Hydrogen [4-[4-(p-ethoxyanilino)-4'-[ethyl(m-sulphonatobenzyl)amino]-2'-methylbenzhydrylene]-3-methylcyclohexa-2,5-dien-1-ylidene](ethyl)(m-sulphonatobenzyl)ammonium, monosodium salt

Endpoint summary

Description of key information

FAT 20085/A is not irritating to the skin and eyes of the albino rabbit.

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:

May 1975

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: "Hazardous Substances Regulations" under the U.S. Federal Hazardous Substances Labelling Act Sect. 191.11 (February 1965).

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Animals:
Healthy New Zealand White rabbits (Porton strain) aged 3-4 months with average body weights of 2.71 kg (males) and 2.66 kg (females), bred on the premises and maintained under a semi-barrier system. Six rabbits (3 males and 3 females) were acclimatized in the test area for one week prior to the start of the trial.

Husbandry:
The rabbits were caged singly in an experimental room maintained at a temperature of 20 °C. (± 1 °C) and a relative humidity of 50-70 %. Animals were exposed to artificial light for 10 hours daily from 08.00 -18..00 hours.
A commercial irradiated diet (Styles-Oxoid) was fed ad lib. Sterile filtered water was available at all times.

Type of coverage:

occlusive

Preparation of test site:

other: shaved or abraded

Vehicle:

water

Controls:

no

Amount / concentration applied:

10 g of the test compound was mixed with 10 ml of water to make a solution of 15 ml. 0.75 ml of which was applied to each test site (0.5 g).

Duration of treatment / exposure:

24 hours

Observation period:

72 hours

Number of animals:

six rabbits (3 males and 3 females).

Details on study design:

Twenty-four hours prior to the dermal application, the backs of the rabbits were shaved over an area consisting of at least 10 % of the total body surface. Two test sites lateral to the mid line of the back were used on each rabbit. Immediately before the application of the test compound, the right hand site was abraded with the point of a sterile hypodermic needle. The abrasions were sufficiently deep to penetrate the
stratum corneum but not to damage the dermis. The left hand site remained intact.

10 g of the test compound was mixed with 10 ml of water to make a solution of 15 ml 0.75 ml- of which was applied to each test site
on a 2.5 cm square gauze pad.
These were covered with aluminium foil secured with "Sleek"* adhesive tape. The test sites were then enclosed by a 6" wide "Coban"** self-adhesive bandage the edges of which were fixed to the skin by strips of "Sleek" in order to retain the test substance in close contact with the skin.

After 24 hours the dressing was removed and the intact and abraded application sites were assessed for oedema and erythema.
A further assessment was made at 72 hours.

SCORING SYSTEM:

Erythema and Eschar Formation:
*No erythema: 0
*Slight erythema (barely perceptible): 1
*Well defined erythema: 2
*Moderate to severe erythema: 3
*Severe erythema (beet red) to slight eschar formation: 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 mm): 3
*Severe-oedema (raised more than 1 mm and extending beyond the area of exposure): 4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

3 males and 3 females

Time point:

other: 24 hours

Score:

0.83

Max. score:

2

Reversibility:

fully reversible within: 3 days

Remarks on result:

other: Intact skin

Irritation parameter:

erythema score

Basis:

mean

Remarks:

3 males and 3 females

Time point:

other: 72 hours

Score:

0

Max. score:

0

Remarks on result:

other: Intact skin

Irritation parameter:

erythema score

Basis:

mean

Remarks:

3 males and 3 females

Time point:

other: 24 hours

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 3 days

Remarks on result:

other: Abraded skin

Irritation parameter:

erythema score

Basis:

mean

Remarks:

3 males and 3 females

Time point:

other: 72 hours

Score:

0

Max. score:

0

Remarks on result:

other: Abraded skin

Irritation parameter:

edema score

Basis:

mean

Remarks:

3 males and 3 females

Time point:

other: 24 hours

Score:

2.17

Max. score:

3

Reversibility:

fully reversible within: 3 days

Remarks on result:

other: Intact skin

Irritation parameter:

edema score

Basis:

mean

Remarks:

