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Administrative data

Description of key information

skin irritation

Reconstructed human Epidermis (RhE), OECD Guideline 439, result: negative

eye irritation

Reconstructed human Cornea-like Epidermis (RhCE), OECD Guideline 492, result: negative

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
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)
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
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
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
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017/12/11 - 2018/01/25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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
Species:
human
Strain:
other: not applicable, human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability : good water solubility
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular (TM) tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea.It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The sterility of the tissue was confirmed.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
120 min
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used : This in vitro study was performed in order to evaluate the eye hazard potential of Hydroxyacetone. The EpiOcular (TM) Eye Irritation Test (EIT) predicts the acute ocular irritation potential of chemicals by measurement of its irreversible tissue damage caused by cytotoxic effects in the human cornea model. Within a testing strategy, the EpiOcular (TM) EIT is used as a replacement of the Draize Eye Irritation Test. It is utilized for the classification and labelling of chemicals concerning their eye irritation potential. The EpiOcular (TM) EIT is intended to differentiate substances that are “not eye irritant” from those that require labelling as either GHS category 1 or 2 for serious eye damage resp. eye irritation potential. Eye irritant materials are identified by their ability to produce a decrease in cell viability as determined. The cell viability is measured by dehydrogenase conversion of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison of untreated negative controls is used to predict the eye irritation potential. The test was performed according to the protocol provided from MatTek Corporation.
- RhCE tissue construct used, including batch number : EpiOcular (TM) tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Day of delivery: 12. Dec. 2017
Batch no.: 27017
- Doses of test chemical and control substances used: 50 µL (100 %)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) :
exposure: 30 min at 37°C
post-exposure: 120 min at 37°C
- Description of any modifications to the test procedure : no modifications identified
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : The test item was tested for the ability of direct MTT reduction. To test for this ability, 50 μL of the liquid test item were added to 1 mL of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 and 80 – 100 % relative humidity for 3 hours. 1 mL of MTT solution plus 50 μL of H2O demin. were used as negative control. The test item showed MTT reduction as a colour change was observed. In addition the test item was classified as non-irritant in the main test. Therefore, it was necessary to perform a functional test with freeze killed tissue that possess no metabolic activity but absorb and bind the test item like viable tissues. Freeze killed tissues were prepared by placing untreated tissues in the freezer (- 20±5 °C) and stored in the freezer until use. In addition to the normal test procedure described, the functional check employed two freeze-killed tissue treated with the MTT reducing test item and one untreated killed tissue that shows the small amount of MTT reduction due to residual NADH and associated enzymes within the killed tissue. 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.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : 570 nm
- Description of the method used to quantify MTT formazan : A 24-well-plate was prepared with 300 μL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into the empty 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was sealed and stored in the refrigerator overnight. On the next day, the plate was shaken for 2 hours at room temperature, protected from light.
The inserts were pierced with an injection needle, taking care that all colour is extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well plate which was read in a plate spectrophotometer at 570 nm.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : If the viability is greater than 60 %, the substance is non eye irritant. If the tissue viability is equal or less than 60 %, the substance is at least eye irritant (cut-off point as specified for this test system in OECD test guideline 492).
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.
- Complete supporting information for the specific RhCE tissue construct used : All cells used to produce EpiOcular (TM) are purchased or derived from tissue obtained by MatTek Corporation from accredited institutions. In all cases, consent was obtained by these institutions from the donor or the donor's legal next of kin, for use of the cells or derivatives of the tissue for research purposes. The cells are screened for potential biological contaminants using the methods given below:
HIV-1 virus (Oligonucleotide-directed amplification)
Hepatitis B virus (Oligonucleotide-directed amplification)
Hepatitis C virus (Oligonucleotide-directed amplification)
Bacteria, yeast and other fungi (long term antibiotic, antimycotic free culture)
- Reference to historical data of the RhCE tissue construct :
Tissue viability (MTT Assay, n=3): OD (540 - 570 nm) = 1.604 ± 0.08 (acceptance criteria: 1.1 - 3.0)
Barrier function (ET-50 assay, n=2): 27.7 min (acceptance criteria: 12.2 - 37.5 min)
Sterility: sterile
- Positive and negative control means and acceptance ranges based on historical data :
OD negative control: mean: 1.838 (range: 1.167 - 2.437), in study: 1.961
Relative Tissue Viability positive control: mean: 31 % (range: 12.4 - 57.2 %), in study: 44 %
- Acceptable variability between tissue replicates for positive and negative controls : 2 % (negative control), 1 % (positive control) (acceptance criterion: < 20 % variability)
- Acceptable variability between tissue replicates for the test chemical: 2.5 % (test item) (acceptance criterion: < 20 % variability)
Irritation parameter:
other: Tissue viability (%)
Run / experiment:
corrected mean of 2 tissues
Value:
88.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: Tissue viability (%)
Run / experiment:
Tissue 1
Value:
87.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: Tissue viability (%)
Run / experiment:
Tissue 2
Value:
90
Vehicle controls validity:
not applicable
Negative controls validity:
not examined
Positive controls validity:
not examined
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not specified

DEMONSTRATION OF TECHNICAL PROFICIENCY: passed

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline:
OD negative control: mean: 1.838 (range: 1.167 - 2.437), in study: 1.961
Relative Tissue Viability positive control: mean: 31 % (range: 12.4 - 57.2 %), in study: 44 %
Interpretation of results:
other: EU GHS criteria not met
Conclusions:
One valid experiment with two 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. After the treatment with the test item, the mean value of relative tissue viability corrected for the substance-mediated MTT reduction was 86.6 % (reduction of 13.4%). This value is well above the threshold for eye irritation potential (≤ 60% viability).
Executive summary:

Eye irritation of Hydroxyacetone was determined using the EpiOcular (TM) Reconstructed human Cornea-like Epithelium (RhCE) test method following OECD 492. One valid experiment with two 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. The test item Hydroxyaceton was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 28 minutes. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 2.0. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 44.0% (< 50%). Variation within tissue replicates was acceptable (well below 20%). 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 86.6 % (reduction of 13.4%). This value is well above the threshold for eye irritation potential (≤ 60% viability).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Hydroxyacetone was negative in the Reconstructed human Epidermis (RhE), as well as in the Reconstructed human Cornea-like Epithelium (RhCE) test method. As a conclusion, Hydroxyacetone is non-irritant to skin and eyes. Therefore, the substance is not classified for skin and eye irritation in accordance with Regulation (EC) No 1272/2008.