regulations under the FHSA. Undiluted C.I Direct Blue 218 when instilled into the conjunctival sac of 6 New Zealand White rabbits produced moderate eye damage at 24 hours in the form of slight to moderate erythema of the palpebral and bulbar conjunctivae, slight to marked chemosis of the lids, slight to moderate accumulation of watery to watery-mucoid discharge and moderate corneal opacity (4 of 6 test eyes) involving approximately 1/4 of-the corneal surface. Some improvement was noted by 72 hours, but all retained some erythema and 3/6 test eyes showed corneal opacity.

A maximum average score of 13.5 was recorded at 24 hours.

Based on the scores, C.I. Direct Blue 218 can be considered to be moderately irritating to rabbit eyes.

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation:

In different studies,C.I Direct Blue 218 [CAS: 28407 -37 -6] has been investigated for potential for dermal irritation to a greater or lesser extent. The studies are based on in vivo experiments in rabbits for the target chemical and its structurally similar read across substances, Disodium 5-acetamido-4-hydroxy-3-(phenyldiazenyl)naphthalene-2,7-disulfonate(RED 2G)[CAS: 3734-67-6] and trisodium 5-amino-3-[(E)-2-(4-{4-[(E)-2-(7-amino-1-hydroxy-3-sulfonatonaphthalen-2-yl)diazen-1-yl]phenyl}phenyl)diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate(Chlorazol Black BH)[CAS: 2429-73-4].

A study was performed in rabbits to determine (OTS0206237, NTRL report, last updated 1982) the dermal irritation potential of C.I. Direct Blue 218 [CAS: 28407 -37 -6]. The study was conducted as per 16 CFR 1500.41 &16 CFR 1500.3 Guidelines.6 New Zealand white rabbits were used for the study. After at least seven days of equilibration, the back and sides of each rabbit were clipped free of fur with an electric clipper. Rabbits with dermal lesions or irritation were replaced. One day after clipping, the left side of the spinal column was abraded (minor incisions which did not disturb the derma or produce bleeding). 0.5 g of the test material was applied to the intact and abraded skin of the back of the rabbits. The material was covered with dry surgical gauze, one inch square, two layers thick. The patches were secured with adhesive tape; the trunk was wrapped with impervious material. After 24 hours, the patches were removed. The dermal reactions were scored by the Draize technique at 24 and 72 hours. The primary dermal index was calculated.

No signs of erythema or edema were observed on the intact and abraded skin of the 6 rabbits after 24 and 72 hours of patch removal. 

The Primary Irritation Index was calculated to be 0.0.

Based on the scores and observations, it was concluded that C.I. Direct Blue 218 [CAS: 28407 -37 -6] was not irritating to skin.

This is supported by another experimental study summarized in OTS0215154, NTRL report, last updated 1983; for the target chemical. 6 New Zealand white rabbits were used for the study. The backs of the animals were clipped free of hair and the skin examined before testing. Only those animals without skin defects or irritation were used. Abrasions (minor incisions through the stratum corneum, but not sufficiently deep to disturb the derma) were made on one area of the backs while other area was left intact. The animals were immobilized in animal holders. 0.5 ml of a 50% w/v aqueous dilution of Direct Blue 218 [CAS: 28407 -37 -6] was applied under each of two one-inch square gauze patches to the prepared areas on the back of the animals. After the patches had been secured by adhesive tape, the entire trunk of each animal was wrapped with a plastic binder to keep the material in position and in contact with the skin for 24 hours. Following the 24 hours of exposure, the patches were removed; any remaining material was washed off the treated areas. The test sites were observed at 24 and 72 hours after removal of patches. The dermal reactions were scored by the Draize technique at 24, 72 hours and the Primary Irritation Index (PII) was calculated. The average values for erythema and eschar formation at 24 hours and 72 hours for the intact skin areas of exposure were added to the average values of the abraded skin areas of exposure at 24 hours and 72 hours (four values). Similarly, the average values for edema formation at 24 hours and at 72 hours for the intact and abraded skin areas of exposure were added (four values). The Primary Irritation Index is the total of the eight average values divided by four. According to the F.H.S.A method of testing, a Primary Irritant is a substance which produces a skin reaction resulting in a score of five or greater.C.I Direct Blue 218 when applied as a 50% w/v dilution with deionized water to the intact and abraded skin of New Zealand albino rabbits was found to produce minimal skin damage. The test chemical caused minor skin irritation in the form of slight edema at the abraded sites in 6/6 rabbits at the 72-hour observation period. The Primary Irritation Index was calculated as 0.8.

