Reaction mass of 6-Chloro-2-{4[ethyl(2-hydroxyethyl)amino]phenyl}-1-methylbenzo[cd]indolium hydroxide and 6-chloro-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium acetate

Endpoint summary

Description of key information

The substance did not lead to skin irritation, however it needs to be classified as eye irritant category 2.

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:

Test start date: 15 December, 1990; Test end date: 22 December, 1990; Study completion date: 13 March, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Specific details on test material used for the study:

- Substance identified as: FAT 31064/F
- Batch No.: EN 158496.82 / HEW 133/6
- Description: powder
- Stability of test article: stable; expiration date: November 1994
- Stability of test article dilution: stable for at least 2 hours
- Safety Precautions: Gloves, goggles and face mask were sufficient to assure personnel health and safety.

Species:

rabbit

Strain:

New Zealand White

Remarks:

females were used

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wolferstrasse 4, CH-4414 Füllinsdorff
- Age at study initiation: 23 weeks
- Weight at study initiation: 3.6 - 3.8 kgs
- Housing: individually in steel cages
- Diet: Pelleted standard Kuba 341, Batches 49/89 and 50/89 rabbit maintenance diet ("Kuba", Klingentalmuehle AG, CH-4303 Kaiseraugst), ad libitum
- Water: ad libitum
- Acclimation period: 4 days under test conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
Standard Laboratory Conditions.
Air-conditioned with 10-15 air changes per hour and hourly monitored environment with temperature 20±3 degrees centigrade, relative humidity 40-70%, 12 hours artificial fluorescent light/12 hours dark, music/light period.

Type of coverage:

occlusive

Preparation of test site:

shaved

Remarks:

Approximately 24 hours before treatment, the dorsal fur was shaved with an electric clipper, exposing an area of approximately 100 square centimeters (10 cm x 10 cm). The skin of the animals was observed 1 day prior to test article administration.

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3 females

Details on study design:

- On test day 1, 0.5 g of the test article was applied to approx. 6 square cm of the intact skin of the shaved area. It was covered with a 3.0 cm x 3.0 cm patch of surgical gauze. The gauze was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with an elastic bandage. The duration of treatment was 4 hours.

Observations:
Viability / Mortality: daily.
Body Weights: Start of acclimatization, Day 1 of test (application day) and at termination of observation.

Symptoms:
Four hours after the application, the dressing was removed and the skin was flushed with lukewarm tap water. The skin reaction was assessed according to the OECD numerical scoring system.
The skin reaction was assessed at 1, 24, 48 and 72 hours intervals after the removal of the dressing, gauze patch and test article.
The corrosive properties of the test article and the staining of the treated skin area related to the test article were described and recorded.

Necropsy
The observation was terminated 72 hours after application of the test article.
All rabbits were killed by an intravenous injection of T61 (Hoechst AG) into the ear vein and discarded.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

COLORATION: In the area of application, blue staining of the treated skin by pigment or coloring of the test article was observed in all animals from 1 hour to 72 hours after treatment (termination).

BODY WEIGHTS: The body weight gain of all rabbits was similar.

CLINICAL SYMPTOMS / MORTALITY

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

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 31064/F was considered to be a "non-irritant" to the skin.

Executive summary:

The irritation potential of FAT 31064/F was evaluated in a study conducted according to OECD Guideline 404.

3 female New Zealand whiate rabbits were used for this study. A gauze patch (20 cm²) bearing 0.5 g of the test substance was applied to the shaved area on flank for 4 hours. The skin reactions were evaluated 24, 48, and 72 hours after removing the gauze patches according to the OECD scoring system.

No erythema or edema was observed at any timepoints. Hence, FAT 31064/F was considered to be a non-irritant to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Test start date: 16 June, 1980; Test end date: 23 June, 1980; Study completion date: 30 June 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: the United States Environmental Protection Agency (EPA) § 163.81-4 "Primary eye irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978.

Deviations:

no

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Code No.of test material: FAT 31064/C
- Purity: 42 %

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: In-house
- Age at study initiation: adult ones
- Weight at study initiation: 2 to 3 kgs
- Housing: individually in metal cages, numbered by ear tags
- Diet: ad libitum, standard rabbit food - NAFAG, No. 814, Gossau SG
- Water: ad libitum
- Acclimation period: minimum of 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 10 %
- Photoperiod: 10 hour light cycle day

IN-LIFE DATES: From: 4 February, 1980; To: 12 February, 1980

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL

Duration of treatment / exposure:

In 3 of the 6 rabbits approximately 30 seconds after treatment the treated eye was flushed with 10 mL of physiological saline. No flushing was performed in remaining animals.

