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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reliable GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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)
Remarks:
BASF SE, Ludwigshafen, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
700-860-3
EC Number:
700-860-3
Cas Number:
1419401-88-9
Molecular formula:
C17 H26 N2 O4
IUPAC Name:
700-860-3

In vitro test system

Test system:
human skin model
Remarks:
EpiDerm(TM) model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
other:
Remarks:
25 μL sterile PBS was applied first, then bulk volume of 25 μL of the solid test material was applied
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number(s): 28696
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
At room temperature: 25 minutes (NC); 28 minutes (PC and test substance)
At 37°C: 35 minutes (NC) or 32 minutes (PC and test substance)
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
All tissues were washed with sterile PBS to remove residual test material 1 hour after start of application.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test substance (50 μL bulk volume (solid test substance)) was added to 0.9 mL MTT solution. The mixture was incubated in the dark at about 37°C for 3 hours. A negative control
(deionized water) was tested concurrently. If the color of the MTT solution or (in case of water-insoluble test substances) the border to the water-phase turned blue / purple, the test substance was presumed to reduce MTT directly. In case of direct MTT reduction, three freeze-killed control tissues (KC) were treated additionally with each the test article and the negative control. Based on the results of a previous pretest it was judged, that application of killed control tissues (KC) is not necessary.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Mean tissue viability (% of negative control): ≤ 50 -> No prediction can be made (UN GHS Category 2 or Category 1). Further testing with the Skin Corrosion Test (SCT) is required, because the SIT cannot resolve
between UN GHS Categories 2 or 1.
Mean tissue viability (% of negative control): > 50 -> Non-irritant (No UN GHS Category)

A borderline range (50 ± 5%) was statistically determined by using historic test laboratory data and hence considers the variance of the test method. This evaluation is confirming the borderline range provided in OECD Guideline 439.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
25 μL sterile PBS were applied first. Thereafter, approx. 25 mg (corresponding to about 39 mg/cm2) undiluted solid test material was applied directly onto the tissue surface with the syringe and distributed together with the fluid, so that the surface of the epidermis model was uniformly and completely covered.

NEGATIVE CONTROL
30 μL sterile PBS

POSITIVE CONTROL
30 μL 5% SDS
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean viability (% of NC)
Value:
124.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
mean viability (% of NC) = 3.6
Other effects / acceptance of results:
The test substance is not able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was not introduced.

Application of the positive control 5% SDS showed a relative mean viability of the tissues of 3.6% and reflects the expected sensitivity of the tissues
The mean OD570 of the negative control (PBS) fulfill the acceptance criteria and demonstrate the validity of the assay.

The OD570 values determined for the tissues treated with the test substance are higher than OD570 values of the negative control. However, as all quality criteria of the test were met, the study is considered to be valid despite the higher viability values of the test substance.

Any other information on results incl. tables

Table 1: Relative viability of EpiDermTM tissues samples

 

Mean

SD

NC

Mean OD570

1.729

 

Viability [% of NC]

100.0

4.2

Test substance

Mean OD570

2.158

 

Viability [% of NC]

124.8

5.3

PC

Mean OD570

0.062

 

Viability [% of NC]

3.6

0.3

NC: negative control

OD: optical density

PC: positve control

SD: standard deviation

Applicant's summary and conclusion