Registration Dossier

Administrative data

Description of key information

Skin irritation / corrosion: not irritating (in vitro).

Eye irritation / corrosion: not irritating (in vitro).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
18 April 2017 - 20 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
no
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: LabCyte EPI-MODEL24 RHE Model
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
LabCyte EPI-MODEL is a 3-dimensional reconstructed human epidermis model, produced by culturing normal human epidermal cells to become multilayered. The structure of LabCyte EPI-MODEL is morphologically similar to that of human epidermis, consisting of the basal layer, the spinous layer, the granular layer and the cornified layer (http://www.jpte.co.jp/english/business/LabCyte/LabCyte_EPI-MODEL_Instruction_Manual_Mar2017.pdf).

TEST SYSTEM
Reconstructed Human Epidermis (RHE) Model
Name : LabCyte EPI-MODEL24, Human Epidermal Model
Manufacturer : Japan Tissue Engineering Co., Ltd. (J-TEC)
Supplier : S&T Corp. (Jongno-gu, Seoul, Republic of Korea)
Diameter : 0.3 cm2
Lot No. : LCE24-170626-B
Tissue number : receipt - 24 Tissues : use - 12 Tissues : freeze - 12 Tissues (disuse 12 Tissues)

RHE tissue model freeze:
The rest of the RHE tissus was removed from the agarose gel and transfer to the 24-well plate. RHE tissue was frozen at -20 °C for more than 48 hours.

Quality control
According to the acceptance criterion acceptable LabCyte EPI-MODEL24 tissue model was used. The quality assurance results such as viability, barrier function and morphology were provided by the manufacturer.

Culture condition:
Temperature : (37 ± 1) °C
CO2 gas : (5 ± 1) %
Humidity : Under moist atmosphere
Incubator : CO2 incubator (Thermo, Forma 3121)
The temperature and humidity during the test period were measured by a COBEX recorder of a CO2 incubator.

Culture medium:
The culture medium used during the test period was provided by the manufacturer (Japan Tissue Engineering Co., Ltd. (J-TEC)). Assay medium was stored in cold (4 °C) and warmed to 20 - 25 °C before use.

TEST SUBSTANCE INTERFERE CHECK
Check-method for possible direct non-specific coloring with test substances:
Test substances were evaluated for their intrinsic color or ability to become coloured in contact with water. 25 uL of test substance was incubated for 1 hour in a 24-well plate with 0.5 mL of sterilized distilled water at room temperature and the change of color was observed. The color was not changed and additional experiments were omitted.

Check-method for possible direct MTT reduction with test substances:
Test substances were evaluated for the confirmation of the non-specific reduction of MTT. 25 uL of test substance was incubated for 3 hour in a 24-well plate with 0.5 mL of MTT solution at 37 °C, 5 % CO2 incubator the change of color was observed. The color was not changed and additional experiments were omitted.

TEST METHOD
RHE tissue model observation:
Before the beginning of incubation, each insert containing the RHE tissue was checked whether tissue surface were even and excess moisture were present and then selected a tissue that was free of defects.

RHE tissue pre-incubation:
Each insert containing the RHE tissue was removed from the agarose gel and transferred to the 6-well plate with 1 mL of medium. The plate was incubated at 37 °C , 5 % CO2 incubator for 20 ±1 hour.

Application of test substances:
25 uL of the un-diluted liquid was topically applied to the upper epithelial surface of the tissue with interval of 1 minute.

Rinsining of test substances:
Treated RHE tissues was kept for 15 minutes exposure in the ventilated cabinet sterile conditions at room temperature and then washed with PBS solution. RHE tissue surface was rinsed 15 times (1 mL/times) to remove all residual test substance.

Post incubation:
Washed RHE tissue was transferred to the 24-well plate with 1 mL of medium. The plate was incubated at 37 °C , 5 % CO2 incubator for 42 hour.

CELL VARIABILITY TEST
Application of MTT solution:
After removing all medium, RHE tissues was transferred to the 24-well plate with 0.5 mL of MTT solution(0.5 mg/mL). The plate was incubated in the 37 °C, 5 % CO2 incubator for 3 hours.

