4,11-diamino-2-[3-(2-methoxyethoxy)propyl]-1H-naphth[2,3-f]isoindole-1,3,5,10(2H)-tetrone

Endpoint summary

Description of key information

Substance was considered to not induce skin or eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

yes

Remarks:

See below

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on REACH

Deviations:

yes

Remarks:

See below

Principles of method if other than guideline:

None

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Justification for test system used:

Recommended by the OECD guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier: MatTek
Date received: 14 April 2015
EpiDermTM Tissues (0.5cm2) lot number: 21654
Assay Medium lot number: 040915TMA
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was placed into a refrigerator.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

two exposure periods of 3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Irritation / corrosion parameter:

% tissue viability

Remarks:

relative mean

Run / experiment:

60 minute exposure

Value:

105.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Remarks:

relative mean

Run / experiment:

3 minute

Value:

108.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosion

Other effects / acceptance of results:

Direct MTT Reduction
The direct MTT reduction test was inconclusive due to the dark blue color of the test item. Therefore, an additional procedure using freeze killed tissues was performed to assess for the possibility of direct MTT reduction. An additional procedure was also performed using viable tissues to assess for the possibility of color interference. The results of the additional procedures showed a negligible degree of interference due to possible direct reduction of MTT or color interference. It was therefore considered unnecessary to use the results of the additional procedures for quantitative correction of results and reporting purposes.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 2.4 % relative to the negative control treated tissues following the 3-minute exposure period. The positive control acceptance criterion was therefore satisfied. The mean OD562 for the negative control treated tissues was 2.004 for the 3-minute exposure period and 2.005 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

 

Mean OD₅₆₂ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item	Exposure Time	Mean OD562^1	Percentage Viability
Negative Control†	3 minutes	2.004	100*
	60 minutes	2.005	100*
Positive Control†	3 minutes	0.049	2.4^2
	60 minutes	0.048	2.4^3
Test Item	3 minutes	2.180	108.8^2
	60 minutes	2.122	105.8^3

*=   The mean viability of the negative control tissues is set at 100%

1=   Mean of EpiDerm^TMtissues tested in duplicate

2=   Viability expressed as a percentage of the 3 minute negative control tissues

3=   Viability expressed as a percentage of the 60 minute negative control tissues

†= Control data was shared with Harlan Laboratories Ltd. study number 41500303

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

The corrosivity potential of the test item was evaluated using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. The direct MTT reduction test was inconclusive due to the dark blue color of the test item. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT or cause color interference and could have given rise to a false negative result. Therefore, an additional procedure using freeze killed tissues was performed to assess for the possibility of direct MTT reduction. An additional procedure was also performed using viable tissues to assess for the possibility of color interference. At the end of the exposure period the test item was rinsed from each tissue before the tissues were taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The results of the additional procedures showed a negligible degree of interference due to possible direct reduction of MTT or color interference. It was therefore considered unnecessary to use the results of the additional procedures for quantitative correction of results or for reporting purposes. The relative mean viabilities of the test item treated tissues were as follows:

60 minute exposure: 105.8 %

3 minute exposure: 108.8 %

The quality criteria required for acceptance of results in the test were satisfied. The test item was considered to be non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

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

Principles of method if other than guideline:

None

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH-4414 Fuellinsdorf
- Age at study initiation:approx. 12-14 weeks old
- Weight at study initiation: 2150 to 2220 g
- Housing: The animals were housed individually in metal cages, identified with individually numbered ear tags.
- Diet (e.g. ad libitum): The rabbits received ad libitum standard rabbit pellet - Nafag No. 814, Gossau, Switzerland.
- Water (e.g. ad libitum): Fresh water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±3
- Humidity (%):30-70
- Air changes:
- Photoperiod: 12 hours light cycle day

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

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

Duration of treatment / exposure:

4 hours

Observation period:

1, 24, 48, and 72 hours

Number of animals:

3 female rabbits

Details on study design:

TEST SITE
- Area of exposure: A gauze patch (20 cm²) bearing 0.5 ml of the test substance was applied to the flank.
An area of at least 6 cm² was shaved on both flanks of the animals approximately 24 hours before treatment. A gauze patch (20 cm² bearing 0.5 ml of the test substance was applied to the flank. A control gauze patch was applied to the contralateral flank. The patches were loosely covered with an aluminum foil (36 cm² and held in place for 4 hours by an adhesive tape (Isoplast, Isoplast AG, CH-5200 Bruggl). The animals were checked daily for systemic symptoms and mortality.

