Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 10 to 13, 2017
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on test system:
PREPARATION
- Procedure used: on day of receipt, tissues were conditioned by incubation to release transport stress related compounds and debris. Duration of pre-incubation before treatment was 60 min.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 min at room T, 35 min at 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: after 60 ± 1 min from start of exposure, tissues were rinsed with PBS and transferred to a fresh medium; after 24 ± 20 min of post-incubation period, the medium was replaced by a fresh-one
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Libra S22
- Wavelength: 570 nm
- Filter: no external filter
- Filter bandwidth: 2-3 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment, composed of 3 replicate tissues

PREDICTION MODEL / DECISION CRITERIA
- In case the test chemical is found to be non-corrosive (e.g. based on guideline 430, 431 or 435) and tissue viability after exposure and post-treatment incubation is less than (<=) 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS category 2.
- The test test chemical may be considered as non-irritant to skin in accordance with UN GHS no category, if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 mg atop previously moistened tissue with 25 µl PBS

POSITIVE CONTROL
- Concentration: 5 % SDS in aqueous solution
Duration of treatment / exposure:
60 minutes: 25 minutes at room temperature and 35 minutes at culture conditions
Duration of post-treatment incubation (if applicable):
24 h and 20 minutes, then medium was replaced by a fresh one and tissues were incubated for 19 h and 7 minutes.
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
90.3
Negative controls validity:
valid
Positive controls validity:
valid
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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (OD570 is 1.682)
- Acceptance criteria met for positive control: yes (% of negative control tissues in 2.9 %)
- Acceptance criteria met for variability between replicate measurements: yes (SD of 3 identically treated replicates is < 18 % in all cases)

Any other information on results incl. tables

Average viability of treated tissues was 91.4 % of negative control viability. Negligible OD570 was observed in colorant control tissues extract though correction could be done.

True tissue viability = 91.4 % - 1.1 % = 90.3 %.

OD570 values obtained at MTT test, averages, standard deviations (%) and relative viabilities

treatment OD570 avg SD average viability (% NC)
1 2 3
NC PBS 1.752 1.728 1.567 1.682 0.082 100
% viab. NC 104.1 102.7 93.1 100 4.882
C1 PBS 1.529 1.601 1.483 1.538 0.049 91.4
% 90.9 95.2 88.2 91.4 2.887

C1

CC

medium, no MTT 0.019 0.018 - 0.018 0.001 1.1
% 1.129 1.011 - 1.1 0.059
PC 5 % SDS 0.065 0.069 0.066 0.067 0.002 4.0
% 3.9 4.1 3.9 4.0 0.101

C1 test substance

CC colorant control

NC negative control

PC positive control

SD standard deviation

Applicant's summary and conclusion

Interpretation of results:
other: non classified according to the CLP Regulation (EC 1272/2008)
Conclusions:
Average tissue viability upon treatment with test substance is 90.3 %.
Effect of test substance in EpiDermTM model was negative.
Executive summary:

Method

In vitro skin irritation study according to OECD guideline 439. One single experiment was run, using 3 replicates for test substance, positive and negative control. After pre-incubation of tissues, 25 mg test substance were spread on the tissue surface. Exposure duration was 60 minutes. After substance removal, tissues were post-incubated for ca. 42 hours. Afterwards, 3 hours incubation with MTT and 3 hours extraction followed. Optical density (OD570) of isopropyl alcohol was measured on a spectrophotometer.

Controls for colour interference and direct MTT reduction were also carried out.

Results

Slight colour interfernce and no direct MTT reduction were found.

Average cell viability was 90.3 %, i.e. above 50 %. Therefore, effect of the substance in EpiDermTM model was negative.