3 males and 3 females

Time point:

other: 72 hours

Score:

0

Max. score:

0

Remarks on result:

other: Intact skin

Irritation parameter:

edema score

Basis:

mean

Remarks:

3 males and 3 females

Time point:

other: 24 hours

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 3 days

Remarks on result:

other: Abraded skin

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 72 hours

Score:

0

Max. score:

0

Remarks on result:

other: Abraded skin

Irritant / corrosive response data:

Slight to well defined erythema, and slight to moderate oedema were seen in 4/6 and 6/6 rabbits respectively 24 hours after application of the compound. All sites were normal by 72 hours. The primary irritation score was 1.4.

Scoring tables

Table: Erythema scores- intact skin

Erythema
Animal	Observation time
	24 hours	72 hours
11	1	0
12	1	0
13	0	0
14	0	0
15	1	0
16	2	0
	0.83	0

Table: Erythema scores: abraded skin

Animal	Observation time
	24 hours	72 hours
11	1	0
12	0	0
13	0	0
14	0	0
15	1	0
16	2	0
	0.67	0

Table: Oedema scores - intact skin

Animal	Observation time
	24 hours	72 hours
11	2	0
12	2	0
13	2	0
14	2	0
15	3	0
16	2	0
	2.17	0

Table: Oedema scores: abraded skin

Animal	Observation time
	24 hours	72 hours
11	2	0
12	2	0
13	2	0
14	2	0
15	2	0
16	2	0
	2.00	0

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 20085/A is not a skin irritant in the albino rabbit.

Executive summary:

The primary skin irritation test in Albino rabbits with compound FAT 20085/A was conducted according to the method "Hazardous Substances Regulations" under the U.S. Federal Hazardous Substances Labelling Act Sect. 191.11 (February 1965). 3 males and 3 females were used in the test.

Twenty-four hours prior to the dermal application, the backs of the rabbits were shaved over an area consisting of at least 10 % of the total body surface. Two test sites lateral to the mid line of the back were used on each rabbit. Immediately before the application of the test compound, the right hand site was abraded with the point of a sterile hypodermic needle. The abrasions were sufficiently deep to penetrate the stratum corneum but not to damage the dermis. The left hand site remained intact.

10 g of the tost compound was mixed with 10 ml of water to make a solution of 15 ml. 0.75 ml of which was applied to each test site on a 2.5 cm square gauze pad. These were covered with aluminium foil secured with "Sleek" adhesive tape. The test sites were then enclosed by a 6" wide "Coban" self-adhesive bandage the edges of which were fixed to the skin by strips of "Sleek" in order to retain the test substance in close contact with the skin.

After 24 hours the dressing was removed and the intact and abraded application sites were assessed for oedema and erythema. A further assessment was made at 72 hours.

EXPERIMENTAL RESULTS:

Slight to well defined erythema, and slight to moderate oedema were seen in 4/6 end 6/6 rabbits respectively 24 hours after application of the compound. All sites were normal by 72 hours. The primary irritation score was 1.4. However, the scores fail to meet the threshold criteria of classification as set forth by the Regulation (EC) No. 1272/2008. In conclusion, FAT 20085/A is considered to be not a skin irritant in the albino rabbit.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

May 1975

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: "Hazardous Substances Regulations" under the U.S.A. Federal Hazardous Substances Labelling Act Sect. 191.12 (February 1965.)

Deviations:

not applicable

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animals:
Healthy New Zealand White rabbits (Porton strain) aged 10-13 weeks with average body weights of 2.71 kg (males) and 2.66 kg (females), bred on the premises and maintained under a semi-barrier system. Six rabbits (3 males and 3 females) were acclimatised in the test area for one week prior to the start of the trial.

Husbandry:
The rabbits were caged singly in an experimental room maintained at a temperature of 20 °C. (+/- 1.0 °C) and a relative humidity of 50-70 %. Animals were exposed to artificial light for 10 hours daily from 08.00 - 18.00 hours. A commercial irradiated diet (Styles-Oxoid) was fed ad lib. Sterile filtered water was available at all times.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

100 mg of the test compound

Observation period (in vivo):

The rabbits were examined 1 and 6 hours, 1, 2, 3, 6, and 10 days after application of the test compound.