SCORING SYSTEM:
The skin reactions were evaluated 1, 24, 48, and 72 hours after removing the gauze patches according to the OECD scoring system.
The irritant/corrosive potency of FAT 36152/B was classified according to the EEC commission directive No. 83/467/1983.

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: The application site was blue-stained (substance-related) in animals No. 89 and 91 up to 72 hours after the application; therefore the evaluation of a possible erythema formation was impeded.

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

The application site was blue-stained in animals No. 89 and 91 up to 72 hours after the application; therefore the evaluation of a possible erythema formation was impeded.

Other effects:

None

Individual Skin Reaction Score:

Animal No.		ERYTHEMA			EDEMA
		89/F   CF/TF	90/F  CF/TF	91/F   CF/TF	89/F   CF/TF	90/F  CF/TF	91/F   CF/TF
After 1 hour		0/*	0/1	0/*	0/0	0/0	0/0
After 24 hours		0/*	0/0	0/*	0/0	0/0	0/0
After 48 hours		0/*	0/0	0/*	0/0	0/0	0/0
After 72 hours		0/*	0/0	0/*	0/0	0/0	0/0
Mean 24-72 hours		0/*	0/0	0/*	0/0	0/0	0/0
After 7 days		0/0	0/0	0/0	0/0	0/0	0/0

CF - Control flank

TF - Test flank

M - Male

F - Female

* - Blue staining (substance related)

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 36152/B can be classified as non-irritant according to the EEC directive No. 83/467/1983.

Executive summary:

A study was performed to determine the skin irritation of FAT 36152/B in rabbits according to OECD Guideline 404 (Acute Dermal Irritation / Corrosion).

The test was performed on 3 female rabbits, checked for normal skin conditions. An area of at least 6 cm'was shaved on both flanks of the animals approximately 24 hours before treatment. A gauze patch (20 cm² bearing 0.5 ml of the test substance was applied to the flank. A control gauze patch was applied to the contralateral flank. The skin reactions were evaluated 1, 24, 48, and 72 hours after removing the gauze patches according to the OECD scoring system. The application site was blue-stained in some animals up to 72 hours after the application; therefore the evaluation of a possible erythema formation was impeded. Since distinct erythema reactions do not occur without edema reactions FAT 36152/B can be classified as non-irritant in albino rabbits. Under the experimental conditions employed, FAT 36152/B can be classified as non-irritant according to the EEC directive No. 83/467/1983.

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

Study period:

1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

no

Specific details on test material used for the study:

None

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH-4414 Fuellinsdorf
- Age at study initiation: 2160 to 2350 g
- Weight at study initiation: approx. 12-14 weeks old
- Housing: The animals were housed individually in metal cages, identified with individually numbered ear tags.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20±3
- Humidity (%): 30-70
- Photoperiod: 12 hours light cycle day

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eye remained untreated and served as a control

Amount / concentration applied:

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

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 male rabbits

Details on study design:

0.1 ml of FAT 36152/B was placed into the conjunctival sac of the right eye of each animal, after gently pulling away the lower lid from the eyeball. The lids were then held together for about one second in order to prevent loss of the test material. The left eye remained untreated and served as a control. The animals were checked daily for systemic symptoms and mortality. The ocular reactions were evaluated 1, 24, 48, and 72 hours after the instillation of FAT 36152/B according to the OECD scoring system. A slit-lamp was used to facilitate the evaluation. The irritant/corrosive potency of FAT 36152/B was classified according to the EEC commission directive No. 83/467, 1983

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

fully reversible within: 24 h

Remarks on result:

other: Conjunctival redness (score = 1) observed at 1 h observation in animals # 76 and 77, was fully reversible by 24 h observation.

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Because no reactions were observed at 72 hours after instillation of FAT 36152/B, the test was ended after the 72 hours evaluation.