Formazan extraction:
RHE tissues was transferred to 1.5 mL micro tube with 0.3mL of isopropanol. Micro tube was incubated in a dark cold place overnight in order to completely extract formazan.

Absorbance:
Formazan solution was transferred to the 96-well plate 200 uL aliquots. 96-well plate was read by spectrophotometer using a wavelength 570 nm and 650 nm.

Acceptance criteria:
The suitability of this test was determined according to the following criteria.
- The absorbance value of the negative control group (Sterilized distilled water) was measured as 0.999 The result value was included in the range from 0.7 to 2.5 or less.
- The cell viability of the positive control group (5 % SDS) was calculated as 4.8 %. The result value was included in the range from less than 40%.
- The variation of cell viability per substance (n=3 tissues) was calculated 18 or less.
Control samples:
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
Preparation of test substance:
Two test samples were prepared with the Test substance provided by the sponsor. One was used 100 % without conversion of purity and the other was diluted to concentration of 20 % with sterilized distilled water. The dose volume per tissue was 25 µL.

Preparation of positive control substance:
SDS was diluted to final concentration of 5 %(w/v) with PBS solution.

Positive control substance:
Name : 5 % Sodium dodecyl sulfate(SDS)
Manufacturer : Sigma-Aldrich Corporation

Negative control substance:
Name : Sterilized distilled water
Manufacturer : Daihan Pharm CO., LTD.

Preparation of MTT solution:
MTT stock solution of 5 mg/mL is commonly used in conjunction with other tests. Before use, MTT stock solution of 5 mg/mL was diluted to concentration of 0.5 mg/mL with medium and proceeded 0.22 um filtration.

MTT substance:
Name : 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
Manufacturer : Sigma-Aldrich Corporation
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour
Value:
102
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
test conc. 20 %
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour
Value:
100.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
test conc. 100 %
Other effects / acceptance of results:
Cell viability for the positive group was 4.8 ± 0.7 % in comparison with the negative control group. For the test substance groups, test substance solution 20 % and 100 % was calculated as 102.0 ± 3.8 % and 100.2 ± 3.0 % cell viability, respectively.

Summary table: Evaluation of cell viability

Substance Dose [%] - Optical
Density
Cell viability
[%]
Sterilized distilled water - Mean 0.999 100
S.D. 0.058 5.8
N 3 3
SDS 5 Mean 0.048 4.8
S.D. 0.007 0.7
N 3 3
(2R,3R)-butane-2,3-diol 20 Mean 1.019 102
S.D. 0.037 3.8
N 3 3
100 Mean 1.001 100.2
S.D. 0.03 3
N 3 3

N: Number of well, S.D.: Standard deviation

Optical Density: ODsample = (570 ODsample-570 ODblank)-(650 ODsample-650 ODblank)

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the in vitro study, the test material is considered to be not irritating to skin.
Executive summary:

The study was conducted to assess the toxic potential of skin irritation of (2R,3R)-butane-2,3-diol in LabCyte EPI-MODEL24 RHE Model. The study was performed according to OECD 439 (2015).

The absorbance value of the negative control substance was measured 0.999. The result value was within the acceptable range (0.7 ≤ OD ≤ 2.5) under the skin irritation test using the LabCyte EPI-MODEL24 RHE model. It was concluded that all tissues used in the test was stably incubated during the period of the test Cell viability for the positive group was 4.8 ± 0.7 % in comparison with the negative control group. For the test substance groups, test substance solution 20 % and 100 % was calculated as 102.0 ± 3.8 % and 100.2 ± 3.0 % cell viability, respectively.

As a result of in vitro skin irritation test of the test item in LabCyte EPI-MODEL24 RHE Model, cell viability exceeded the reference value of 50 %. Therefore it was considered to be a non-irritant substance. The study was considered reliable and adequate for hazard assessment.