Number of animals or in vitro replicates:

6 rabbits (3 males and 3 females)

Details on study design:

The test compound were instilled into the conjunctival sac of the left eye 6 rabbits. The eyelids were then held closed for 1 second. The right eye served as a control. After 30 seconds, the compound was, as far as possible, flushed out of the eyes of three of the rabbits with warm water for 1 minute.


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

AxBx5 (Total maximun= 80)

IRIS:
(A) Value
*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

Ax5 (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 #1

Time point:

other: mean 24-72h

Score:

0

Max. score:

4

Remarks on result:

other: eyes rinsed after 30 seconds

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

other: mean 24-72h

Score:

0

Max. score:

4

Remarks on result:

other: eyes rinsed after 30 seconds

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

other: mean 24-72h

Score:

0

Max. score:

4

Remarks on result:

other: eyes rinsed after 30 seconds

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: mean 24-72h

Score:

0

Max. score:

2

Remarks on result:

other: eyes rinsed after 30 seconds

Irritation parameter:

iris score

Basis:

animal #2

Time point:

other: mean 24-72h

Score:

0

Max. score:

2

Remarks on result:

other: eyes rinsed after 30 seconds

Irritation parameter:

iris score

Basis:

animal #3

Time point:

other: mean 24-72h

Score:

0

Max. score:

2

Remarks on result:

other: eyes rinsed after 30 seconds

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: mean 24-72h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 10 days

Remarks on result:

other: eyes rinsed after 30 seconds

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

other: mean 24-72h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 10 days

Remarks on result:

other: eyes rinsed after 30 seconds

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

other: mean 24-72h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 10 days

Remarks on result:

other: eyes rinsed after 30 seconds

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: mean 24-72h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 10 days

Remarks on result:

other: eyes rinsed after 30 seconds

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

other: mean 24-72h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 10 days

Remarks on result:

other: eyes rinsed after 30 seconds

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

other: mean 24-72h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 10 days

Remarks on result:

other: eyes rinsed after 30 seconds

Irritant / corrosive response data:

A mild to moderate conjunctival reaction was seen in all treated eyes one hour after application of the compound. This slowly subsided until all eyes were normal by day 10.
A transient corneal reaction consisting of damage to the surface epithelium was seen in one eye at one hour only.
Unwashed eyes generally showed both a slightly more severe reaction than washed and staining of the conjunctivae by the compound for up to 48 hours.

Scoring tables

Table: Corneal opacity

Cornea opacity
Animal	Observation time			Mean value
	24 hours	48 hours	72 hours
11	0	0	0
13	0	0	0
15*	0	0	0
12	0	0	0
14*	0	0	0
16*	0	0	0	0.0

Table: Iris

Iris
Animal	Observation time			Mean value
	24 hours	48 hours	72 hours
11	0	0	0
13	0	0	0
15*	0	0	0
12	0	0	0
14*	0	0	0
16*	0	0	0	0.0

Table: Conjunctival redness

Conjunctivae (redness)
Animal	Observation time			Mean value
	24 hours	48 hours	72 hours
11	2	2	2
13	2	2	2
15*	1	1	1
12	2	2	1
14*	1	1	1
16*	2	1	0	1.4

Table: Chemosis

Conjunctivae (chemosis)
Animal	Observation time			Mean value
	24 hours	48 hours	72 hours
11	2	2	2
13	2	1	1
15*	1	1	0
12	2	2	1
14*	1	1	0
16*	0	1	0	1.1

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 20085/A is not an irritant to the rabbit eyes.

Executive summary:

The purpose of this eye irritation study was to assess the possible irritation potential when FAT 20085/A were placed in the conjunctival sac of rabbit left eyes (3 males and 3 female, 0.1 g/animal). This experiment was performed according to the "Hazardous Substances Regulations" under the U.S.A. Federal Hazardous Substances Labelling Act Sect. 191.12 (February 1965.).