Other effects:

None

Individual eye scores:

Animal No. 75/M	Cornea	Iris	Cinjuctiva
	CE/TE	CE/TE	Redness CE/TE	Chemosis CE/TE
After 1 h	0/0	0/0	0/0	0/0
After 24 h	0/0	0/0	0/0	0/0
After 48 h	0/0	0/0	0/0	0/0
After 72 h	0/0	0/0	0/0	0/0
Mean (24-72 hours)	0/0	0/0	0/0	0/0

Animal No. 76/M	Cornea	Iris	Cinjuctiva
	CE/TE	CE/TE	Redness CE/TE	Chemosis CE/TE
After 1 h	0/0	0/0	0/0	0/0
After 24 h	0/0	0/0	0/0	0/0
After 48 h	0/0	0/0	0/0	0/0
After 72 h	0/0	0/0	0/0	0/0
Mean (24-72 hours)	0/0	0/0	0/0	0/0

Animal No. 77/M	Cornea	Iris	Cinjuctiva
	CE/TE	CE/TE	Redness CE/TE	Chemosis CE/TE
After 1 h	0/0	0/0	0/0	0/0
After 24 h	0/0	0/0	0/0	0/0
After 48 h	0/0	0/0	0/0	0/0
After 72 h	0/0	0/0	0/0	0/0
Mean (24-72 hours)	0/0	0/0	0/0	0/0

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 36152/B is classified as non-irritant to albino rabbit's eye.

Executive summary:

A study was performed to determine eye irritation potential of FAT 36152/B according to OECD Guideline 405 (Acute Eye Irritation / Corrosion). 0.1 ml of FAT 36152/B was placed into the conjunctival sac of the right eye of each animal, after gently pulling away the lower lid from the eyeball. The lids were then held together for about one second in order to prevent loss of the test material. The left eye remained untreated and served as a control. The animals were checked daily for systemic symptoms and mortality. The ocular reactions were evaluated 1, 24, 48, and 72 hours after the instillation of FAT 36152/B according to the OECD scoring system. A slit-lamp was used to facilitate the evaluation. The irritant/corrosive potency of FAT 36152/B was classified according to the EEC commission directive No. 83/467, 1983. Because no reactions were observed at 24, 48 and 72 hours after instillation of FAT 36152/B, the test was ended after the 72 hours evaluation, and FAT 36152/B was considered as non-irritant to albino rabbit's eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

The corrosivity potential of the test item was evaluated using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. At the end of the exposure period the test item was rinsed from each tissue before the tissues were taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). The relative mean viabilities of the test item treated tissues were as follows:

60 minute exposure: 105.8 %

3 minute exposure: 108.8 %

Based on these findings, the test item was considered to be non-corrosive to the skin.

Further, in vivo studies performed according to OECD Guideline 404 with FAT 36152/A (1983) and FAT 36152/B (1986) found the target chemical did not induce irritation on shaven rabbit skin. Hence based on the available data, it can be concluded that the substance under evaluation is not irritating to the skin.

 

Eye:

A bovine corneal opacity test was performed to identify if the target chemical requires classification for eye irritation or serious eye damage. In this study, the test item was applied at a concentration of 20 % w/v in 0.9 % w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). Following treatment, In Vitro Irritancy Score for the test item was determined to be 0.2. Hence, FAT 36152/N can be classified as not irritanting to eyes. In an in vivo study performed according to OECD Guideline 405 (Acute Eye Irritation / Corrosion), 0.1 ml of FAT 36152/B was placed into the conjunctival sac of the right eye of each animal, after gently pulling away the lower lid from the eyeball. The lids were then held together for about one second in order to prevent loss of the test material. The left eye remained untreated and served as a control. The animals were checked daily for systemic symptoms and mortality. The ocular reactions were evaluated 1, 24, 48, and 72 hours after the instillation of FAT 36152/B according to the OECD scoring system. A slit-lamp was used to facilitate the evaluation. The irritant/corrosive potency of FAT 36152/B was classified according to the EEC commission directive No. 83/467, 1983. Because no reactions were observed at 24, 48 and 72 hours after instillation of FAT 36152/B, the test was ended after the 72 hours evaluation and FAT 36152/B was considered as non-irritant to albino rabbit's eye. FAT 36152/A was considered as not irritating when instilled in rabbit eyes (1983). Hence, based on the available data, it was concluded that the substance under evaluation is not irritating to the eyes.

Justification for classification or non-classification

Based on the above stated assessment of the skin as well as eye irritation potential, the substance does not have to be classified for skin or eye irritation according to CLP (Regulation (EC) No 1272/2008 Of the European parliament and of the Council.