According to F.H.S.A method of testing, C.I Direct Blue 218 [CAS: 28407 -37 -6] was considered to be not irritating to skin.

Both the experimental results are in agreement with each other, indicating a strong possibility of C.I Direct Blue 218[CAS: 28407 -37 -6] being not irritating to skin.

 

The above studies are supported by the experimental study conducted inan OECD GLP laboratory (Sustainability Support Services (Europe) AB has the letter of access, 2016) for the structurally similar read across substance,Disodium 5-acetamido-4-hydroxy-3-(phenyldiazenyl)naphthalene-2,7-disulfonate(RED 2G)[CAS: 3734-67-6]. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures.

The test item was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days. Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment.

The overall irritation score of the substance was determined to be 0 and no erythema and edema (skin irritation) were observed at the end of 14 days after patch removal. Hence, it was concluded that disodium 5-acetamido-4-hydroxy-3-(phenyldiazenyl)naphthalene-2,7-disulfonate was not-Irritating to the skin of rats under the experimental conditions tested .Thus it can be concluded that the substance, disodium 5-acetamido-4-hydroxy-3-(phenyldiazenyl)naphthalene-2,7-disulfonate can be classified under the category "Not Classified" as per CLP regulation.

These studies are also supported by the experimental study summarized in OTS0215154, NTRL report, last updated 1983; for the structurally similar read across substance, trisodium 5-amino-3-[(E)-2-(4-{4-[(E)-2-(7-amino-1-hydroxy-3-sulfonatonaphthalen-2-yl)diazen-1-yl]phenyl}phenyl)diazen-1-yl]-4 -hydroxynaphthalene-2,7-disulfonate(Chlorazol Black BH)[CAS: 2429 -73-4]. 6 New Zealand white rabbits were used for the study. The backs of the animals were clipped free of hair and the skin examined before testing. Only those animals without skin defects or irritation were used. Abrasions (minor incisions through the stratum corneum, but not sufficiently deep to disturb the derma) were made on one area of the backs while other area was left intact. The animals were immobilized in animal holders. 0.5 gm of a 50% w/v aqueous dilution of Chlorazol Black BH [CAS: 2429 -73-4] was applied under each of two one-inch square gauze patches to the prepared areas on the back of the animals. After the patches had been secured by adhesive tape, the entire trunk of each animal was wrapped with a plastic binder to keep the material in position and in contact with the skin for 24 hours. Following the 24 hours of exposure, the patches were removed; any remaining material was washed off the treated areas. The test sites were observed at 24 and 72 hours after removal of patches. The dermal reactions were scored by the Draize technique at 24, 72 hours and the Primary Irritation Index (PII) was calculated. The average values for erythema and eschar formation at 24 hours and 72 hours for the intact skin areas of exposure were added to the average values of the abraded skin areas of exposure at 24 hours and 72 hours (four values). Similarly, the average values for edema formation at 24 hours and at 72 hours for the intact and abraded skin areas of exposure were added (four values). The Primary Irritation Index is the total of the eight average values divided by four. According to the F.H.S.A method of testing, a Primary Irritant is a substance which produces a skin reaction resulting in a score of five or greater.