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
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
Qualifier:
according to
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. certificate)
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
Vehicle:
unchanged (no vehicle)
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).
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 of50%. 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.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22 May 2017 - 24 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to
Guideline:
other: MFDS Animal alternative test guideline (III) for cosmetic toxicity
Version / remarks:
Korean Ministry of Food and Drug Safety (MFDS) Animal alternative test guideline (III) for cosmetic toxicity: Bovine corneal opacity and permeanility test method
GLP compliance:
yes
Species:
cattle
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
Bovine eyes were collected by slaughterhouse on the day of slaughter and were transported in cooling condition.
Supplier : Hwajung-Food (Nonsan-si, Chungcheongnam-do, Korea)
Eyes excised date : 2017 - 05 - 30
Transfer condition : Cold (5.4 – 7.0) °C
Number of arrived Eyes : 14 ea
Number of used cornea : 12 ea (3 ea/group)
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Undiluted, 100 %
Diluted, 20 % (w/v) in water
Duration of treatment / exposure:
The corneas were incubated in the presence of either the test substance, positive control or negative control for 10 minutes at 32 ± 1 °C in an incubator.
Duration of post- treatment incubation (in vitro):
The corneas were incubated for 2 hours at 32 ± 1 °C in an incubator.
Number of animals or in vitro replicates:
3
Details on study design:
COLLECTION, SELECTION AND PREPARATION OF CORNEAS
Collection and Selection of Bovine eyes
1) Collection: Bovine eyes were collected by slaughterhouse on the day of exposure.
2) Transport : Collected bovine eyes were placed in 1X Hanks’ Balanced Salt Solution (containing penicillin (100 IU/mL) and 100 ug/mL streptomycin) and transported to the laboratory on ice container.
3) Selection : The eyes were grossly examined for damage (e.g., increased opacity, scratches, neovascularization and pigmentation) and those exhibiting defects were discarded in the laboratory. Corneas with a horizontal diameter of >= 27.5 and <= 28.5 mm were selected using vernier calipers (KG-EQM-429) for measuring.

Collection and Selection of Corneas
1) Isolation of corneas : Selected eyes were stored in a dish containing 1X HBSS (containing penicillin/streptomycin solution) until all corneas were dissected. The cornea was dissected from eyes such that approximately 2 to 3 mm of sclera was present around the cornea. The isolated corneas were also re-stored in a dish containing 1X HBSS (containing penicillin/streptomycin solution) until all corneas were
dissected.
2) Pre-incubation : The isolated corneas were mounted immediately in the corneal holders and then the corneal holder were filled with cMEM (without phenol red). The holders were pre-incubated for 1 hour at 32 ± 1°C in an incubator.
3) Initial opacity measurement and cornea selection : After pre-incubation, the cMEM in the holders were replaced with fresh cMEM (without phenol red). Initial opacity (lux) of corneas were measured using opacitometer (KG-EQM-424). Before measurement of initial opacity of corneas, the opacitometer was calibrated using opacitometer standard filters.
4) Cornea allocation : Initial opacity unit was calculated according to the guideline. The corneas above 7 opacity unit were discarded in order to ensure the quality of the corneas. After cornea selection by initial opacity unit, suitable corneas which showed below 7 initial opacity unit were allocated according to mean and standard deviation using allocation program (KG-AAT-008).
5) Cornea identification : Corneal holders mounted cornea were marked using a permanent marker.

APPLICATION DOSE AND EXPOSURE TIME
1) Treatment : The cMEM was removed from the anterior chamber of holder and the corneas were treated with the test substance, positive control or negative control. An aliquot of 750 uL of either the positive control, negative control, or test substance was introduced over the cornea.
2) Exposure : The corneas were incubated in the presence of either the test substance, positive control or negative control for 10 minutes at 32 ± 1°Cin an incubator.


REMOVAL OF TEST SUBSTANCE
Washing and Gross observation
1) Washing and post-incubation : After 10 minutes of exposure time, the negative control, positive control and test substance were removed. The corneas were washed more than three times with cMEM (with phenol red). The corneas were then given a final rinse with cMEM (without phenol red) and refilled with cMEM (without phenol red) in anterior and posterior chamber of holder. The corneas were incubated for 2 hours at (32 ± 1)°C in an incubator.
2) Quality evaluation of sodium fluorescein solution : Sodium fluorescein solution was evaluated in post-incubation period. Sodium fluorescein solution was incubated for I hour at (32 ± 1)°C in an water bath. Standard solution of sodium fluorescein solution was prepared as in the following, and then, absorbance value of standard solution at 490 nm was measured using 96-well microtiter plate reader (KG-EQM-412). The standard solution volume in the 96 well plate was 300 uL/well and each standard solution were filled in 3 wells in the 96 well plate. cMEM (without phenol red) was used as Blank.
3) Gross observation : After post-incubation, the corneas were observed visually for tissue peeling, residual test substance, opaque spots and other irregularities.

