methyl 2-hydroxybut-3-enoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-03-31 to 2021-05-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2019-07-31

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2020-06-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

methyl 2-hydroxybut-3-enoate

EC Number:

831-109-9

Cas Number:

5837-73-0

Molecular formula:

C5H8O3

IUPAC Name:

methyl 2-hydroxybut-3-enoate

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: Adult human donors

Justification for test system used:

The Reconstructed Human Epidermis test method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD 404).

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin Small Model (EpiSkin SM), manufactured by EPISKIN Laboratories Lyon, France
- Tissue batch number: 21-EKIN-013 (main experiment), 21-EKIN-010 (killed tissues to check for direct MTT reduction)
- Expiry date: 05 April 2021 (main experiment), 15 March 2021 (killed tissues to check for direct MTT reduction)
- Date of initiation of testing: 31 March 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (23.9-24.5 °C)
- Temperature of post-treatment incubation: 37±1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Units were rinsed thoroughly with approx. 25 mL 1x PBS solution
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: Varioskan™ LUX Type 3020
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 200-1000 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The IC50 of the batch was determined using SDS in the MTT test. An IC50 between 1.5 mg/mL and 3 mg/mL was considered acceptable. The IC50 of the batch was determined to be 2.3 mg/mL and thus within the acceptance range. For HCD see "Attached background material".
- Barrier function: A number of cell layers of 4 or more is considered acceptable. A representative reconstructed epidermis from each batch was stained with HES and showed 7.5 cell layers and was thus considered acceptable.
- Contamination: The product was prepared and packaged under aseptic techniques. The absence of HIV1 and HIV2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs as well as bacteria, fungi or mycoplasma was verified by blood and cellular analysis of donor blood and keratinocytes.
- Reproducibility: Yes, the obtained results for positive and negative controls were within the acceptance criteria of the assay and within the laboratory's HCD (see "Attached background material")

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: Three killed test item treated tissues and three killed negative control treated tissues were used for the MTT evaluation in one run (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues (batch No. of killed epidermis: 21-EKIN-010). The same treatment steps were followed with these tissues and the living tissues.
- Procedure used to prepare the killed tissues: Fresh tissues were placed in a 12 well plate with 2 mL of distilled water and incubated at 37±1 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere for 48 h +/- 1 hours. Killed epidermis were kept in a freezer at -15 to -30 °C until use.
- N. of replicates: 3 (for each test item and negative control treatment)
- Method of calculation used: See "Any other information on materials and methods incl. tables"

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One independent experiment was used with three tissue replicates.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if the viability after 15 minutes exposure and 42 hours post incubation is less or equal to 50%.
- The test substance is considered to be non-irritating to skin if the viability after 15 minutes exposure and 42 hours post incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 μL

NEGATIVE CONTROL
- Amount applied: 10 μL

POSITIVE CONTROL
- Amount applied: 10 μL
- Concentration: 5%

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

Three tissue replicates in one independent experiment

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 3 tissue replicates

Run / experiment:

1

Value:

10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Direct MTT reduction: Yes. During the check-method for possible direct MTT reduction, a colour change was observed after three hours of incubation. The test item interacted with MTT, therefore additional controls and data calculations were necessary. The non-specific MTT reduction (NSMTT) was determined to be 3%. As the NSMTT were below 50%, the true MTT metabolic conversion for all samples was calculated and the correction of viability percentages determined.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is slightly yellow and therefore considered not to be able to significantly stain the tissues and lead to a false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method, the laboratory demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

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:

study cannot be used for classification

Remarks:

This test method cannot resolve between UN GHS Categories 1 and 2, thus classification is only possible in combination with OECD guideline 431.

Conclusions:

In an in vitro skin irritation test according to OECD guideline 439, the test item showed irritating properties.

Executive summary:

An EpiSkin^TM SM test according to OECD guideline 439 and GLP has been performed to predict the irritation potential of the test item by measurement of its cytotoxic effect, as reflected in the MTT assay. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with 1x PBS solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 h) in an incubator with 5±1 % CO₂, ≥95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % CO₂, ≥95 % humidified atmosphere, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5 % aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. The test item is MTT-reducer, therefore additional controls (three test item treated killed tissues and three negative control treated killed tissues) were used to detect and correct for test item interference with the viability measurement. The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.  In this in vitro skin irritation test using the EPISKIN model, the test item showed significantly reduced cell viability in comparison to the negative control (mean corrected relative viability value: 10 %). All obtained test item viability results were far below 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected OD and cell viability values within acceptable limits. Standard deviation of all calculated % viability values (test item and controls) was below 18. All acceptance criteria of the assay were fulfilled. The results obtained from this in vitro skin irritation test, using the EPISKIN model, with the test item indicate that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2.