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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 January 2019 - 31 January 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

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)

Version / remarks:

25th June 2018

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Source and lot/batch No.of test material: GSC180130-110
- Serial No.: GSC181119B
- Expiration date of the lot/batch: 28 January 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not applicable

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcular™ tissue consists of normal, human-derived epidermal 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 which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium.

Test system

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 (83.3 μL/cm² according to guideline)

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:

- RhCE tissue construct used, including batch number: EpiOcular™ kits, Lot No.: 27089 (MatTek Corporation, 82105, Bratislava, Slovakia)
- Doses of test chemical and control substances used: 50 µL undiluted test substance, positive (PC) and negative control (NC)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): 30 min exposure at 37 ± 1.5°C, 120 min post-incubation in medium at 37 ± 1.5°C
- 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): The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.
- Description of the method used to quantify MTT formazan:
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Since the test item was colourless inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2 mL isopropanol in each well so that isopropanol was flowing into the insert. The plates were sealed with parafilm and a standard plate sealer, and were stored about 18 h at 2-8 °C in the dark. To extract the MTT, the tissues were pierced and the plates were placed on an orbital plate shaker and shaken for 2.5 hours at room temperature. The corresponding negative, positive, and additional viable tissues (without MTT addition) were treated identically with piercing.
The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
- Prediction model:
If the test item-treated tissue viability is > 60% relative to the negative control-treated tissue viability, the test item is labeled non-irritant (no Category according to UN GHS).
If the test item-treated tissue viability is ≤ 60% relative to negative control-treated tissue viability, the test item is labeled irritant (Category 2 or Category 1 according to UN GHS; no differentiation between the categories possible).
A single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: yes
- Reference to historical data of the RhCE tissue construct: yes
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: yes
- Acceptability of the Assay
1) The negative control mean OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items).
4) The positive and negative control data shall fall within the historical control data.
- Positive and negative control means and acceptance ranges based on historical data
PC and NC data fell within the historical control data.
- Acceptable variability between tissue replicates for test chemical, positive and negative controls
The difference of viability between the two relating tissues of a single item is < 20% (values between 0.45 p.p and 2.67 p.p) in the same run (for positive and negative control tissues and tissues of single test items).

Results and discussion

In vitro

Results

Irritation parameter:

other: % viability

Run / experiment:

30 min exposure/180 min post-incubation

Value:

85.31

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

29.91%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The viability value of the test item exposed tissues was 85.31% when compared to negative control and hence did not decrease below 60%.

Any other information on results incl. tables

Test Group	Tissue No.	Well 1 [OD570]	Well 2 [OD570]	Mean [OD570] (Well 1 and well 2)	Mean [OD570] blank corr. (Well 1 and well 2)	Mean [OD570] of T1 and T2	Tissue viabil.* [%]	rel. viabil. of T1 and T2**	Diff. of viabil. between T1 and T2 [p.p.]
Blank		0.035	0.035	0.035
Negative Control	1	2.481	2.438	2.459	2.424	2.457	100.0	98.7	2.67
	2	2.559	2.491	2.525	2.490			101.3
Positive Control	1	0.796	0.768	0.782	0.747	0.735	29.91	30.4	0.98
	2	0.763	0.753	0.758	0.723			29.4
Test Item	1	2.218	2.033	2.126	2.090	2.096	85.31	85.1	0.45
	2	2.173	2.101	2.137	2.101			85.5

* Tissue viability = [100 × meanOD blank corrected of T1&T2test item / positve control / negative control]/[meanOD of T1&T2negative control]

** Relative Tissue viability = [100 × meanOD blank correctedtest item / positive control / negative control]/[meanOD of T1&T2negative control]

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) does not need to be classified according UN GHS.

Executive summary:

This in vitro study was performed to assess the eye irritation potential of 2,3-butanediol ((2R,3R)-rich) by means of the Human Cornea Model Test. The study was conducted according to OECD 492 (adopted 2018) and in compliance with GLP. Tissues of the human cornea model EpiOcular™ were treated with 50 μL of the test item, the positive and the negative control. The viability was measured after 30 min exposure and 2 hours post-incubation.

Since the viability value of the test item exposed tissues was 85.31% when compared to negative control and hence did not decrease below 60%, the test item is not considered to possess an eye irritating potential. This study results were considered acceptable, because treatment with the positive control induced a decrease in the mean tissue viability compared with the negative control to 29.91%. The study was considered reliable and adequate as part of the hazard assessment of eye irritation.