(R)-(-)-butane-1,3-diol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 May 2017 to 04 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

02 May 2017 to 04 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This endpoint study record is additional supporting in vitro data for the source substance.

Reason / purpose for cross-reference:

reference to same study

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)

GLP compliance:

yes

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:

91.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Remarks:

% viability

Run / experiment:

Tissue # 2

Value:

88.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Remarks:

% viability

Run / experiment:

Mean of Tissue # 1 & 2

Value:

90.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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/S)-Butane-1,3-diol is considered to be a non-irritant to eyes as the mean tissue viability is 90.1% which is greater than 60%.

Executive summary:

The test substance (R/S)-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 90.1% which is greater than 60%.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(R)-(-)-butane-1,3-diol

EC Number:

228-532-0

EC Name:

(R)-(-)-butane-1,3-diol

Cas Number:

6290-03-5

Molecular formula:

C4H10O2

IUPAC Name:

butane-1,3-diol

Test material form:

liquid

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 animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

Reconstructed Human Cornea-like Epithelium (RhCE)

Test system

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%).

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

% viability

Run / experiment:

Tissue #1

Value:

83.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Remarks:

% viability

Run / experiment:

Tissue # 2

Value:

80.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

in vitro irritation score

Remarks:

% viability

Run / experiment:

Mean of Tissue # 1 & 2

Value:

82.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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%).

Applicant's summary and conclusion

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%.