Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-05 to 2017-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde für Gesundheit und Verbraucherschutz

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: human donors
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EpiDerm (MatTek)
- Tissue batch number(s): 25829

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: yes
- Wavelength: 540 nm
- Filter: no

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.857 ± 0.107
- Barrier function: 5.4 h
- Contamination: Screened for HIV-1, hepatits B and C, bacteria, yeast, and other fungi. No contaminates were detected.

NUMBER OF REPLICATE TISSUES: triplicate

PREDICTION MODEL / DECISION CRITERIA
The irritant potential was predicted from the relative mean tissue viabilities compared to the negative control. If the mean relative tissue viability is > 50% the test material is not considered an irritant. If the mean relative tissue viability is ≤ 50%, the test material is considered an irritant.

The test meets acceptance criteria if:
- mean absolute OD570 of the three negative control tissues is ≥ 1.0 and ≤ 2.5
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 mg
- Concentration: 25 mg test item were mixed with 300 μL sterile deionised water

NEGATIVE CONTROL
- Amount(s) applied: 30 μL

POSITIVE CONTROL
- Amount(s) applied: 30 μL
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
ca. 111
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: The standard deviation determined for all triplicates was below the limit of acceptance of 18 %. Hence, all acceptance criteria required were fulfilled.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1. Summary of Results

 

Optical density [OD540]

mean tissue (n = 3)

Mean Viability

(n = 3) [%]

CV

[%]

Dimelamine pyrophosphate

1.550

111.0

1.2

D-PBS (negative control)

1.396

100.0

7.6

5% SDS (positive control)

0.092

6.5

10.9

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria are not met, no classification is required according to Regulations (EC) No 1272/2008.
Conclusions:
In an in vitro skin irritation human skin model test (EpiSkin) according to OECD guideline 439 and in compliance with GLP, a cell viability of 111% was measured when compared to the untreated control. Therefore, the test item should not be classified.