Fatty acids, coco, tetraesters with bis[2,2-bis(hydroxymethyl)butyl] adipate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 08 November 2011 and 11 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Principles of method if other than guideline:

The principle of the assay is based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-la and IL-8 in the culture medium retained following the incubation period could be measure to determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,6-bis({2,2-bis[(undecyloxy)methyl]butyl}) hexanedioate

EC Number:

700-772-5

Cas Number:

1190961-28-4

Molecular formula:

N/A - too complex

IUPAC Name:

1,6-bis({2,2-bis[(undecyloxy)methyl]butyl}) hexanedioate

Test material form:

other: liquid

Details on test material:

- Substance type: UVCB
- Physical state: clear extremely pale straw coloured viscous liquid
- Analytical purity:100% UVCB
- Lot/batch No.: 010154379
- Expiration date of the lot/batch: 06/17/2012
- Storage condition of test material: room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

The SkinEthic RHE model incorporates several features which make it advantageous in the study of skin irritancy potential. The model consists of an airlifted, living, multi-layered epidermal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium, featuring normal ultra-structure and functionally equivalent to human epidermis in vivo. Test items are applied directly to the culture surface, at air interface, so that undiluted and/or end use dilutions can be tested directly.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE Model by SkinEthic Laboratories, Nice, France
- Tissue batch number(s): 11 022A 1006
- Delivery date: 08 November 2011

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

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing DPBS. Rinsing was achieved by filling and emptying each tissue insert, over approximately 40 seconds, using a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated 'holding plate' containing 300 pl of maintenance medium until all the tissues were rinsed (including the duplicate negative and positive control tissues).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.5mg/ml
- Incubation time: 3 hours at 30C, 5% CO2 in air
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540nm

NUMBER OF REPLICATE TISSUES:

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
50 pl of the test item was added to 1 ml of a 0.5 mg/ml MTT solution freshly prepared in maintenance medium and incubated at 370C, 5% C02 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution colour turned blue relative to the control, the test item was presumed to have reduced the MTT. Water insoluble test items may show direct reduction (darkening) only at the interface between the test item and the medium.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The mean tissue viabilities obtained after the 4-hour and 24-hour exposure periods were compared to the respective negative control and classified according to the following:
Mean tissue Viability Prediction
4hrs: <60 Irritant
4 hours: 60-90 Mild-moderate Irritant
24 hours: >60 Mild-moderate Irritant
24 hours >90 Mild-moderate Irritant

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

4 hours/24 hours

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

3

Test system

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control and Triton X-100 (0.1% w/v) was used as the positive control (this is an in vitro test)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 ul test material
VEHICLE: none

Duration of treatment / exposure:

4 and 24 hours

Number of animals:

Triplicate tissues for test material;
Duplicate tissues for positive and negative control groups.

Details on study design:

TEST Tissue was cultured in a 6 well plate.
REMOVAL OF TEST SUBSTANCE
- Washing (if done): 4 an d24 hours after applications
SCORING SYSTEM: MTT assay

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

4 hour

Value:

98.1

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:

24 hour

Value:

91.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Assessment of Direct Test Item Reduction of MTT

The solution did not turn blue. This was taken to indicate that the test item was not able to directly reduce MTT.

Assessment of Skin Irritation Potential

The relative mean viability of the test item treated tissues was 98.1 % after the 4-Hour exposure period and 91.6 % after the 24-Hour exposure period.

It was  considered unnecessary  to  proceed  with  tissue  histology  or analysis  of inflammatory mediator levels.

Assay Acceptance Criterion

The positive control induced a response of <60% (mild-moderate irritation) after the 24- Hour exposure period relative to the negative control treated tissues and therefore the results of the study were considered acceptable.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test material was considered to be Non-Irritant.

Executive summary:

Introduction

The purpose of this study was to determine the skin irritation potential of the test item using the SkinEthic Reconstructed Human Epidermal model (RHE, SkinEthic Laboratories, Nice, France) following exposure periods of 4 and 24 hours. The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHE model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.

Methods

The experimental design of the study consists of a test for direct reduction of MTT by the test item, followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with approximately 50ul of test item and exposed for 4 hours and 24 hours. The tissues were incubated at 37°C in a humidified atmosphere of 5% C02 in air for the appropriate exposure times.

Duplicate tissues treated with sterile water were used for each exposure period to serve as negative controls. Duplicate tissues treated with 50 I of Triton X-100 (0.1% w/v) were used for the 24-Hour exposure period to serve as a positive control.

At the end of the 4-Hour exposure period each SkinEthic tissue was rinsed using Dulbecco's Phosphate Buffered Saline (DPBS) and placed into a 'holding plate' until all the tissues had been rinsed. The rinsed tissues (2 per group) were then transferred to an MTT 'loading plate' and incubated at 37°C for 3 hours in a humidified atmosphere of 5% C02 in air. At the end of this time, each SkinEthic tissue was blotted dry and placed into an MTT 'extraction plate' in order to extract all of the reduced MTT from the tissues. The remaining test item treated tissue was retained for possible histology.  The same rinsing, loading, extraction and retention procedures were repeated for the 24-Hour tissues once the 24-Hour exposure period was complete. The maintenance medium in each well of the 4 and 24-Hour exposure period treatment plates were retained for possible analysis of inflammatory mediator levels of IL-1a and IL-8.

At the end of the extraction period, the extracted MTT solution was mixed for each SkinEthic tissue and 3 x 200 !JI samples representing each tissue were transferred to the appropriate wells of a 96 well plate. The optical density at 540nm (00540) of each well was measured. Data are presented in the form of percentage viability (MTT conversion relative to negative controls) for each of the two exposure periods.

The results were used in order to make a prediction of skin irritation potential.

 

 

Results

The relative mean viability of the test item treated tissues was 98.1 % after the 4-Hour exposure period and 91.6% after the 24-Hour exposure period.

It was considered unnecessary to proceed with tissue histology or analysis of inflammatory mediators.

Conclusion

 The test item was considered to be a Non-Irritant.