Reaction products of Dihydro-3-(tetrapropenyl)furan-2,5 dione with propane-1,2,diol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 February 2016 to 22 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

2-(tetrapropenyl)succinic acid, monoester with propane-1,2-diol

EC Number:

257-836-6

EC Name:

2-(tetrapropenyl)succinic acid, monoester with propane-1,2-diol

Cas Number:

52305-09-6

Molecular formula:

C17H30O5 - C21H38O5

IUPAC Name:

2-[2-(2-hydroxy-1-methylethoxy)-2-oxoethyl]tetradecanoic acid

Test material form:

solid

Details on test material:

- Apperance: Yellow solid block
- Storage: Room temperature in the dark

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was heated in a water bath to 37 °C, taken up into a sufficient number of syringes and allowed to cool to room temperature prior to dosing.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: EPISKIN™ reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes.

Cell source:

other: Not specified

Source strain:

not specified

Details on animal used as source of test system:

SOURCE ANIMAL
Model: EPISKIN™ three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
- Supplier: SkinEthic Laboratories, Lyon, France

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ reconstructed human epidermis model from SkinEthic Laboratories, Lyon, France.
- Tissue batch number(s): 16-EKIN-007
- Delivery date: 16 February 2016
- Pre-incubation (Day 0: Tissue Arrival):
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Colour Satisfactory: Yes
Agar Medium Colour Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5 % CO2 in air overnight.
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5 % CO2 in air overnight.
- Application of Test Item and Rinsing (Day 1):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with approximately 10 μL of the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. An infinite dose of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5 % w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature.
- Temperature of post-treatment incubation: The rinsed tissues were incubated at 37 °C, 5 % CO2 in air for 42 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. 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 material. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5 % CO2 in air for 42 hours.
- Observable damage in the tissue due to washing: None specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
- MTT concentration: 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates.
- Incubation time: The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5 % CO2 in air.
At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’.
- Spectrophotometer: The optical density was measured (quantitative viability analysis) using the Anthos 2001 microplate reader.
- Wavelength: 562 nm
- Filter: Without a reference filter

NUMBER OF REPLICATE TISSUES: Triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
A test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
Approximately 10 mg of test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue or purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
A test item may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. Approximately 10 mg of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the colour was made.

QUANTITATIVE MTT ASSESSMENT 9PERCENTAGE TISSUE VIABILITY
For the test item the relative mean tissue viabilities obtained after the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = (mean OD562 of test item / mean OD 562 of negative control) x 100

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be an irritant to skin if the viability after 15 minutes exposure is less than, or equal to 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 μL

NEGATIVE CONTROL
- Amount applied: 10 μL

POSITIVE CONTROL
- Amount applied: 10 μL

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours.

Number of replicates:

Triplicate tissues were treated with the test material and with the positive and negative controls.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three runs/experiments

Value:

9.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS
- Direct-MTT reduction: The MTT solution containing the test material did not turn blue or purple which indicated that the test material did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test material was a very pale yellow colour. This colour was attributed to the intrinsic color of the test item itself and was not considered to affect the MTT endpoint. It was therefore unnecessary to run colour correction tissues.
It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD562 for the negative control treated tissues was 0.908 and the standard deviation value of the viability was 3.5 %. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: Yes. The relative mean tissue viability for the positive control treated tissues was 5.7 % relative to the negative control treated tissues and the standard deviation value of the viability was 0.5 %. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test material treated tissues was 1.7 %. The test material acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Material

Material	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative Individual tissue viability (%)	Relative Mean viability (%)	± SD of Relative Mean viability (%)
Negative Control Material	0.943	0.908	0.031	103.9	100*	3.5
	0.882			97.1
	0.889			99.0
Positive Control Material	0.056	0.052	0.005	6.2	5.7	0.5
	0.053			5.8
	0.047			5.2
Test Material	0.070	0.085	0.016	7.7	9.4	1.7
	0.085			9.4
	0.101			11.1

OD = Optical Density

SD = Standard deviation

∗ = The mean viability of the negative control tissues is set at 100 %

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of this study, the test material was determined to be a skin irritant.

Executive summary:

A study was performed in vitro to assess the irritancy potential of the test material. The study was conducted in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

Triplicate tissues were treated with the test material 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-labelled 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-labelled 96-well plate. The optical density was measured at 562 nm.

The relative mean tissue viability for the positive control treated tissues was 5.7 % relative to the negative control treated tissues and the standard deviation value of the viability was 0.5 %. The positive control acceptance criteria were therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.908 and the standard deviation value of the viability was 3.5 %. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.7 %. The test item acceptance criterion was therefore satisfied.

The relative mean viability of the test material treated tissues was 9.4 % which is below the threshold of 50 %, therefore under the conditions of this study the test material was determined to irritating to the skin.