(R,R)-(-)-butane-2,3-diol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 January 2019-15 February 2019

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 28, 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(R,R)-(-)-butane-2,3-diol

EC Number:

246-186-9

EC Name:

(R,R)-(-)-butane-2,3-diol

Cas Number:

24347-58-8

Molecular formula:

C4H10O2

IUPAC Name:

(R,R)-(-)-butane-2,3-diol

impurity 1

Reference substance name:

(2R,3S)-butane-2,3-diol

Cas Number:

5341-95-7

Molecular formula:

CH3CH(OH)CH(OH)CH3

IUPAC Name:

(2R,3S)-butane-2,3-diol

impurity 2

Reference substance name:

(2S,3S)-butane-2,3-diol

Cas Number:

19132-06-0

Molecular formula:

C4H10O2

IUPAC Name:

(2S,3S)-butane-2,3-diol

Test material form:

liquid

Details on test material:

Identification: 2,3-butanediol ((2R,3R)-rich)
Batch: GSC180130-110
Serial No.: GSC181119B
Appearance: Colourless liquid
Expiry Date: 28 January 2020

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis (MatTek Corporation, 82105 Bratislava, Slovakia)
- Lot number(s): 28683
- Delivery date: February 12, 2019
- Date of initiation of testing: February 12, 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 ± 1.5 °C and 25 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): 42 hours at 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: gently rinsed with PBS for at least 15 times
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3-hour (37 ± 1.5 °C)
- Spectrophotometer: Microplate reader (Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 2.047
- Barrier function: 4.79 hrs
- Morphology: The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, 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 arranged in patterns analogous to those found in vivo.
- Contamination: absence of HIV1 virus, hepatitis B virus, hepatitis C virus, bacteria, yeast, and other fungi
- Reproducibility: After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also, its intrinsic colour was not intensive.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean percent tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
- The test substance is considered to be non-irritant to skin if the tissue viability after exposure and post-treatment incubation is more 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

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

triplicate

Test system

Vehicle:

unchanged (no vehicle)

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean relative viability

Value:

91.72

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

3.35%

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: None
- Colour interference with MTT: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: Laboratory technical proficiency with the test system according to OECD 439 was demonstrated.

ACCEPTANCE OF RESULTS:
Concurrent negative controls (NC) and positive controls (PC) were within a defined historical acceptance range (NC: OD ≥ 1.28 and ≤ 2.0; PC: OD ≥ 0.03 and ≤ 0.11). Mean OD values were within historically established boundaries for the NC (OD = 1.897) and PC (OD = 0.063).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, 2,3-butanediol ((2R,3R)-rich) is non-irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of 2,3-butanediol ((2R,3R)-rich) by means of the Human Skin Model Test according to OECD 439 (adopted 2015). Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS). The viability was measured after 60 min exposure and 42 hours post-incubation.

After treatment with the test item 2,3-butanediol ((2R,3R)-rich) the mean relative viability value decreased to 91.72% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of≤50%. Therefore, the test item is not considered to possess an irritant potential. This study results were considered acceptable, because the treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval. The study was considered reliable and adequate as part of the hazard assessment of skin irritation.