4,11-diamino-2-(3-methoxypropyl)-1H-naphth[2,3-f]isoindol-1,3,5,10(2H)-tetrone

Endpoint summary

Description of key information

FAT 36152/M is considered to be non-irritant to skin and eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

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

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.

Amount/concentration applied:

25 mg of the test item

Duration of treatment / exposure:

3-Minute and 60-Minute exposure periods

Duration of post-treatment incubation (if applicable):

3 hours

Observation period:

None

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min incubation time

Value:

101.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes incubation time

Value:

103.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

The relative mean viabilities of the test item treated tissues were as follows:
60 minute exposure: 101.1%
3 minute exposure: 103.3%

Other effects:

None

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 control 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	2.074	103.3^2
	60 minutes	2.026	101.1^1

 

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

1 = Mean of EpidermTM tissues 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 41500291

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 36152/M was considered to be non-corrosive to the skin.

Executive summary:

A key study was performed to evaluate the corrosivity potential of FAT 36152/M 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.

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: 101.1%

3 minute exposure: 103.3%

The quality criteria required for acceptance of results in the test were satisfied.

In conclusion, FAT 36152/M was considered to be non-corrosive to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: 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 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Vehicle:

other: 0.9% w/v sodium chloride solution

Controls:

yes

Amount / concentration applied:

20 % w/v

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

None

Number of animals or in vitro replicates:

Three corneas were allocated to the test item

Details on study design:

Test Item Formulation and Experimental Preparation
For the purpose of this study the test item was prepared as a 20 % w/v solution in 0.9 % w/v sodium chloride solution.
The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.


Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.

The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (MEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.


Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete MEM.

A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.

Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.


Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

The negative and positive control data was shared with Harlan Laboratories Ltd. study numbers 41500304 and 41500337.


Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.


Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.


Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.


Evaluation of Results
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.


Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.


Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.


In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

Visual Observation
The condition of the cornea was visually assessed post treatment.

Irritation parameter:

in vitro irritation score

Run / experiment:

main test

Value:

0.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritant / corrosive response data:

The In Vitro irritancy scores are summarized as follows:
The test item In Vitro Irritancy Score was 0.9.
The negative control In Vitro Irritancy Score was 0.8.
The positive control In Vitro Irritancy Score was 73.6.

Other effects:

Corneal Epithelium Condition
The corneas treated with the test item or negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of ≤4.1 and permeability ≤0.105. The negative control acceptance criteria were therefore satisfied.

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD)		In VitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control†	5	5	5	0		0.004
	17	4	4	0		0.018
	20	3	5	2		0.007
				0.7*		0.010♦		0.8
Positive Control†	12	3	71	68	67.3	0.679	0.669
	27	4	62	58	57.3	0.754	0.744
	28	5	70	65	64.3	0.721	0.711
					63.0●		0.708●	73.6
Test Item	16	3	3	0	0.0	0.024	0.014
	19	3	2	-1	0.0	0.006	0.000
	26	3	6	3	2.3	0.014	0.004
					0.8●		0.006●	0.9

OD= Optical density            

* = Mean of the post-treatment -pre‑treatment values            

♦ = Mean permeability                      

● = Mean corrected value

† = Control data was shared with Harlan Laboratories Ltd. study numbers 41500304 and 41500337

Corneal Epithelium Condition Post Treatment

Treatment	Cornea Number	Observation Post Treatment
Negative Control†	5	clear
	17	clear
	20	clear
Positive Control†	12	cloudy
	27	cloudy
	28	cloudy
Test Item	16	clear
	19	clear
	26	clear

† Control data was shared with Harlan Laboratories Ltd. study numbers 41500304 and 41500337

Opacitometer Calibration

The opacitometer was calibrated on the day of the test.

 

Calibration of Opacitometer
Balance dial = 0	Target	Reading displayed	Acceptable
Calibrator number 1	75	75	Yes
Calibrator number 2	150 ±2%	150	Yes
Calibrator number 3	225 ±2%	225	Yes

 

Calibration of the opacitometer was considered to be acceptable.

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 36152/M requires no classification to UN GHS or EU CLP.

Executive summary:

A supporting test was performed to identify whether FAT 36152/M can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

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). The negative and positive control data was shared with Harlan Laboratories Ltd. study numbers 41500304 and 41500337.

Interpretation

The test item is classified according to the prediction model below:

IVIS	UN GHS	European Regulation (EC) 1272/2008
≤3	No category	Not classified for irritation
>3;≤ 55	No prediction can be made	No prediction can be made
> 55	Category 1	Category 1 H318: Causes serious eye damage

The In Vitro irritancy scores are summarized as follows:

Treatment	In vitro irritancy score
Test item	0.9
Negative control	0.8
Positive control	73.6

In conclusion, FAT 36152/M requires no classification to UN GHS or EU CLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin: 

An in vitro study was performed to evaluate the corrosivity potential of FAT 36152/M 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.

The relative mean viabilities of the test item treated tissues were as follows:

60 minute exposure: 101.1 %

3 minute exposure: 103.3 %

In conclusion, FAT 36152/M 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 GLP-compliant in vitro test was performed to identify whether FAT 36152/M can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage.

The test item was applied at a concentration of 20 % w/v in 0.9 % w/v sodium chloride solution for 240 minutes. 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). The in vitro irritancy scores for the test substance is 0.9 In conclusion, FAT 36152/M requires no classification to UN GHS or EU CLP.

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 irritation potential, the substance does not have to be classified for skin irritation according to CLP (Regulation (EC) No 1272/2008 Of the European Parliament and of the Council.

Based on the above assessment of the eye irritation potential, the substance does not have to be classified as for risk of serious damage to eyes according to CLP (Regulation (EC) No 1272/2008 Of the European Parliament and of the Council.