The test article (0.1 g per animal) was placed in the conjunctival sac of the left eye of each animal. The right eye served as a control. After 30 seconds the compound was, as far as possible, flushed out of the eyes of three of the rabbits with warm water for 1 minute. The rabbits were examined 1, 6, 24, 48 and 72 hours after application of the test compound and for any further period that was considered necessary.

A mild to moderate conjunctival reaction was seen in all treated eyes one hour after application of the compound. This slowly subsided until all eyes were normal by day 10. A transient corneal reaction consisting of damage to the surface epithelium was seen in one eye at one hour only. Unwashed eyes generally showed both a slightly more severe reaction than washed and staining of the conjunctivae by the compound for up to 48 hours. The scores for conjunctival redness and chemosis fail to meet the threshold scores required for classification as set forth by the Regulation (EC) No. 1272/2008. Hence it can be concluded that, FAT 20085/A is not irritating when applied to the rabbit eye mucosa.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

The skin irritation potential of Acid Blue 090 was evaluated in Albino rabbits in a study conducted according to the method "Hazardous Substances Regulations" under the U.S. Federal Hazardous Substances Labelling Act Sect. 191.11 (February 1965). 3 males and 3 females were used in the test.

Twenty-four hours prior to the dermal application, the backs of the rabbits were shaved over an area consisting of at least 10 % of the total body surface. Two test sites lateral to the mid line of the back were used on each rabbit. Immediately before the application of the test compound, the right hand site was abraded with the point of a sterile hypodermic needle. The abrasions were sufficiently deep to penetrate the stratum corneum but not to damage the dermis. The left hand site remained intact.

10 g of the tost compound was mixed with 10 ml of water to make a solution of 15 ml. 0.75 ml of which was applied to each test site on a 2.5 cm square gauze pad. These were covered with aluminium foil secured with "Sleek" adhesive tape. The test sites were then enclosed by a 6" wide "Coban" self-adhesive bandage the edges of which were fixed to the skin by strips of "Sleek" in order to retain the test substance in close contact with the skin.

After 24 hours the dressing was removed and the intact and abraded application sites were assessed for oedema and erythema. A further assessment was made at 72 hours.

EXPERIMENTAL RESULTS:

Slight to well defined erythema, and slight to moderate oedema were seen in 4/6 end 6/6 rabbits respectively 24 hours after application of the compound. All sites were normal by 72 hours. The primary irritation score was 1.4. However, the scores fail to meet the threshold criteria of classification as set forth by the Regulation (EC) No. 1272/2008. In conclusion, FAT 20085/A is considered to be not a skin irritant in the albino rabbit.

Eye:

The eye irritation potential of Acid Blue 090 was assessed in an experiment that was performed according to the "Hazardous Substances Regulations" under the U.S.A. Federal Hazardous Substances Labelling Act Sect. 191.12 (February 1965.).

The test article (0.1 g per animal) was placed in the conjunctival sac of the left eye of each animal. The right eye served as a control. After 30 seconds the compound was, as far as possible, flushed out of the eyes of three of the rabbits with warm water for 1 minute. The rabbits were examined 1, 6, 24, 48 and 72 hours after application of the test compound and for any further period that was considered necessary.

A mild to moderate conjunctival reaction was seen in all treated eyes one hour after application of the compound. This slowly subsided until all eyes were normal by day 10. A transient corneal reaction consisting of damage to the surface epithelium was seen in one eye at one hour only. Unwashed eyes generally showed both a slightly more severe reaction than washed and staining of the conjunctivae by the compound for up to 48 hours. The scores for conjunctival redness and chemosis fail to meet the threshold scores required for classification as set forth by the Regulation (EC) No. 1272/2008. Hence it can be concluded that, FAT 20085/A is not irritating when applied to the rabbit eye mucosa.

Justification for classification or non-classification

Acid Blue 090 was considered to be not irritating to the skin and eyes from the available results, hence it does not warrant classification as per the criteria of the Regulation (EC) No. 1272/2008 (CLP).