METHODS FOR MEASURED ENDPOINTS
Measurement of opacity
After gross observation, the anterior and the posterior chambers were refilled with fresh cMEM (without phenol red) and the final measurement of opacity (lux) was performed immediately. Final opacity unit was calculated according to the following formula:

Final opacity unit calculation : (I0/I-0.9894)/0.0251
*I0 is the illuminance(lux) through the reference holder
**I is the illuminance through the cornea holder

Measurement of permeability
1) After the final opacity measurement (lux) was performed, the medium was removed from the anterior chamber of the holder and replaced with 1 mL of a 4 mg/mL sodium fluorescein solution.
2) The corneas were then incubated for 90 ± 5 minutes at (32 ± 1) °C in an incubator.
3) At the end of the 90 ± 5 minutes incubation, the medium was collected from the posterior chamber of holder on a per cornea basis. Absorbance value of medium at 490 nm was measured using 96-well microtiter plate reader (KG-EQM-412). The medium volume in the 96 well plate was 300 uL/well and each media were filled in 3 wells in the 96 well plate. cMEM (without phenol red) was used as Blank.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The IVIS which was calculated by opacity score and permeability score was as in the following (unit : score):

Negative control : -1.7 ± 1.2
Positive control : 42.0 ± 3.0
Test substance (undiluted) : 20.4 ± 1.9
Test substance (20 % diluted) 2.0 ± 0.2

DECISION CRITERIA:
The following formula was used to determine the in vitro irritancy score (IVIS):
IVIS = mean opacity value + (15 x mean OD490 value).
Eye irritation was evaluated according to “Classification system’ according to the guideline and UN GHS criteria:

IVIS ≤ 3: No Category
IVIS > 3 ≤ 55: No prediction can be made
IVIS: > 55 Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
10 min
Value:
20.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
test conc. 100 %
Irritation parameter:
cornea opacity score
Run / experiment:
10 min
Value:
15
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
test conc. 100 %
Irritation parameter:
other: permeability score
Run / experiment:
10 min
Value:
0.361
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
test conc. 100 %
Other effects / acceptance of results:
The test substance-related opacity and permeability change was observed in undiluted group but not 20 % diluted group during study period.
As a result or calculating the IVIS by changes in opacity and permeability, undiluted-test substance group was calculated as 20.4 ± 1.9 and 20 % dilution group was calculated as 2.0 ± 0.2. The IVIS of negative control and positive control was -1.7 ± 1.2 and 42.0 ± 3.0 respectively.
This study results were considered acceptable because the positive control gave an IVIS that falls within two standard deviations of the current historical mean.

Summary Data for Eye Irritation - ex vivo

Opacity score

Negative control : -1.7 ± 1.2

Positive control : 23.1 ± 2.6

Test substance (undiluted) : 15.0 ± 0.8

Test substance (20 % diluted) : 1.9 ± 0.2

Permeability score (OD490 value, absorbance)

Negative control : 0.000 ± 0.001

Positive control : 1.259 ± 0.041

Test substance (undiluted) : 0.361 ± 0.088

Test substance (20 % diluted) 0.009 ± 0.006

In vitro irritancy score (IVIS)

