Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Aug - 03 Sep 2012

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

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)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Test material

Test material information

Constituent 1

Reference substance name:

Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

EC Number:

260-257-1

EC Name:

Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

Cas Number:

56554-53-1

Molecular formula:

C30H56O6

IUPAC Name:

1,3-bis[(3,5,5-trimethylhexanoyl)oxy]propan-2-yl 3,5,5-trimethylhexanoate

Test animals

Species:

human

Strain:

other: EPISKIN™ reconstructed human epidermis model

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: SkinEthic Laboratories, Nice, France
- Description: The EPISKIN™ model is a 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. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

ADAPTATION TO CELL CULTURE CONDITIONS
2 mL of maintenance medium, warmed to appr. 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.

INCUBATION CONDITIONS (Incubator)
- Temperature (°C): 37
- CO2 gas concentration (%): 5

Test system

Type of coverage:

other: open in vitro system

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent control tissues treated with Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+ served as negative controls, positive controls were exposed to 5% SDS

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 µL

NEGATIVE CONTROL
- Negative control: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+
and Mg2+
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Positive control substance: Sodium Dodecyl Sulphate (SDS), 5% w/v

Duration of treatment / exposure:

15 min

Observation period:

Not applicable. Post-treatment incubation period: 42 h

Number of animals:

Not applicable. The test was performed in triplicates for each treatment and control group.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: 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 Ca2+ and Mg2+. 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 item.
- Time after start of exposure: 15 min

CELL VIABILITY MEASUREMENTS
- Method: colourimetric MTT reduction assay
- Principles of method: Cell viability is measured by enzymatic reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells. The formazan product is extracted from the tissue with acidified isopropanol and the optical density of the extracts is measured at 540 nm using acidified isopropanol as blank.
- Time after exposure: 42 h (MTT loading/Formazan extractin) and on Day 6 (Absorbance/Optical density measurements)

INTERPRETATION OF RESULTS
For the test item, the relative mean tissue viabilities obtained after the 15 min exposure period followed by the 42 h post-exposure incubation period were compared to the mean of the negative control tissues (n = 3). The relative mean viabilities were calculated as follows:

Relative mean viability (%) = (mean OD540 of test item / mean OD540 of negative control) x 100

Classification of irritation potential was based upon relative mean tissue viability following the 15 min exposure period followed by the 42 h post-exposure incubation period according to the following criteria:

Relative mean tissue viability ≤ 50%: Irritant (I) R38
Relative mean tissue viability ≥ 50%: Non-irritant (NI)

The results were evaluated according to EU Commission Directive 2001/59/EC.

QUALITY CRITERIA
The results of the assay were considered acceptable if the following assay acceptance criteria are achieved:
- Positive control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤ 40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤ 18%.
- Negative control: The assay establishes the acceptance criterion for an acceptable test if the mean OD540 for the negative control treated tissues was ≥ 0.6, and the standard deviation value of the percentage viability is ≤ 18%.
- Test item: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tisues is ≤ 18%.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

101.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

QUALITY CRITERIA

The relative mean tissue viability for the positive control treated tissues was 12.2% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 2.6%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.784 and the standard deviation value of the percentage viability was 6.7%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 7.9%. The test item acceptance criterion was therefore satisfied.

The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viability values and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test item treated tissues was 101.6% after a 15-minute exposure period.

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Table 1. Mean OD540 values and percentage viabilities for the negative control item, positive control item and test item

Item	OD540 of tissues	Mean OD540 of triplicate tissues	± SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of relative mean viability
Negative control	0.754	0.784	0.053	96.2	100*	6.7
	0.845			107.8
	0.754			96.2
Positive control	0.115	0.096	0.020	14.7	12.2	2.6
	0.097			12.4
	0.075			9.6
Test item	0.801	0.796	0.062	102.2	101.6	7.9
	0.856			109.2
	0.732			93.4

 

SD: standard deviation

*: The mean viability of the negative control tissue is set at 100%

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.