Reaction mass of (8R)-(11S)-11-[(4-hydroxyphenyl)methyl]-8-alkyl-polyoxo-1-phenyl-polyaza-2-oxadodecane and (8R)-(11R)-11-[(4-hydroxyphenyl)methyl]-8-alkyl-polyoxo-1-phenyl-polyaza-2-oxadodecane

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 September 2009 – 22 September 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted in compliance with GLP. A draft OECD Test Guideline (2008) was followed, and the test method used has been accepted as a replacement to the in vivo test by the European Centre for the Validation of Alternatve Models (ECVAM, 2007).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline For The Testing of Chemicals (2008), Draft proposal for a new guideline: In Vitro Skin Irritation: Human Skin Model Test. Year 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: L’Oreal. In Vitro Skin Irritation Test: Human Epidermis Model EPISKIN, EPISKIN Skin Irritation Test-42 hours, Standard Operating Procedure: January 2005 Version 1.6.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The study report included a current certificate of GLP compliance for the test facility, issued by the MHRA.

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: Off-white solid
- Storage condition of test material: aprox. -20 degrees in the dark.

Test animals

Species:

other: EPISKIN three-dimensional human skin model.

Details on test animals or test system and environmental conditions:

The test involves the application of the test substance for 15 minutes to the EPISKIN three-dimensional human skin model. The model consists of
normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1 matrix coated with
type IV collagen. After 13 days in culture, a multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum
corneum has formed. The epidermis surface area supplied is 0.38cm2. The EPISKIN kits include assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar.

The principle of the assay is that irritant substances are sufficiently cytotoxic to cause cell death in the cell layers. The cell viability is determined by
mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) to a
soluble, coloured, formazan product. The prediction model (L’Oreal 2005) uses the percentage viability values (compared to negative control
viability) to identify irritant and non-irritant substances. The test includes acceptance criteria for both negative and positive controls.

Test system

Vehicle:

other: Water - see comment below.

Controls:

other: Refer to above entry.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10±2 mg of test material (ground using a pestle and mortar) was dispensed over each tissue using glass weighing boats. The tissues were wetted with 5 µL of distilled water prior to application of the test substance.

Duration of treatment / exposure:

15 minutes

Observation period:

Each insert (tissue) was incubated for 42±1 hours at 37±2°C in a humidified atmosphere after exposure and rinsing but prior to analysis (incubation with MTT).

Number of animals:

Triplicate tissues each for test substance, negative control (sterile Dulbecco's Phosphate Buffered Saline (DPBS) with magnesium and calcium, 10 µL), and positive control (5% Sodium Dodecyl Sulphate (SDS) in purified water, 10 µL).

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Non-irritant

Remarks on result:

other:

Remarks:

Basis: other: No interaction with the MTT. Time point: 3 hours . Remarks: The test substance has a mean tissue viability of 97.0 ± 4.4% and is predicted as a non-irritant to the skin. . (migrated information)

In vivo

Irritant / corrosive response data:

The mean value for tissue viability found for the test substance was 97.0% with a standard deviation of 4.4. On the basis that the viability value was
greater than 50%, this results therefore predicts a non irritant outcome for this endpoint.

Any other information on results incl. tables

Assessment of Results

Assessment of viability

The mean Optical Density (OD) for the 6 replicate blanks was subtracted from the individual substance and control tissues OD.

The viability of each tissue was expressed as a percentage of the mean negative control value.

Assay acceptance criteria

Negative control

The OD from the negative control tissue in the MTT assay is an indicator of tissue viability after the shipping and storage procedure and under the specific conditions of the assay. The mean absorbance of the triplicate negative control values should be ≥0.6 and the Standard Deviation (SD) value of the % viability should be ≤18.

Positive control

The OD of the positive control is an indicator of the sensitivity of the tissues. The mean viability should be ≤30% of the negative control and the SD ≤18.

Data interpretation - Prediction model

If the mean tissue viability was less than 50% of the negative control value, the sample was classed as Irritant R38 (EU classification).

Results

Possible reduction of MTT by test substance

There was no change in the colour of the test substance/MTT solution or the water control/MTT solution after three hours incubation in the dark at 37 ± 2°C in a humidified atmosphere of 5% CO₂ in air. The test substance had not interacted with the MTT.

Assay validity

Negative control

The mean absorbance of the triplicate negative control values was 0.906 which was above the minimum acceptance value of 0.6. The standard deviation of the % viability was 7.5 which was below the maximum acceptance value of 18.

Positive control

The mean % viability of the positive control was 16.4 ± 5.6 of the negative control. These were below the maximum acceptance values of 30% viability and SD of 18.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Prediction based on EPISKIN model Criteria used for interpretation of results: EU

Conclusions:

It was concluded that the test substance with a mean tissue viability of 97.0 ± 4.4%, was predicted as a non irritant to the skin.

Executive summary:

An EPISKIN skin irritation test was conducted by Huntingdon Life Sciences, UK, to assess the potential of the test substance to cause skin irritation, by means of an in-vitro test. The EPISKIN test has been accepted as a replacement to the in vivo test (Draize Skin Irritation Test), by the European Centre for the Validation of Alternative Methods (ECVAM), and a draft proposal for a new OECD Test Guideline (2008) was followed; the study was also conducted in accordance with GLP. The test substance was applied to EPISKIN human epidermis skin constructs which consisted of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum. The cell viability of the multi layers was determined by mitochondrial dehydrogenase activity. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances. The test substance elicited a mean tissue viability of 97.0% and it was concluded that the test substance is predicted as a non irritant to the skin.