Chlorazol Black BH [CAS: 2429 -73-4] when applied as a 50% w/v suspension with deionized water to the intact and abraded skin of New Zealand albino rabbits produced mild skin damage (abraded skin only) which persisted till 72 hours. No gross signs of local irritation were observed in the intact skin throughout the study. A deep bluish- purple was present at the abrasion of each anima at each reading; a light stain of similar color of the intact skin was observed at the 24-hour reading (all animals) and 48 hour reading (4 animals).

The Primary Irritation Index was calculated to be 0.50. According to F.H.S.A method of testing, Chlorazol Black BH [CAS: 2429 -73-4] was considered to be not irritating to skin.

Available data for the target and its structurally similar read across substances indicate a strong possibility of C.I Direct Blue 218 being not irritating to skin.Comparing the above annotations with the criteria of CLP regulation,C.I Direct Blue 218 [CAS: 28407 -37 -6] can be classified under the category “Not Classified”.

Eye Irritation:

In different studies,C.I Direct Blue 218 [CAS: 28407 -37 -6] has been investigated for potential for ocular irritation to a greater or lesser extent. The studies are based on in vivo experiments in rabbits for the target chemical and its structurally similar read across substances, trisodium 5-amino-3-[(E)-2-(4-{4-[(E)-2-(7-amino-1-hydroxy-3-sulfonatonaphthalen-2-yl)diazen-1-yl]phenyl}phenyl)diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate(Chlorazol Black BH)[CAS: 2429-73-4] and Disodium 5-[[4'-[(2,6-diamino-3-methyl-5-sulphonatophenyl)azo]-3,3'-dimethyl[1,1'-biphenyl]-4-yl]azo]salicylate(Direct Orange 6)[CAS: 6637-88-3].

A study was performed in rabbits to determine (OTS0206237, NTRL report, last updated 1982) the ocular irritation potential of C.I. Direct Blue 218 [CAS: 28407 -37 -6]. 6 New Zealand white rabbits were used for the study. The rabbits were equilibrated in the laboratory for at least seven days. Rabbits with irritated or damaged eyes were not used in this test. 0.1g test was instilled in the lower conjunctival sac of one eye of each rabbit and the lids held closed for approximately one second to insure even distribution of the test material over all surfaces of the eye. The other eye served as an untreated control. The eyes were examined and graded for irritation and corneal damage at 1, 24, 48 and 72 hours after instillation. Eyes not returning to a score of zero by 72 hours were graded on the seventh day after instillation. Eyes still not returning to a score of zero were graded on the fourteenth day after instillation. All eyes were examined by applying 1 drop of 2% fluorescein sodium ophthalmic solution, U.S.P., to the cornea pre-test, 24, 72 hours and 7 and 14 days after instillation. The fluorescein was rinsed from the eye with physiological saline. The ocular reactions were graded and interpreted as described in –Appraisal of the Safety of Food. Drugs, and Cosmetics", Association of Food and Drug Officials of the United States, 1959 with the assistance of the "Illustrated Guide for Grading Eye Irritation by Hazardous Substances".

The Primary Irritation Index calculated at 1, 24, 48, 72 hours, 7 and 14 days were 11, 9.7, 14, 8, 3 and 1 respectively.

Based on the scores, C.I. Direct Blue 218 [CAS: 28407 -37 -6] can be considered to be moderately irritating to rabbit eyes.

