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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:
2018/01/15 - 2018/02/02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydroxyacetone
EC Number:
204-124-8
EC Name:
Hydroxyacetone
Cas Number:
116-09-6
Molecular formula:
C3H6O2
IUPAC Name:
1-hydroxypropan-2-one
Test material form:
liquid
Specific details on test material used for the study:
Stability in solvents: H2O > 1 week, EtOH some days, pH rises because of reaction of alcohols with Hydroxyacetone, CH3CN expectedly > 1 week, DMSO no experience
Solubility: H2O good, (commercial product 65% HA in water), EtOH good soluble, DMSO no experience

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Source strain:
other: not applicable (human)
Justification for test system used:
This in vitro study was performed in order to evaluate the potential of Hydroxyacetone to evoke skin irritation in a reconstructed human epidermis (RhE) test method. Skin irritation refers to the production of reversible damage to the skin following the application of a test chemical. The test system is a commercially available EpiDerm (TM)-Kit, procured by MatTek. The EpiDerm (TM) tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo. The EpiDerm (TM) tissues are cultured on specially prepared cell culture inserts.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm (TM)-Kit, procured by MatTek
- Tissue batch number(s): 25874
- Delivery date: 2018/01/16
- Date of initiation of testing: 2018/01/17

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 washing step
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Microtiter plate photometer
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD (540 - 570) = 1.71 ± 0.09
- Barrier function: 5.28 h (ET-50 Assay)
- Morphology: Tissue viability and the barrier function test are within the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: no

NUMBER OF REPLICATE TISSUES:

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Procedure used to prepare the killed tissues (if applicable): freeze-killing
- N. of replicates : 3
- Method of calculation used: Calculations were performed as follows:
- Calculation of mean OD of the blank isopropanol (ODBlk)
- Subtraction of mean ODBlk of each value of the same experiment (corrected values)
- Calculation of mean OD of the two replicates for each tissue
- Calculation of mean OD of the three relating tissues for controls and test item
Note: Corrected OD value of negative control corresponds to 100 % viability. The photometric absorbance of the negative controls is considered as 100%. For each replicate of test item and positive control, tissue viability is calculated as % photometric absorbance compared with the mean of the negative controls:
% Tissue viability = [(ODreplicate test item resp. positive control)/(ODmean of negative controls)] * 100
OD = Optical Density

Data Correction with additional Tests
Test with Freeze-killed Tissues
OD test item (freeze-killed) = corrected OD test item (freeze-killed) – corrected OD negative control (freeze-killed)

“% Viability (freeze-killed)”:
% Viability (freeze − killed) = [(OD test item (freeze killed)/(ODcorrected mean negative control)] * 100
The value of “% Viability (freeze-killed)” was subtracted from “% Viability” of the main test. The corrected mean OD of the negative control (freeze-killed tissue) was subtracted from the corrected mean value of the OD of the test item (freeze-killed tissues). The difference is 0.028. This value was subtracted from the absorbance value of the test item in the main test to achieve the corrected absorbance value

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be skin irritant if the viability after 1 hour exposure is equal or less than 50%
- The test substance is considered to be non-irritante to skin if the viability after 1 hour exposure is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): 100 %

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL DPBS buffer

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL SDS
- Concentration (if solution): 5 %
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours 30 minutes
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 1
Value:
100.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 2
Value:
100.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 3
Value:
92.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of Tissue 1, 2, 3
Value:
97.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: The test item showed MTT reduction as a colour change was observed when adding 30 µL of the test item to 1 mL MTT solution. As the direct reduction of MTT by the test item was ≤ 50% of the negative control, a valid test could be performed. To cover for the direct interaction, the net OD of the test item treated killed tissues was subtracted from the net OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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

Applicant's summary and conclusion

Interpretation of results:
other: EU GHS criteria not met
Conclusions:
The test item Hydroxyacetone is considered as non-irritant to skin. After the treatment with the test item, the mean value of relative tissue viability corrected for the substance-mediated MTT reduction was 97.9 % (reduction of 2.1%). This value is well above the threshold for a skin irritation potential (50% viability). Test items that induce viability values above the threshold of 50% are considered non-irritant to skin. The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8. The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system. Variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18%). For these reasons, the result of the test is considered valid.
Executive summary:

Skin irritation of Hydroxyacetone was determined with the Reconstructed human Epidermis (RhE) Test Method following OECD Guideline 439. One valid experiment with three tissues and two independent MTT measurements was performed. In the pre-test, potential MTT reduction by the test item was observed. Therefore, an additional test with two tissues was performed to allow a correction for this possible direct MTT reduction by the test item. In the main test, three tissues of the human skin model EpiDerm(TM) were treated with Hydroxyaceton for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control. After treatment with the negative control, the mean absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.8. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 1.9% (required: ≤ 20%). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%). Thus, confirming the validity and sensitivity of the test system. After the treatment with the test item, the mean value of relative tissue viability corrected for the substance-mediated MTT reduction was 97.9 % (reduction of 2.1%). This value is well above the threshold for a skin irritation potential (50% viability). Test items that induce viability values above the threshold of 50% are considered non-irritant to skin. Therefore, Hydroxyacetone is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.