1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2017-12-12 to 2018-12-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 28 july 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

EC Number:

813-811-7

Cas Number:

253265-97-3

Molecular formula:

C11H13NO7

IUPAC Name:

1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: powder
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16FD3167
- Expiration date of the lot/batch: 2018-06-30 (retest date)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C), in tightly closed container
- Stability under test conditions: not stable in water
- Solubility and stability of the test substance in the solvent/vehicle: not applicable, the test item was applied directly on top of the skin tissue.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The solid test item was applied directly on top of the skin tissue. The test item was spread to match the size of the tissue.

OTHER SPECIFICS:
- The correction factor was not applicable for the in vitro skin irritation test, therefore no correction was made for the purity/composition of the test compound.

In vitro test system

Test system:

human skin model

Remarks:

of human-derived keratinocytes

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: not applicable

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small model (EPISKIN-SMTM, 0.38 cm²) supplied by SkinEthic Laboratories, Lyon France
- Tissue batch number(s): 17-EKIN-050
- This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 22 hours at 37°C.
- The skin was moistened with 5 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and the solid test item was added into 12-well plates on top of the skin tissues.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 60 - 85%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.3°C).
- Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline (PBS) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-medium (0.3 mg/mL). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 66 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.
- Test for reduction of MTT by test item: A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed. The test item was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (project 520042). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability (≥ 19.5%): 22.5 +/- 0.3
- Barrier function (IC50 determination, ≥ 1.5 mg/mL): 2.1
- Morphology: well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: - on blood of the same donors, the following was verified: the absence of HIV1 and 2 antibodies, the absence of hepatitis C antibodies, the absence of hepatitis B antigen HBs; - on epidermal cells of the same donors, the following was verified: the absence of bacteria, fungus and mycoplasma.
- Expiration date: 2017-12-18

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

INTERPRETATION
- A test item is considered to be irritant in the skin irritation test (category 2) if: the relative mean tissue viability of three individual tussues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered to be non-irritant in the in vitro skin irritation test (no category) if the relative mean tissue viability after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST ITEM : 25.7 to 30.6 mg; The skin was moistened with 5 μl Milli-Q water to ensure close contact of the test item to the tissue.

NEGATIVE CONTROL (PBS): 25 µL

POSITIVE CONTROL (5% SDS): 25 µL in PBS

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 per test item together with negative and positive controls

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: 92 to 103%

Remarks:

SD: +/-5.4

Results 2

Irritation / corrosion parameter:

other: Optical density

Run / experiment:

1

Value:

1.251

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: 1.185 to 1.324

Remarks:

SD: +/-0.069

Other effects / acceptance of results:

mean tissue viability (percentage of control):
negative control: 100 +/- 5.1
positive control: 6.6 +/- 2.2
mean optical density:
negative control: 1.282 +/- 0.065
positive control: 0.085 +/- 0.029

The test item was checked for possible direct MTT reduction and colour interference in the in vitro skin corrosion test using EpiDerm as a skin model (project 520042). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.

Interpretation:
The viability of all replicates was within one category.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 98% (92 to 103%). Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 6.6% (4.1 to 8.3%). The absolute mean OD570 of the negative control tissues was within 0.6 and 1.5. The standard deviation values of the percentage viability of three tissues treated identically were 5.1%, 2.2% and 5.4% for the negative, positive control and the test item respectively, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that this test is valid and that the test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.