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

6 (3 males and 3 females)

Details on study design:

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

SCORING SYSTEM: the United States Environmental Protection Agency (EPA) § 163.81-4 "Primary eye irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978.

TOOL USED TO ASSESS SCORE: Reading of reaction is facilitated by use of a binocular loupe, hand slit-lamp, or other expert means.

SCORING SYSTEM: Scale for scoring Ocular Lesions.
1.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 maximum=80

2.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, haemorrhage, gross destruction (any or all of these): 2
Ax5 Total maximum=10

3.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
Score (a+b+c)x 2 Total maximum=20


TOOL USED TO ASSESS SCORE: Slit lamp
The eye irritation was appraised with a slit-lamp on day 1, 2, 3, 4 and 7 and was scored for each individual rabbit.

Irritation parameter:

cornea opacity score

Remarks:

Rinsed eyes

Basis:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 7 d

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Remarks:

Rised eyes

Basis:

mean

Time point:

24/48/72 h

Score:

0.78

Max. score:

2

Reversibility:

fully reversible within: 7 d

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Rinsed eyes

Basis:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 7 d

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Remarks:

Rinsed eyes

Basis:

mean

Time point:

24/48/72 h

Score:

2.78

Max. score:

4

Reversibility:

not fully reversible within: 7 d

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Remarks:

Eye not rinsed

Basis:

mean

Time point:

24/48/72 h

Score:

1.66

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Remarks:

Eye not rinsed

Basis:

mean

Time point:

24/48/72 h

Score:

0.66

Max. score:

2

Reversibility:

not fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Eye not rinsed

Basis:

mean

Time point:

24/48/72 h

Score:

2.44

Max. score:

3

Reversibility:

not fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Remarks:

Eye not rinsed

Basis:

mean

Time point:

24/48/72 h

Score:

2.66

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

FAT 31064/C was found to cause a moderate irritation when applied to the rabbit eye mucosa. Rinsing the eyes following instillation was of little but assessable effect.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

FAT 31064/C can be classified as an irritant to the eye.

Executive summary:

The eye irritation potential of the test item was evaluated in a study conducted according to procedure described in the United States Environmental Protection Agency (EPA) § 163.81-4 "Primary eye irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978. 6 New Zealand rabbits (3 males; 3 females) were used in this study. 0.1 mL of test material was inserted into the conjunctival sac of the left eye of the rabbits and the lids were gently closed for a few seconds. The right eye was not treated and served as an untreated control. In 3 of the 6 rabbits approximately 30 seconds after treatment the treated eye was flushed with 10 mL of physiological saline. The eye irritation was appraised with a slit-lamp on day 1, 2, 3, 4 and 7 and was scored for each individual rabbit. The mean reaction scores for cornea, iris and conjunctiva observed for the rabbits with rinsed eyes were 1.00, 0.78, 2.00 and 2.78, respectively. The instillation was found to cause moderate irritation when applied to the rabbit eye mucosa. Hence, the test item can be classified as an irritant to the eye, classified as eye irritant category 2 as per CLP (Regulation EC No. 1272/2008) criteria.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Test start date: 21 July, 1980; Test end date: 28 July, 1980; Study completion date: 21 August 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: the United States Environmental Protection Agency (EPA) § 163.81-4 "Primary eye irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978.

Deviations:

no

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Code No.of test material: FAT 31064/D
- Appearance: powder
- Purity: 69 %

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: In-house
- Age at study initiation: Adult ones
- Weight at study initiation: 2 to 3 kgs
- Housing: Individually in metal cages, numbered by ear tags
- Diet: ad libitum, standard rabbit food - NAFAG, No. 814, Gossau SG
- Water: ad libitum
- Acclimation period: Minimum of 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 10 %
- Photoperiod: 10 hour light cycle day

IN-LIFE DATES: From: 4 February, 1980; To: 12 February, 1980

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g

Duration of treatment / exposure:

In 3 of the 6 rabbits approximately 30 seconds after treatment the treated eye was flushed with 10 mL of physiological saline. No flushing was performed in remaining animals.

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

6 (3 males and 3 females)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: Yes, 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: Reading of reaction is facilitated by use of a binocular loupe, hand slit-lamp, or other expert means.

SCORING SYSTEM: the United States Environmental Protection Agency (EPA) § 163.81-4 "Primary eye irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978.

