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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:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2008, amended 20 July 2012
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
484-470-6
EC Name:
-
Cas Number:
623-40-5
Molecular formula:
C5H11NO
IUPAC Name:
2-Pentanone oxime

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EPISKIN Small Model TM
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-SM TM, 0.38 cm² (SkinEthic Laboratories, Lyon, France)
- Tissue batch number(s): 19-EKIN-024
- Date of initiation of testing: 11 Jun 2019

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.
- Observable damage in the tissue due to washing: Not reported.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h (at 37°C)
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: IC50 = 2.1 mg/mL (1.5 mg/mL≤ IC50 ≤ 3.0 mg/mL
- Morphology: HES stained paraffin section: Multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and a multilayered stratum corneum (result is satisfactory) / number of cell layers ≥ 4
- Contamination: On blood of the donors the absence of HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs has been verified; on cells from donors, the absence of bacteria, fungus and mycopasma has been varified.

NUMBER OF REPLICATE TISSUES: triplicate

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

PREDICTION MODEL / DECISION CRITERIA :
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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): 5%
The positive control was re-spread after 7 minutes contact time.
Duration of treatment / exposure:
15 ± 0.5 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
triplicate for each treatment and control group

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
15 min exposure
Value:
96
Vehicle controls validity:
not applicable
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: Not reported.
- Direct-MTT reduction: Not reported.
- Colour interference with MTT: Because no color changes were observed it was concluded that the test item did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues (1.376 ± 0.056) was within the laboratory historical control data range (0.422 - 1.547; mean: 0.96, SD: 0.17, n = 174).
- Acceptance criteria met for positive control: The absolute mean OD570 of the positive control tissues (0.123 ± 0.020) was within the laboratory historical control data range (0.023 - 0.449; mean: 0.12, SD: 0.09, n = 173).
The standard deviation value of the percentage viability of three tissues treated identically was < 5% (negative control 4.1%, positive control 1.5% and test item 3.9%), indicating that the test system functioned properly.

Any other information on results incl. tables

Table 1: MTT assay after 15 min exposure

  Negative control Positive control Test item
Tissue sample 1 2 3 1 2 3 1 2 3
OD570 1.388 1.426 1.315 0.107 0.146 0.115 1.258 1.346 1.355
OD570(mean of 3 tissues) 1.376 ± 0.056 0.123 ± 0.020 1.320 ± 0.053
Viability (%) 100 8.9 96
SD 4.1 1.5 3.9

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified