Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 January 2017 - 27 February 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:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
20 July 2012
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
solid: crystalline
Details on test material:
Appearance: white crystalline powder
Storage conditions: at room temperature protected from light
Specific details on test material used for the study:
Stable at higher temperatures: Yes, with a maximum temperature of 40 °C and for a maximum duration of 2 days.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
- Procedure used: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 22 hours at 37°C

ENVIRONMENTAL CONDITIONS
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 36.0-37.1 °C
- Humidity (%): 64-87

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1, with phosphate buffered saline
- Observable damage in the tissue due to washing: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent no treatment
Amount/concentration applied:
TEST ITEM:
18.9 to 32.0 mg on skin moistened with 5 μl Milli-Q water

NEGATIVE CONTOL:
- Amount applied: 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate
- Re-spread after 7 minutes contact time
Duration of treatment / exposure:
15 ±0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Test system

Details on study design:
TEST SITE
- EPISKIN Standard Model (TM) (EPISKIN-SM (TM), 0.38 cm^2, Lot no.: 17-EKIN-008).This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days , which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
- The test item was checked for possible direct MTT reduction and colour interference

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of 3 replicates
Value:
94
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
(100%)
Positive controls valid:
yes
Remarks:
(22%)
Remarks on result:
other: SD of test item: 2.6%
Other effects / acceptance of results:
- The positive control had a mean cell viability of 22% after 15 minutes of exposure. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly.
- Since all acceptability criteria are met, the study is considered to be valid.

Any other information on results incl. tables

The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. No colour change was observed, therefore it was concluded that the test substance did not interact with MTT.

The mean optical densities at 570 nm (OD570) were:

Negative control: 0.835 ±0.033

Test item: 0.784 ±0.021

Positive control: 0.186 ±0.043

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
(CLP criteria also not met)
Conclusions:
An in vitro skin irritation test with Bicin was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the substance is not irritating in the in vitro skin irritation test according to the GHS and CLP criteria, under the experimental conditions described in this report.