Scale for scoring Ocular Lesions.
1.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 maximum=80

2.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, haemorrhage, gross destruction (any or all of these): 2
Ax5 Total maximum=10

3.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

Score (a+b+c)x 2 Total maximum=20

Irritation parameter:

cornea opacity score

Remarks:

Rinsed eyes

Basis:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 7 d

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Remarks:

Rinsed eyes

Basis:

mean

Time point:

24/48/72 h

Score:

0.78

Max. score:

2

Reversibility:

fully reversible within: 7 d

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Remarks:

Rinsed eyes

Basis:

mean

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: 7 d

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Remarks:

Rinsed eyes

Basis:

mean

Time point:

24/48/72 h

Score:

2.89

Max. score:

4

Reversibility:

not fully reversible within: 7 d

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Remarks:

Eye not rinsed

Basis:

mean

Time point:

24/48/72 h

Score:

1.66

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Remarks:

Eye not rinsed

Basis:

mean

Time point:

24/48/72 h

Score:

1.55

Max. score:

2

Reversibility:

not fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Eye not rinsed

Basis:

mean

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Remarks:

Eye not rinsed

Basis:

mean

Time point:

24/48/72 h

Score:

3.77

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

FAT 31064/D was found to cause a moderate irritation when applied to the rabbit eye mucosa. Rinsing the eyes following instillation was of little but assessable effect.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

FAT 31064/D can be classified as an irritant to the eye.

Executive summary:

The eye irritation potential of the test item was evaluated in a study conducted according to procedure described in the United States Environmental Protection Agency (EPA) § 163.81-4 "Primary eye irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978. 6 New Zealand rabbits (3 males; 3 females) were used in this study. 0.1 g of test material was inserted into the conjunctival sac of the left eye of the rabbits and the lids were gently closed for a few seconds. The right eye was not treated and served as an untreated control. In 3 of the 6 rabbits approximately 30 seconds after treatment the treated eye was flushed with 10 mL of physiological saline. The eye irritation was appraised with a slit-lamp on day 1, 2, 3, 4 and 7 and was scored for each individual rabbit. The mean reaction scores for cornea, iris and conjunctiva observed for the rabbits with rinsed eyes were 1.00, 0.78, 3.00 and 2.89, respectively. The instillation was found to cause moderate irritation when applied to the rabbit eye mucosa. Hence, the test item can be classified as an irritant to the eye, classified as eye irritant category 2 according to CLP (Regulation EC No. 1272/2008) criteria.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The irritation potential of FAT 31064/F was evaluated in a study (1990) conducted according to OECD Guideline 404. 3 female rabbits were used in this study. A gauze patch (20 cm²) bearing 0.5 g of the test substance was applied to the shaved area on flank for 4 hours. The skin reactions were evaluated 24, 48, and 72 hours after removing the gauze patches according to the OECD scoring system. No erythema or edema was observed. Hence, FAT 31064/F was considered to be a non-irritant to the skin. The substance was further evaluated in a (four) number of studies and the outcome of these studies supports the conclusion that the substance is not a askin irritant.

Eye irritation:

The eye irritation potential of the test item was evaluated in a key study (1980) conducted according to procedure described inthe United States Environmental Protection Agency (EPA) § 163.81-4 "Primary eye irritation study", Federal Register, Vol. 43, No. 163, August 22, 1978. 6 rabbits (3 males; 3 females) were used in this study. The test material in an amount of 0.1 g was inserted into the conjunctival sac of the left eye of the rabbits and the lids were gently closed for a few seconds. The right eye was not treated and served as an untreated control. In 3 of the 6 rabbits approximately 30 seconds after treatment the treated eye was flushed with 10 mL of physiological saline. The eye irritation was appraised with a slit-lamp on day 1, 2, 3, 4 and 7 and was scored for each individual rabbit. The mean reaction scores for cornea, iris and conjunctiva observed for the rabbits with rinsed eyes were 1.00, 0.78, 3.00 and 2.89, respectively. The instillation was found to cause moderate irritation when applied to the rabbit eye mucosa. Hence, the test item can be classified as an irritant to the eye, classified as eye irritant category 2 according to CLP (Regulation EC No. 1272/2008) criteria. The substance was further evaluated in number of studies, and 3 out of 4 studies support the conclusion that the substance exposure in rabbit eyes leads to irritation. In the most recently conducted study (1990), the substance did not cause irritation in the eyes. However, using the conservative approach, the substance needs to be classified as eye irritant cat. 2 according to CLP (Regulation EC No. 1272/2008) criteria.

Justification for classification or non-classification

Based on the available information, Basic Blue 145 does not need to be classified for skin irriation, however it needs to be classified as eye irritant cat. 2 according to CLP (Regulation EC No. 1272/2008) criteria.