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Description of key information

Results from the in vitro skin irritation study indicate an absence of skin irritation potential, the in vitro skin corrosion study need not be conducted

 

The test substance (R)-(-)-butane-1,3-diol was assessed by OECD 439 (in vitro skin irritation: reconstructed human epidermis test method) to predict dermal irritation potential under GLP conditions. MatTek EpiDerm™ tissue samples were treated in triplicate with the test article, negative control and positive control for 60 minutes. Following treatment and subsequent incubation time, the viability of the tissues was determined using methyl thiazole tetrazolium (MTT) uptake and reduction. The absorbance of each sample was measured at 540 nm. The viability of tissues was then expressed as a percent of negative control values. All acceptance criteria were met. The test material is considered to be a non-irritant to skin as the mean tissue viability is 85.5% which is greater than 50%.

 

The test substance (R)-(-)-butane-1,3-diol was assessed by OECD 492 (in vitro reconstructed human cornea-like epithelium (RhCE) test method for chemicals not requiring classification and labelling for eye irritation or serious eye damage) to provide classification of chemicals concerning their eye irritation potential under GLP conditions. MatTek EpiOcular™ tissue samples were treated in duplicate with the test article, negative control and positive control for 30 minutes. Following treatment and subsequent incubation time, the viability of the tissues was determined using methyl thiazole tetrazolium (MTT) uptake and reduction. The absorbance of each sample was measured at 570 nm. The viability was then expressed as a percent of control values. the negative control was determined to be a non-irritant and the positive control was determined to be an irritant. The difference in viability between identically treated tissues were less than 20% (0.10 -2.69%). The test material is considered to be a non-irritant to eye as the mean tissue viability is 82.3% which is greater than 60%.

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:
02 May 2017 to 05 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
LOT# DSP-48-BD-P1
Assay (%), GCMS, dry-basis: 99.10%
Water (%): 1.56%
Acid Number (mg KOH/g): 0.004
Color (APHA): <3.0
Test system:
human skin model
Details on animal used as source of test system:
Epidermal Model, MatTek EpiDerm™ (EPI-200-SIT) kit.
Justification for test system used:
The EpiDerm Skin Model closely parallels human skin, thus providing a useful in vitro means to assess dermal irritancy and toxicology.
Vehicle:
unchanged (no vehicle)
Details on test system:
- Model used: MatTek EpiDerm™

- Tissue batch number(s): Lot No. 25785 Kit E

- Delivery date: 02 May 2017

- Date of initiation of testing: 03 May 2017; Before use, the tissues were incubated with assay medium for a one-hour equilbration. The tissues were then moved to new wells with fresh medium for an additional overnight equilibrium.

- Exposure time: 60 minutes

- Temperature used during treatment / exposure: The dosed tissues were placed in an incubator at 37 °C ± 1 °C, 5 ± 1% CO2 for 35 ± 1 minutes, and then returned to the sterile hood for the remainder of the 60-minute exposure period.

-Volume and number of washing steps: thoroughly rinsed with sterile phosphate buffered saline

- Temperature of post-treatment incubation: 37 °C ± 1 °C for an additional 18 ± 2 hours

- MTT concentration: 1 mg/mL methyl thiazole tetrazolium (MTT) in Dulbecco's Modified Eagle's Medium (DMEM)

- Incubation time: 3 hours

- Spectrophotometer: μQuant Plate Reader

- Wavelength: 540 nm

- Acceptance criterion: The assay meets the acceptance criterion if the mean OD540 of the negative control tissues is between 1.0 and 2.5, inclusive, and the mean viability of positive control tissues, expressed as percentage of the negative control tissues, is less than or equal to 20%. In addition, the standard deviation (SD) calculated from individual percent tissue viabilities of the three identically-treated replicates is less than 18%.

- Number of replicate tissues: Each treatment with test article or control was conducted in triplicate

- The test substance is considered to be non-irritant to skin if mean tissue viability of the test substance is greater than 50% of the mean viability of the negative controls
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
For each test article, 30 μL of the test article were applied to the EpiDerm™ tissue. A negative control (30 μL of phosphate buffered saline [PBS]) and a positive control (30 μL of 5% sodium dodecyl sulfate [SDS] solution) were each tested concurrently, with a nylon mesh placed on top to facilitate even distribution of the material.
Duration of treatment / exposure:
The exposure period for the test articles and controls was 60 minutes.
Duration of post-treatment incubation (if applicable):
After dosing and incubation, the tissues were rinsed with PBS, blotted to remove the test substance and dry the tissue, and transferred to fresh medium. The rinsed EpiDerm™ tissues were returned to the incubator for an additional 18 ± 2 hours.
Number of replicates:
Each treatment with test article or control was conducted in triplicate.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue Number 1
Value:
96.7
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue Number 2
Value:
77.5
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue Number 3
Value:
82.3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Average of tissues 1, 2 and 3
Value:
85.5
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Remarks:
[mean ± standard deviation: 85.5% ± 9.98%]
Other effects / acceptance of results:
- Direct-MTT reduction: The test substance did not reduce MTT directly

- Colour interference with MTT: The test substance is clear colourless, there was no colour interference

- Acceptance criteria met for negative control: Yes, OD540 of the negative control tissues was 1.808 ± 0.175

- Acceptance criteria met for positive control: Yes, the mean viability of the positive control tissuesexpressed as a percentage of negative control tissue, was 2.4%

- Acceptance criteria met for variability between replicate measurements: Yes, the standard deviation of the individual percent tissue viability of the three identically-treated replicates is 9.98% for the test substance, 9.69% for the negative control and 0.19% for the positive control.
Interpretation of results:
GHS criteria not met
Conclusions:
(R)-(-)-Butane-1,3-diol is considered to be a non-irritant to skin as the mean tissue viability is 85.5% which is greater than 50%.
Executive summary:

The test substance (R)-(-)-butane-1,3-diol was assessed by OECD 439 (in vitro skin irritation: reconstructed human epidermis test method) to predict dermal irritation potential under GLP conditions. MatTek EpiDerm™ tissue samples were treated in triplicate with the test article, negative control and positive control for 60 minutes. Following treatment and subsequent incubation time, the viability of the tissues was determined using methyl thiazole tetrazolium (MTT) uptake and reduction. The absorbance of each sample was measured at 540 nm. The viability of tissues was then expressed as a percent of negative control values. All acceptance criteria were met. The test material is considered to be a non-irritant to skin as the mean tissue viability is 85.5% which is greater than 50%.