This is supported by another experimental study summarized in OTS0215154, NTRL report, last updated 1983; for the target chemical. 6 New Zealand white rabbits were used for the study. Both eyes of each animal were examined with Fluorescein Sodium Ophthalmic solution U.S.P. 96 hours before testing and only those animals without observable eye defects or irritation were used. 0.1 ml undiluted C.I Direct Blue 218 was instilled into the conjunctival sac of 6 New Zealand White rabbits. The lids were held closed for 1-second for even distribution of the test substance. The other eye served as an untreated control. The eyes were not washed following instillation of the test material. The animals were immobilized in a suitable animal restrainer 1 hour subsequent to treatment. After the initial examination at 24 hours all eyes were washed with sufficient clean tap water at body temperature to remove the residual test sample. All eyes were further examined by placing one drop of Fluorescein Sodium Ophthalmic solution U.S.P. on the cornea. After 5 minutes the excess fluorescein was flushed out with sufficient tap water. Fluorescein Sodium in 2% aqueous solution when applied topically to the eye is used as an ophthalmic diagnostic aid for the detection of corneal lesions, injured areas of the cornea appear greenish yellow following application of fluorescein. The eyes were examined and the grade of ocular reaction was recorded at 1, 24, 48 and 72 hours, using the scale for scoring ocular lesions as outlined by Lehman. The evaluation of the data was done in accordance with Section 191.12 Chapter 1, Title 21, Code of Federal Regulations under the FHSA. Undiluted C.I Direct Blue 218 [CAS: 28407 -37 -6] when instilled into the conjunctival sac of 6 New Zealand White rabbits produced moderate eye damage at 24 hours in the form of slight to moderate erythema of the palpebral and bulbar conjunctivae, slight to marked chemosis of the lids, slight to moderate accumulation of watery to watery-mucoid discharge and moderate corneal opacity (4 of 6 test eyes) involving approximately 1/4 of-the corneal surface. Some improvement was noted by 72 hours, but all retained some erythema and 3/6 test eyes showed corneal opacity.

A maximum average score of 13.5 was recorded at 24 hours.

Based on the scores, C.I. Direct Blue 218[CAS: 28407 -37 -6] can be considered to be moderately irritating to rabbit eyes.

Both the experimental results are in agreement with each other, indicating a strong possibility of C.I Direct Blue 218 [CAS: 28407 -37 -6] being moderately irritating to eyes.

The above studies are supported by the experimental study summarized in OTS0215154, NTRL report, last updated 1983; for the structurally similar read across substance, trisodium 5 -amino-3-[(E)-2-(4-{4-[(E)-2-(7-amino-1-hydroxy-3-sulfonatonaphthalen-2-yl)diazen-1-yl]phenyl}phenyl)diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate(Chlorazol Black BH)[CAS: 2429 -73-4]. 6 New Zealand white rabbits were used for the study. Both eyes of each animal were examined with Fluorescein Sodium Ophthalmic solution U.S.P. 96 hours before testing and only those animals without observable eye defects or irritation were used. 0.1 ml undiluted C.I Direct Blue 218 was instilled into the conjunctival sac of 6 New Zealand White rabbits. The lids were held closed for 1-second for even distribution of the test substance. The other eye served as an untreated control. The eyes were not washed following instillation of the test material. The animals were immobilized in a suitable animal restrainer 1 hour subsequent to treatment. After the initial examination at 24 hours all eyes were washed with sufficient clean tap water at body temperature to remove the residual test sample. All eyes were further examined by placing one drop of Fluorescein Sodium Ophthalmic solution U.S.P. on the cornea. After 5 minutes the excess fluorescein was flushed out with sufficient tap water. Fluorescein Sodium in 2% aqueous solution when applied topically to the eye is used as an ophthalmic diagnostic aid for the detection of corneal lesions, injured areas of the cornea appear greenish yellow following application of fluorescein. The eyes were examined and the grade of ocular reaction was recorded at 1, 24, 48 and 72 hours, using the scale for scoring ocular lesions as outlined by Lehman. The evaluation of the data was done in accordance with Section 191.12 Chapter 1, Title 21, Code of Federal Regulations under the FHSA.

Since the results from the initial test were unsatisfactory to classify the substance according to F.H.S.A Guidelines, an additional group of 6 rabbits were selected and tested using the same procedure to obtain suitable information to classify the test chemical. The eyes of animals of Group II were examined with fluorescein approximately 4 hours prior to treatment.

