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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
Study period:
30 May 2017 - 26 June 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
Test material form:
liquid
Details on test material:
- Physical appearance: pale yellow liquid
- Storage conditions: at room temperature

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 minimize 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:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model(TM)
- Tissue batch number(s): 17-EKIN-025
- Surface: 0.38 cm^2
- Expiration date: 26 June 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 36.3 - 37.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were washed with PBS (1 washing step)
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE: The test substance was checked for possible direct MTT reduction and color interference in a previous skin corrosion test using EpiDerm as a skin model.
- Incubation time: 3 hours
- Measurement method: TECAN Infinite M200 Pro Plate Reader (570 nm)
- MTT concentration: 0.3 mg/mL

NUMBER OF REPLICATE TISSUES: 3 for the test substance, the negative and the positive control each.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Because the test item interacted with the MTT endpoint, three killed tissues treated with test item and three killed non treated tissues were used for the cytotoxicity evaluation with MTT.
- Killed tissues: EPISKIN-SMTM, 0.38 cm^2, Lot no.: 17-EKIN-022
- Procedure used to prepare the killed tissues: Living epidermis was transferred to 12 well plates and incubated with 2 ml Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were
thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium. Further use of killed tissues was similar to living tissues.
- N. of replicates : 3
- Method of calculation used: the nonspecific MTT reduction (NSMTT) was calculated as follows: %NSMTT = [(ODkt_t+MTT – ODkt_u+MTT)/ mean ODlt_u+MTT] * 100
with (ODkt_u+MTT) = mean OD of the untreated killed tissues; (ODkt_t+MTT) = mean OD of the test item treated killed tissues; (ODlt_u+MTT) = mean OD of the negative control tissues

True tissue viability is calculated as the difference between the living test item treated tissues incubated with MTT medium (ODlt_t+MTT) and the difference between ODkt_t+MTT and ODkt_u+MTT:
OD= ODlt_t+MTT – (ODkt_t+MTT-ODkt_u+MTT)
%Viability = [OD/ mean ODlt_u+MTT] * 100

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

CELL VIABILITY MEASUREMENT: After incubation, tissues were dried and incubated with 2 mL MTT-solution for 3 hours. After incubation, epidermis was separated from the collagen matrix and both parts were extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for approximately 68 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

PREDICTION MODEL / DECISION CRITERIA (see Table 1)
A test item is considered irritant in the skin irritation test 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.
A test item is considered non-irritant in the in vitro skin irritation test 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.

ACCEPTABILITY CRITERIA
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.
d) The non-specific MTT reduction should be ≤ 30% relative to the negative control OD.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 μL

NEGATIVE CONTROL
- Amount applied: 25 μL

POSITIVE CONTROL
- Amount applied: 25 μL, re-spread afer 7 minutes of contact time
Duration of treatment / exposure:
15 +/- 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours; + 3 hours with MTT
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of 3 replicates
Value:
78
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mean tissue viability: 22%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Interaction with the MTT endpoint: yes
- Non-specific reduction of MTT by the test item: 4.80%. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the negative control tissues was within the laboratory historical control data range and the SD of the % viability was 5.9%.
- Acceptance criteria met for positive control: yes, the mean relative tissue viability was 22% and the SD was 11%
- Acceptance criteria met for variability between replicate measurements: yes, the standard deviation of the three tissue replicates treated with test substance was 6.8%.
- The non-specific MTT reduction should was < 30% relative to the negative control OD.

- OD values were corrected for background absorption, measured using isopropanol.
- Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro skin irritation test, performed according to OECD guideline 439 and under GLP principles, X-19657 showed to be non-irritant (78% tissue viability after 15 minutes exposure, compared to the negative control). Therefore, the substance is not classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.