Decan-4-olide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

other: In vitro study regulatory validated

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From March 13 to April 06, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study: GLP. γ-Nonalactone, as aliphatic γ-lactone, is considered adequate for read-across purpose (see §7.1 "Toxicokinetics").

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other 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:

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

Deviations:

yes

Remarks:

IL-1α assay was added to the standard protocol

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 22-23 November 2011

Test material

Test material information

Constituent 1

Reference substance name:

Nonan-4-olide

EC Number:

203-219-1

EC Name:

Nonan-4-olide

Cas Number:

104-61-0

Molecular formula:

C9H16O2

IUPAC Name:

5-pentyldihydrofuran-2(3H)-one

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): gamma-Nonalactone
- Physical state: clear colourless to pale yellow liquid

Test animals

Species:

human

Details on test animals or test system and environmental conditions:

Three-dimensional human epidermis model ("small" size of 0.38 cm²), supplied by SKINETHIC Laboratories, Lyon, France constituted by:
- a collagen type I matrix, coated with type IV collagen
- a differentiated and stratified epidermis model from human keratinocytes, obtained after 13-day culture period.
All biological components of the epidermis and the kit culture medium have been tested for the absence of viruses, bacteria and mycoplasma.
The quality was assessed by an MTT cytotoxicity test and by histological examination (by SKINETHIC).
Epidermis was treated at D15.

Test system

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 µL

Duration of treatment / exposure:

15 ± 0.5 min

Number of animals:

3 epidermis/product

Details on study design:

Before the beginning of the study, the non-specific MTT reduction by the test item was checked by incubating MTT solution and 10 µL test item for 3 hours ± 30 minutes and checking the colour of the solution. The staining power of the test item was checked afer the addition of mineral oil to 10 µL test item and agitation 15 ± 0.5 minutes at room temperature

Application of the test item and control:
- negative control: 10 µL of PBS+ to each epidermis surface
- positive control: 10 µL of SDS solution at 5% (w/v) in distilled water; after 7 minutes contact time, an intermediate re-spreading is done
- test substance: 10 µL of the test item, as supplied
- contact timepoint for all products: 15 ± 0.5 minutes at room temperature

Rinsing:
- epidermis rinsed thoroughly with 25 mL of PBS+
- remaining product removed with a cotton-bud

Incubation:
Plates incubated during 42 ± 1 hours in CO2 incubator with maintenance medium

Medium sampling for IL-1α dosage:
- at the end of the 42 hours incubation period, shake the plates during 15 ± 2 minutes to homogenize the released mediators in the medium
- transfer 1.6 mL of each incubation medium in microtubes
- after realisation of the trial, measurement at 450 nm

MTT colouring:
- epidermis incubated for 3 hours ± 5 minutes in MTT solution in the CO2 incubator

Formazan extraction
- in 500 µL of acidic isopropanol stored at 4 hours ± 30 minutes at room temperature, protected from light or during 70 ± 5 hours at 4 °C
- measurement of OD at 570 nm

Results and discussion

In vivo

Results

Irritation parameter:

other: Viability % (MTT assay)

Basis:

mean

Time point:

other: 15 min

Score:

74.6

Remarks on result:

other: ± 2.8

Irritant / corrosive response data:

Negative control (PBS+): mean O.D. = 0.924
Positive control (SDS): Viability = 15.2 ± 2.0 %

Other effects:

None

Any other information on results incl. tables

Table 7.3.1: MTT conversion assay

		OD1	OD2	Mean	SD	Viability %	Mean viability %	SD
Negative control	Epidermis 1	0.808	0.893	0.851	0.060	92.1	100	7.5
	Epidermis 2	0.956	1.019	0.988	0.045	106.9
	Epidermis 3	0.912	0.953	0.933	0.029	101.0
	Mean	/	/	0.924	/	/
Positive control	Epidermis 1	0.156	0.167	0.162	0.008	17.5	15.2	2.0
	Epidermis 2	0.129	0.121	0.125	0.006	13.5
	Epidermis 3	0.130	0.140	0.135	0.007	14.6
Gamma- Nonalactone	Epidermis 1	0.668	0.667	0.668	0.001	72.3	74.6	2.8
	Epidermis 2	0.670	0.694	0.682	0.017	73.8
	Epidermis 3	0.708	0.729	0.719	0.015	77.8

INTERLEUKINE 1 ALPHA (IL-1α) ASSAY:

	O.D.			Concentration			Mean concentration	Standard deviation	Final conc
	Epidermis 1	Epidermis 2	Epidermis 3	Epidermis 1	Epidermis 2	Epidermis 3
Negative control	0.020	0.031	0.046	-5.8	-4.3	-2.2	-4.1	1.81	/
Positive control	1.609	1.737	1.683	214.9	232.6	225.1	224.2	8.92	228.3
Gamma-Nonalactone	0.802	0.749	0.767	102.8	95.4	97.9	98.7	3.74	102.8

Although IL-1 alpha proved to be useful to acquire additional information on the irritant potential of chemicals, only results from the MTT assay are currently used for classification and labelling according to the EU classification system. With a IL-1α concentration higher than 60 pg/mL, it could be considered that Gamma Nonalactone may have a skin irritant potential.

 

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, Gamma-Nonalactone is not classified as irritating to skin according to the criteria of Annex VI of the Directive 67/548/EEC and the Annex I of the Regulation (EC) No (1272/2008 (CLP).

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the Gamma-Nonalactone using the EPISKIN® reconstructed human epidermis model. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item 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 item treated tissues relative to the negative controls. This method was designed to be compatible with the following:

- OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010)

- Method B.46 of Commission Regulation (EC) No. 440/2008/EC

Triplicate tissues were treated with Gamma-Nonalactone 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. 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 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was74.6 ± 2.8 % after the 15‑Minute exposure period.

The quality criteria required for acceptance of results in the test were satisfied.

γ-Nonalactone was not considered as irritant according to the Directive 67/548/EEC and the Regulation (EC) No. 1272/2008 (CLP).

γ-Nonalactone, as aliphatic γ-lactone, is considered adequate for read-across purpose (see §7.1 "Toxicokinetics").