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Diss Factsheets

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

Constituent 1
Chemical structure
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.

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