Negative control : -1.7 ± 1.2

Positive control : 42.0 ± 3.0

Test substance (undiluted) : 20.4 ± 1.9

Test substance (20 % diluted) 2.0 ± 0.2

Interpretation of results:
other: inconclusive; not causing serious eye damage (Cat 1) but may cause eye irritation (any sub Cat 2)
Conclusions:
Test substance-related opacity and permeability change was observed at test solution concentration of 100%; no change was observed at 20 % (w/v). IVIS was 20.4 ± 1.9 and 2.0 ± 0.2 at test solution 100 % and 20 % (w/v), respectively. According to IVIS cut-off values for UN GHS categories for eye damage, no prediction can be made because the IVIS of 20.4 falls between 3 < IVIS >= 55. Consequently, the test item does not classify for Cat 1 nor for no classification, but cannot be classified as any subcategory of Cat 2 based on the study results.
Executive summary:

The present study investigated the potential ocular irritation of (2R,3R)-butane-2,3 -diol by measuring changes in opacity and permeability in isolated bovine corneas. The study was conducted according to OECD 437 (adopted 2013) and in compliance with GLP. The test item (2R,3R)-butane-2,3 -diol was administered to the test system as undiluted solution (100% test item) and as diluted solution in water of 20 % (w/v). Opacity and permeability were measured after 10 min of exposure and 2 hours post-incubation.

The in vitro irritancy score (IVIS) was determined based on changes in corneal opacity and permeability (relative to the negative control corneas). Test substance-related opacity and permeability change was observed in undiluted test solution of 100 % but not at 20 % (w/v). The IVIS was calculated as 20.4 ± 1.9 and 2.0 ± 0.2 for test solution 100% test item and 20 % (w/v), respectively. The IVIS of negative control and positive control was -1.7 ± 1.2 and 42.0 ± 3.0, respectively. This study results were considered acceptable, because the positive control gave an IVIS that falls within two standard deviations of the current historical mean. The study was considered reliable and adequate as part of the hazard assessment of eye irritation.

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
Qualifier:
according to
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. certificate)
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
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.
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).
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%.

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]

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.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion

Skin irritation / corrosion was performed according to OECD TG 439 (in vitro, adopted 2015) and in compliance with GLP (key study). The in vitro study was conducted to assess the toxic potential of skin irritation of (2R,3R)-butane-2,3-diol in human skin model EpiDerm™. Cell viability exceeded the reference value of 50 % and therefore the test substance was considered to be a non-irritant substance. The study was considered reliable and adequate for hazard assessment.

Eye irritation / corrosion

Two reliable key studies are available for eye irritation / corrosion performed according to OECD TG 437 (in vitro, adopted in 2013) and OECD TG 492 (in vitro, adopted 2018) and in compliance with GLP. OECD TG 437 study investigated the potential ocular irritation of (2R,3R)-butane-2,3 -diol by measuring changes in opacity and permeability in isolated bovine corneas. Opacity and permeability were measured after 10 min of exposure and 2 hours post-incubation in bovine cornea. Change of opacity and permeability was observed at test concentration of 100%; the in vitro irritancy score (IVIS) was 20.4 ± 1.9 SD. OECD TG 492 study was performed to assess the eye irritation potential of 2,3-butanediol ((2R,3R)-rich) by measuring the percent reduction of cell viability in comparison of untreated negative controls. The viability was measured after 30 min exposure and 2 hours post-incubation in the human cornea model. The viability value of the test item exposed tissues was 85.31% when compared to negative control. Both studies were considered reliable and adequate for hazard assessment.

Justification for classification or non-classification

#Skin irritation / corrosion

According to the criteria for the evaluation of dermal reactions specified by the CLP Regulation, the available in vitro study according to OECD TG 439 provides no evidence of either corrosion or irritation. The study was considered reliable and adequate for hazard assessment. Therefore, (2R,3R)-butane-2,3 -diol was not classified for skin irritation / corrosion.

#Eye irritation / corrosion

According to IVIS cut-off values for UN GHS categories for eye damage indicated in OECD TG 437, no prediction can be made because the IVIS of 20.4 falls between 3 < IVIS >= 55. Based on these results, the submission substance does not classify for Cat 1 nor for no classification based in this results. Based on the results of the reliable in vitro study according to OECD TG 492, the submission substance does not need to be classified, because the viability value of the test item exposed tissues is 85.31% and did not decrease below the threshold for irritancy (60%). Taken together, (2R,3R)-butane-2,3-diol does not need to be classified for eye irritation / corrosion according to the CLP regulation.