1,3-dioxolane

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 23 JUNE 2020 to 29 JUNE 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,3-dioxolane

EC Number:

211-463-5

EC Name:

1,3-dioxolane

Cas Number:

646-06-0

Molecular formula:

C3H6O2

IUPAC Name:

1,3-dioxolane

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): 1,3-dioxolane- Physical state: liquid- Analytical purity: 99.9986%- Lot/batch No.: 2003031300R

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Lambiotte & Cie/2003031300R
- Expiration date of the lot/batch: 03/03/2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark on arrival until 22 May 2020 and then stored at approximately 4°C in the dark thereafter

In vitro test system

Test system:

other: Reconstructed epidermis of normal human keratinocytes

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Source strain:

other: Human donors

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin TM
- Tissue batch number(s): 20-EKIN-026
- Delivery date: 23 June 2020
- Date of initiation of testing: 23 June 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer:Labtech LT-4500 microplate reader
- Wavelength: 570
- Filter: without a reference filter
- Filter bandwidth: filter band pass 10 nm
- Linear OD range of spectrophotometer: 0-2.6

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Mean tissue viability in positive control (5% SDS in sterile water) - 6.9% (SD 3.7)
- Barrier function: IC50: 2.0 mg/mL
- Morphology: Multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and a multilayered stratum corneum, 8 cell layers
- Contamination: on cells from the donors, absence of bacteria, fungus and mycoplasma
- Reproducibility: Historical control data in table 1

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if relative mean tissue viability is lower than or equal to 50 %.
- The test substance is considered to be non-irritant to skin if relative mean tissue viability is greater than or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):10 µL (26.3 µL/cm²)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% w/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Test animals

Species:

other: Human donors

Strain:

other: Human donors

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item

Value:

78.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control item

Value:

4.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative control item

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study and under the experimental conditions reported, the test item was classified as non-irritant. The following classifications apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

Introduction
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Method
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm.
Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results
The relative mean viability of the test item treated tissues was 78.8% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.
Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.

Conclusion
In this study and under the experimental conditions reported, the test item was classified as non-irritant. The following classifications apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).