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:
02 May 2017 to 04 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
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)
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
LOT# DSP-48-BD-P1
Assay (%), GCMS, dry-basis: 99.10%
Water (%): 1.56%
Acid Number (mg KOH/g): 0.004
Color (APHA): <3.0
Species:
human
Details on test animals or tissues and environmental conditions:
Reconstructed Human Cornea-like Epithelium (RhCE)
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 µL of test article, negative control (tissue culute water) and positive control (methyl acetate) were each tested concurrently
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
After dosing and incubation, the tissues were rinsed with phosphate-buffered saline (PBS) and soaked in 5 mL of room-temperature assay medium in a 12-well plate for 12 minutes. The soaked tissues were then incubated in fresh assay medium at 37 ± 1°C, 5 ± 1% CO2 for 120 minutes.
Number of animals or in vitro replicates:
Each treatment with test article or control was conducted in duplicate
Details on study design:
- Details of the test procedure used: MatTek EpiOcular™ tissue samples were treated in duplicate with the test articles, negative control and positive control for 60 minutes. Following 30 minute treatment time treatment and subsequent incubation time, the viability of the tissues was determined using methyl thiazole tetrazolium (MTT) uptake and reduction.

- RhCE tissue construct used, including batch number: EpiOcular™ tissues, Lot No. 23479 Kit C

- Doses of test chemical and control substances used: 50 µL (unchanged)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: tissues were then incubated at 37 ± 1 °C, 5 ± 1% CO2 for 30 ± 2 minutes

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: Test articles were not found to have reduced MTT
- Number of tissue replicates used per test chemical and controls: Each treatment with test article or control was conducted in duplicate
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): The optical density of the plate wells was measured at a wavelength of 570 nm (OD570), with no reference wavelength. A regression line and an R-squared value were greater than 0.999.

- Description of the method used to quantify MTT formazan: At the end of the incubation period, each EpiOcular™ tissue was transferred to a 24-well plate containing 300 μL of MTT solution (1 mg/mL MTT in Dulbecco's Modified Eagle's Medium). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiOcular™ tissue was rinsed with PBS and then treated with 2.0 mL of extractant solution (isopropanol) per well overnight at room temperature in the dark. Two aliquots of the extracted MTT formazan from each well were measured at 570 nm using a plate reader (μQuant Plate Reader).

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: Mean tissue viability less than or equal to 60% - GHS Classification Category 1 or 2

- Positive and negative control means and acceptance ranges based on historical data: The assay meets the acceptance criterion if the OD570 of the Negative Control is >0.8 and <2.5 (i.e., 2.325); The assay meets the acceptance criterion if the mean relative viability of the positive control is below 50% of negative control viability (i.e., 41.6%).


- Acceptable variability between tissue replicates for positive and negative controls: The difference in viability between identically treated tissues must be less than 20% (i.e., 0.10 – 3.46%).
Irritation parameter:
in vitro irritation score
Remarks:
% viability
Run / experiment:
Tissue #1
Value:
83.6
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Remarks:
% viability
Run / experiment:
Tissue # 2
Value:
80.9
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Remarks:
% viability
Run / experiment:
Mean of Tissue # 1 & 2
Value:
82.3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- Acceptance criteria met for negative control: Yes, the mean OD570 of the negative control tissues was 2.325
- Acceptance criteria met for positive control: Yes, the mean relative viability of the positive control tissues was 41.6%.

The difference in viability between identically treat tissues were less than 20% (0.10-2.69%).
Interpretation of results:
GHS criteria not met
Conclusions:
(R)-(-)-Butane-1,3-diol is considered to be a non-irritant to eyes as the mean tissue viability is 82.3% which is greater than 60%.
Executive summary:

The test substance (R)-(-)-butane-1,3-diol was assessed by OECD 492 (in vitro reconstructed human cornea-like epithelium (RhCE) test method for chemicals not requiring classification and labelling for eye irritation or serious eye damage) to provide classification of chemicals concerning their eye irritation potential under GLP conditions. MatTek EpiOcular™ tissue samples were treated in duplicate with the test article, negative control and positive control for 30 minutes. Following treatment and subsequent incubation time, the viability of the tissues was determined using methyl thiazole tetrazolium (MTT) uptake and reduction. The absorbance of each sample was measured at 570 nm. The viability was then expressed as a percent of control values. The negative control was determined to be a non-irritant and the positive control was determined to be an irritant. The difference in viability between identically treated tissues were less than 20% (0.10 -2.69%). The test material is considered to be a non-irritant to eye as the mean tissue viability is 82.3% which is greater than 60%.

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

The test substance was assessed by OECD 439 and is considered to be a non-irritant to skin.

 

The test substance was assessed by OECD 492 and is considered to be a non-irritant to eye.

 

Based on the information summarized above, (R)-(-)-butane-1,3-diol does not meet the criteria for classification as a skin corrosion/irritation hazard or a serious eye damage/eye irritation hazard according to sections 3.2 and 3.3. of the European CLP (Regulation (EC) No 1272/2008 as amended).