A maximum average score of 10.2 was recorded at 24 hours in the initial test. This score was not sufficient to classify Chlorazol Black BH[CAS: 2429 -73-4] as per F.H.S.A Guidelines, so a confirmatory test was performed using 6 additional rabbits. In the confirmatory test, ocular damage in the form of erythema, chemosis, discharge and corneal opacity was observed. Minimal to slight conjunctival irritation (6 anims18) and corneal opacity (5 animals) persisted through 72 hours post-treatment.

A maximum average score of 30.3 was recorded at 24 hours.

Based on the observations and score in the confirmatory test, Chlorazol Black BH[CAS: 2429 -73-4] can be considered to be irritating to eyes.

These results are also supported by the experimental summarized inOTS0215154, NTRL report, last updated 1983; for the structurally similar read across substance, Disodium 5 -[[4'-[(2,6-diamino-3-methyl-5-sulphonatophenyl)azo]-3,3'-dimethyl[1,1'-biphenyl]-4-yl]azo]salicylate(Direct Orange 6) [CAS: 6637-88-3].6 New Zealand white rabbits were used for the study. Both eyes of each animal were examined with Fluorescein Sodium Ophthalmic solution U.S.P. 96 hours before testing and only those animals without observable eye defects or irritation were used. 0.1 ml (72 mg) of Direct Orange 6 [CAS: 6637-88 -3]was instilled into the conjunctival sac of 6 New Zealand White rabbits. The lids were held closed for 1-second for even distribution of the test substance. The other eye served as an untreated control. The eyes were not washed following instillation of the test material. The animals were immobilized in a suitable animal restrainer 1 hour subsequent to treatment. After the initial examination at 24 hours all eyes were washed with sufficient clean tap water at body temperature to remove the residual test sample. All eyes were further examined by placing one drop of Fluorescein Sodium Ophthalmic solution U.S.P. on the cornea. After 5 minutes the excess fluorescein was flushed out with sufficient tap water. Fluorescein Sodium in 2% aqueous solution when applied topically to the eye is used as an ophthalmic diagnostic aid for the detection of corneal lesions, injured areas of the cornea appear greenish yellow following application of fluorescein. The eyes were examined and the grade of ocular reaction was recorded at 1, 24, 48 and 72 hours, using the scale for scoring ocular lesions as outlined by Lehman. The evaluation of the data was done in accordance with Section 191.12 Chapter 1, Title 21, Code of Federal Regulations under the FHSA.

Moderate eye damage in the form of slight to moderate erythema, well defined chemosis, slight to moderate accumulation of watery to watery-mucoid discharge and corneal opacity in 6/6 test eyes of a moderate degree involving approximately 1/4 of the corneal surface (2 animals); greater than 1/4, but less than 1/2 (3 animals), and greater than 1/2, but less than 3/4 (1 animal) were observed within 24 hours of exposure to the test chemical. Only slight improvement in the condition of the test eyes was observed by 72 hours. All animals still showed a slight to moderate degree of corneal opacity. A maximum average score of 30.5 was recorded at 24 hours.

Based on the observations and scores, Direct Orange 6 [CAS: 6637-88-3] can be considered to be irritating to eyes.

Available data for the target and its structurally similar read across substances indicate a strong possibility of C.I Direct Blue 218 [CAS: 28407 -37 -6] being moderately irritating to eyes.Comparing the above annotations with the criteria of CLP regulation,C.I Direct Blue 218 [CAS: 28407 -37 -6] can be classified under the category “Category 2”.

Justification for classification or non-classification

Available data for C.I Direct Blue 218 [CAS: 28407 -37 -6] indicates that it is not likely to cause any irritation to skin but causes moderate irritation to eyes..

Hence, C.I Direct Blue 218 [CAS: 28407 -37 -6] can be classified under the category “Not Classified” for skin irritation and “Category 2” for eye irritation as per CLP regulation.