1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

EC Number:

216-122-4

EC Name:

1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

Cas Number:

1502-47-2

Molecular formula:

C6H6N10

IUPAC Name:

1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

Test material form:

solid

Details on test material:

Test Item Name: Exolit 775
Sample Number: Ba-Ha-19-025-1; Deflam 20 20151106 JOB D02441
CAS No.: 1502-47-2

Chemical name (IUPAC): 1,3,4,6,7,9,9b-Heptaazaphenalene-2,5,8-triamine/Melem
CAS No.: 1502-47-2
Sample No.: Ba-Ha-19-025-1; Deflam 20 20151106 JOB D02441
Manufactured Date: 2019-09-06
Expiry Date: 2034-09-06
Purity as per Certificate of Analysis: 95.75%
Physical Appearance: Solid White
Storage Conditions: Cool and Dry (+2 to +8 °C)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

not specified

Details on animal used as source of test system:

Not Applicable

Justification for test system used:

The assessment of skin irritation has typically involved the use of laboratory animals (OECD TG 404). In relation to animal welfare concerns, TG 404 recommends the use of a tiered testing strategy for the determination of skin corrosion and irritation which includes the use of validated in vitro or ex vivo test methods avoiding pain and suffering of animals.

One of the validated in vitro test methods adopted by the OECD TG 439 makes use of reconstructed human epidermis (RhE) which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkin kit

EpiSkin is an in vitro reconstructed human epidermis (RHE) from normal human keratinocytes cultured on a collagen matrix at the air liquid interface. This is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Differentiated and stratified epidermis model comprises the main basal, supra basal, spinous and granular layers and a functional stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.

Amount/concentration applied:

10 mg

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours ± 1

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes exposure

Value:

> 50

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

TABLE 1.       Details of EpiSkin Test Kit

Particulars
Batch No.	20-EKIN-038
Date of receipt	16 September 2020
Shipment medium color	Orange
Temperature indicator	Pale grey
Date of Expiry	21 September 2020
Kit Compliance	Control Certificate No.: E200879 Compliance: Yes
Maintenance Medium	Batch No.: 20-MAIN3-029   Date of Expiry: 23 September 2020   Stored in Refrigerator: BPM 00382
Assay Medium	Batch No.: 20 ESSC-028   Date of Expiry: 23 September 2020   Stored in Refrigerator: BPM 00382

TABLE 2.       Mean OD Values of Individual Epidermis Units

	Absorption (OD570)
	R1	R2	R3	Mean	SD
Negative control	0.653	0.665	0.678	0.665	0.013
Positive control	0.099	0.103	0.103	0.102	0.002
Test Item	0.692	0.686	0.692	0.690	0.003

OD: optical density

SD: Standard deviation

R1, R2 & R3: triplicate exposures

TABLE 3.       True OD Values of Individual Epidermis Units

	Absorption (OD570)
	R1	R2	R3	Mean	SD
Negative control	0.604	0.616	0.629	0.616	0.013
Positive control	0.050	0.054	0.054	0.053	0.002
Test Item	0.643	0.637	0.643	0.641	0.003

 Blank OD Value (mean of 6 replicate values): 0.049

OD: optical density

R1, R2 & R3: triplicate exposures

True OD value = OD Raw – OD Blank

TABLE 4.       Individual Tissue Viability of Epidermis Units (Relative)

	% Individual Viability
	R1	R2	R3	Mean	SD
Positive control	8.12	8.77	8.77	8.55	0.375
Test Item	104.38	103.41	104.38	104.06	0.560

 Negative control mean: 1.0863

R1, R2 & R3: triplicate exposures

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The registration substance was investigated for its skin irritation property according to the OECD Guideline 439. The registration substance is not irritating.

Executive summary:

The registration substance was investigated for its skin irritation property according to the OECD Guideline 439(EpiSkin). 10 mg of the registration substance was applied to the skin tissues for 15 min and the cell viability was determined by the MTT assay. No reduction of the cell viability was found. The registration substance is not irritating to skin.