Methyl 5-deoxy-2,3-O-isopropylidene-β-D-ribofuranoside

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 December 2012 to 09 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully GLP compliant and in accordance with current test guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methylfuranoside

IUPAC Name:

Methylfuranoside

Constituent 2

Reference substance name:

Methyl 5-deoxy-2,3-O-isopropylidene-β-D-ribofuranoside

EC Number:

245-485-1

EC Name:

Methyl 5-deoxy-2,3-O-isopropylidene-β-D-ribofuranoside

Cas Number:

23202-81-5

Molecular formula:

C9H16O4

IUPAC Name:

methyl 5-deoxy-2,3-O-isopropylidene-beta-D-ribofuranoside

Test material form:

other: plae yellow liquid

Details on test material:

Name: Methylfuranoside
CAS number: 23202-81-5
Batch number: 201111082008
Quantitiy received: 1.5 kg
Purity: 99.7%
Expiry date: November 2013
Date of receipt: 19 November 2013
Appearance: pale yellow liquid
Storage details: stored in a sealed container, at room temperature (15 to 25°C) in the dark.

The negative control substance for the corrosivity test was purified water supplied by Baxter Healthcare Ltd, Thetford, UK.
The positive control substance for the corrosivity test was 8N potassium hydroxide supplied by Sigma-Aldrich Co. Ltd., Poole, UK.

The negative control substance for the irritation test was phosphate buffered saline (PBS) (pH 7.4) supplied by MatTek Corporation, Ashland, Massachusetts, USA.
The positive control substance for the irritation test was 5% w/v sodium dodecyl sulphate (SDS) supplied by Severn Biotech Limited, Kidderminster, UK.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum supplied by MatTek Corporation, Ashland, Massachusetts, USA.

Vehicle:

unchanged (no vehicle)

Details on test system:

Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum supplied by MatTek Corporation, Ashland, Massachusetts, USA. Human keratinocytes are used to construct the epithelium. Multiple layers of viable epithelial cells are present under a functional stratum corneum. The stratum corneum is multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevent the passage of material around the stratum corneum to the viable model tissue. The skin model was supplied free of contamination with bacteria, mycoplasma and fungi.

SKIN CORROSION
EpiDermTM inserts were treated with the test item, negative control and positive control over two time points: three minutes and one hour. At the end of the treatment period, the tissues were washed with PBS and cell viability was assessed using the MTT assay. The skin corrosion potential was classified according to the remaining cell viability obtained after test item treatment.
The test item was considered not to be corrosive. The irritation test was therefore conducted.

Skin irritation: After dosing the tissues were incubated at 37 ± 1ºC, 5 ± 1% CO2 for 25 ± 1 minute. Although the tissues were removed from the incubator 10 minutes earlier than required by the protocol, they were left for a total of 1 hour from initiation of treatment to start of wash-off as stated in the protocol. This deviation from protocol was considered not to have affected the integrity or outcome of the study as the data confirms that the controls met the acceptance criteria. The plates were removed from the incubator and placed into a sterile hood until the 60 ± 1 minute treatment period was complete for each tissue. The tissues were washed using PBS and dried using cotton wool buds to remove residual material before being transferred to a new well containing prewarmed
maintenance medium. The tissues were then incubated at 37°C for the 42 ± 2 hour recovery time period.
Upon completion of the 42 hour recovery period, the base of each tissue was rinsed with PBS before being placed on top of 0.3 mL of of MTT solution (1 mg/mL) and incubated for three hours (37°C, 5% CO2).

Skin corrosion: The plates were removed from the incubator and placed into a sterile hood until the 60 ± 1 minute treatment period was complete for each tissue. The tissues were washed using PBS and dried using cotton wool buds to remove residual material before being transferred to a new well
containing pre-warmed maintenance medium. The tissues were then incubated at 37°C for the 42 ± 2 hour recovery time period.
Upon completion of the 42 hour recovery period, the base of each tissue was rinsed with PBS before being placed on top of 0.3 mL of of MTT solution (1 mg/mL) and incubated for three hours (37°C, 5% CO2).

