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

Skin irritation / corrosion

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

02 May 2017 to 05 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes

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:

91.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue Number 2

Value:

83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue Number 3

Value:

85.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Average of tissues 1, 2 and 3

Value:

86.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

[mean ± standard deviation: 86.6% ± 4.29%]

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 tissues, expressed as a percentage of negative control tissues, 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 4.29% 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/S)-Butane-1,3-diol is considered to be a non-irritant to skin as the mean tissue viability is 86.6% which is greater than 50%.

Executive summary:

The test substance (R/S)-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 86.6% which is greater than 50%.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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

In vitro test system

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.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue Number 1

Value:

96.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue Number 2

Value:

77.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue Number 3

Value:

82.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Average of tissues 1, 2 and 3

Value:

85.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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.

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 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%.