Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 December 2017 - 16 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

1
Chemical structure
Reference substance name:
N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
EC Number:
231-034-6
EC Name:
N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
Cas Number:
7417-99-4
Molecular formula:
C19H20N4O2
IUPAC Name:
N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
Test material form:
solid

In vitro test system

Test system:
human skin model
Remarks:
SkinEthic RHE® model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: foreskin (number of donors not specified)
Source strain:
not specified
Justification for test system used:
This study makes use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. Use of reconstructed human epidermis (RhE) is also recommended by OECD and other regulatory authorities. SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin irritation studies.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 17-RHE-127
- Production date:
- Shipping date:
- Delivery date:
- Date of initiation of testing: 12/12/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2
- Spectrophotometer: Synergy™ microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.1 (CV = 4.5%) specification OD > 0.7. Historical negative control mean OD range = 1.964-2.776.
- Barrier function: 4.5 h (Specification 4h < ET50< 10h)
- Morphology: 6 Cell layers, absence of significant histological abnormalities, well differentiated epidermis, specification > 4
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after 42 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the tissue viability after 42 minutes exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg (32mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours ± 60 minutes
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
93.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(DPBS)
Positive controls validity:
valid
Remarks:
1.2% viability (5% SDS)
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, SD of the negative control group was 0.03% (acceptablility criteria, SD ≤ 18%) and OD mean is 2.002 (acceptability criteria, 0.8≤OD≤3).
- Acceptance criteria met for positive control: yes, SD of the positive control group was 0.06% (acceptablility criteria, SD ≤ 18%)
- Acceptance criteria met for variability between replicate measurements: yes. SD of test item was 1.21% (acceptablility criteria, SD ≤ 18%).

Any other information on results incl. tables

Table 1: Data Summary of Percent Viability

Treatment

Tissue Replicate

O.D. at 570 nm

Blank Corrected O.D.

Mean of Corrected O.D.

Mean O.D. of Three Tissues

% Viability/

Tissue

Mean % Viability

S.D. of % Viability

C.V. of % Viability

Corrosivity Class

Negative Control

(Dulbecco’s Phosphate Buffered Saline (DPBS))

1

2.067

2.026

2.004

2.002

100

100

0.03

1.50

NA

2.017

1.976

2.051

2.010

2

2.021

1.980

1.988

2.033

1.992

2.034

1.993

3

2.019

1.978

2.014

2.105

2.064

2.042

2.001

N,N’-(Methylenedi-P-Phenylene)Bis(Aziridine-1-Carboxamide) (Poly-U-01)

1

1.947

1.906

1.889

1.864

94.4

93.1

1.21

1.30

No Category

1.910

1.869

1.932

1.891

2

1.847

1.806

1.842

92.0

1.896

1.855

1.905

1.864

3

1.900

1.859

1.861

93.0

1.895

1.854

1.910

1.869

Positive control

(Sodium dodecyl sulphate (5% aq.))

1

0.068

0.027

0.026

0.025

1.3

1.2

0.06

5.00

Category 2

0.067

0.026

0.066

0.025

2

0.067

0.026

0.025

1.2

0.065

0.024

0.066

0.025

3

0.066

0.025

0.025

1.2

0.065

0.024

0.066

0.025

Key: O.D. = Optical density, S.D. = Standard deviation, C.V. = Coefficient of variation, NA = Not applicable

Note: For Negative control SD and CV of % viability was calculated using OD at 570 nm and for test item and positive control SD and CV of % viability was calculated using % viability/tissue.

Applicant's summary and conclusion

Interpretation of results:
other: No category (CLP Regulation EC no. 1272/2008)
Conclusions:
The test substance was found to not be a skin irritant in a reconstructed human epidermis test.
Executive summary:

An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic RHE® model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol and incubating the tissues for overnight in a refrigerator protected from light, and finally measuring the concentration of formazan by determining the OD at 570 nm. Under the test conditions, the mean percent viability of the treated tissues was 93.1%, versus 1.2% of the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item must be considered as not classified for skin irritation.