Upon completion of the 42 hour recovery period, the base of each tissue was rinsed with PBS before being placed on top of 0.3 mL of of MTT solution (1 mg/mL) and incubated for three hours (37°C, 5% CO2). After incubation, the tissues were washed with PBS Any resultant colour was extracted
by flooding the tissue with 2 mL isopropanol and shaking at 150 rpm for two hours. Upon completion of the extraction, each tissue was pierced using a hypodermic needle so that the extract could run through the tissue. Once drained, the tissue was discarded and the extract mixed by pipette. Two x 200 µL aliquots of each resultant extract were placed into a 96-well plate for spectrophotometric determination of optical density at 570 nm using extraction solution as a blank. Tissue viability was calculated for each tissue as a percentage of the mean of the negative control tissues

ASSESMENT STAINING POTENTIAL FOR NON-COLOURED SUBSTANCES
30 µL of the test item was added to 0.3 mL deionised water in a suitable glass container and incubated for 60 minutes at 37 ± 1ºC, 5 ± 1% CO2. At the end of the incubation, the mixture was shaken. There was no staining, therefore the test item was deemed not to have the potential to stain the tissue.

ASSESSMENT OF MTT INTERACTING SUBSTANCES
In order to assess the potential non-specific reduction of the test item, 30 µL of test item was added to 1.0 mL of 1.0 mg/mL MTT and the colour change was assessed after incubation for 60 minutes at 37 ± 1ºC, 5 ± 1% CO2. There was no change in colour therefore the test item did not interact with MTT.

Amount/concentration applied:

30 µL of the undiluted test item

Duration of treatment / exposure:

Skin corrosion: two exposure times of 3 minutes and 1 hour.
Skin irritation: 25 ± 1 minute.

Duration of post-treatment incubation (if applicable):

42 hour

Number of replicates:

Triplicates

Test system

Type of coverage:

other: Not applicable

Preparation of test site:

other: Not applicalbe

Vehicle:

unchanged (no vehicle)

Controls:

not required

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1- 3 min exposure

Value:

ca. 89

Positive controls validity:

valid

Remarks:

19% viability

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1- 60 min exposure

Value:

ca. 60

Positive controls validity:

valid

Remarks:

9% viability

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

irritation (OECD 439) 2 - 3 min exposure

Value:

90.5

Negative controls validity:

valid

Remarks:

100 % viability

Positive controls validity:

valid

Remarks:

6.7 % viability

Other effects / acceptance of results:

Skin corrosion
Skin viability after a three minute or one hour exposure to the test item was 89% and 60%, respectively.
Skin viability after a three minute or one hour exposure to the positive control item was 19% and 9%, respectively, demonstrating appropriate performance of the assay.

Skin irritation
The group mean viability for the test item was 90.5%, for the negative control was 100% and for the positive control was 6.7%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item, Methylfuranoside, was considered not to be corrosive or irritating to the in vitro skin models.

Executive summary:

The purpose of the study was to determine whether the test item Methylfuranoside caused dermal corrosion or irritation. Dermal corrosion refers to the production of irreversible tissue damage in the skin following the application of a test material. Dermal irritation refers to the production of reversible inflammatory changes in the skin following the application of a test material.

The potential for chemical induced skin corrosion or irritation is an important consideration in establishing procedures for safe handling, packing and transport of chemicals.

The corrosivity potential of the test item was evaluated using the EpiDerm test system. The irritation test was conducted using the EpiDerm SIT (EPI-200) test system.

In the corrosivity test, skin viability after a three minute or one hour exposure to the test item was 89% and 60%, respectively.

In the irritation test, the group mean viability for the test item was 90.5%, for the negative control was 100% and for the positive control was 6.7%.

The test item, Methylfuranoside, was considered not to be corrosive or irritating to skin.