Decan-4-olide

Endpoint summary

Description of key information

- Skin irritation / corrosion: not irritating (K+S)
- Eye irritation: not irritating (WoE)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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").

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

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.

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

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

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.

 

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").

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

16-20 August 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted in compliance with OECD Guideline 405 (1987) with minor deviations: no certificate of analysis of the test substance; age of animals at start of study not reported. γ-Nonalactone, as aliphatic γ-lactone, is considered adequate for read-across purpose (see §"Toxicokinetics").

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

no certificate of analysis of the test substance; age of animals at start of study not reported; non-ocular local and systemic adverse effects not followed

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Specific details on test material used for the study:

- Storage condition of test material: Approximately 4 °C in the dark
- Physical state: Colourless slightly viscous liquid

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River, Deutschland GmbH, Kißlegg, Germany
- Weight at study initiation: 2.1-2.3 kg
- Housing: Animals were housed individually in PPO cages with perforated floor
- Diet: Pelleted complete rabbit diet "Altromin 2123" (Altromin, Lage, Germany), ad libitum
- Water: Domestic quality drinking water acidified with hydrochloric acid to pH 2.5, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 55 ± 15 %
- Air changes: 10/h
- Photoperiod: 12 h dark / 12 h light

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

24 h

Observation period (in vivo):

72 h

Number of animals or in vitro replicates:

4 females

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: After the first 24 h reading fluorescein was instilled. After rinsing with 20 mL 0.9% sodium chloride solution, the eyes were examined again using UV -light to detect possible corneal damage.

SCORING SYSTEM: Draize (1977) scoring system

TOOL USED TO ASSESS SCORE: Eyes of the animals were examined with a hand held inspection lamp fitted with white and UV -light and magnifying glass with 2 X magnifications. The examination was performed before and after instillation of fluorescein.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

4 animals

Time point:

other: Average 24, 48 and 72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Remarks:

4 animals

Time point:

other: Average 24, 48 and 72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

4 animals

Time point:

other: Average 24, 48 and 72 h

Score:

0.17

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

4 animals

Time point:

other: Average 24, 48 and 72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

- One hour after application of the test article, 2 animals showed some conjunctival vessels definitely injected, some swelling above normal and some amount of discharge different from normal. Diffuse, crimson red conjunctiva with individual vessels not easily discernible and some swelling above normal was seen in one animal. One animal showed some conjunctival vessels definitely injected and some amount of discharge different from normal.
- After 24 h, 2 animals showed some conjunctival vessels definitely injected. Remaining 2 animals were free of any signs of eye irritation.
- After 48 and 72 h all animals were free of any signs of eye irritation.

Other effects:

No data

Table 7.3.2/1: Scores for ocular lesions

Rabbit no./ Body weight (kg)	Region of eye	Scores
		1 h	24 h	48 h	72 h	Individual mean score
1546 / 2.1	Corneal opacity	0	0	0	0	0
	Iris	0	0	0	0	0
	Conjunctivae: Redness	1	1	0	0	0.33
	Conjunctivae: Chemosis	1	0	0	0	0
1550 / 2.2	Corneal opacity	0	0	0	0	0
	Iris	0	0	0	0	0
	Conjunctivae: Redness	2	0	0	0	0
	Conjunctivae: Chemosis	1	0	0	0	0
1552 / 2.3	Corneal opacity	0	0	0	0	0
	Iris	0	0	0	0	0
	Conjunctivae: Redness	1	1	0	0	0.33
	Conjunctivae: Chemosis	1	0	0	0	0
1566 / 2.2	Corneal opacity	0	0	0	0	0
	Iris	0	0	0	0	0
	Conjunctivae: Redness	1	0	0	0	0
	Conjunctivae: Chemosis	0	0	0	0	0

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, γ-Nonalactone is not classified as irritating to eyes according to the Directive 67/548/EEC and the Regulation (EC) No 1272/2008 (CLP).

Executive summary:

In an eye irritation study conducted according to the OECD Guideline 405 and in compliance with GLP, 4 healthy female rabbits of the New Zealand White strain were exposed to 0.1 mL of γ-Nonalactone in their left eye, while the right eye remained untreated and served as control. The eyelids were then gently held together for 1 second to avoid any loss of the test material. Eyes were examined 1 and 24 h after the instillation of test material. After the 24 h reading, fluorescein was instilled. After rinsing with 20 mL 0.9 % sodium chloride solution, the eyes were examined again using UV-light to detect possible corneal damage. Eyes were also examined 48 and 72 h after the treatment. Ocular reactions were graded according to the method of Draize (1977).

One hour after application of the test material, all animals showed some conjunctival redness, chemosis and discharge different from normal. After 24 h, two animals showed some conjunctival vessels definitely injected. After 48 and 72 h all animals were free of any signs of eye irritation. The calculated mean scores for each individual lesions for all animal within three scoring times (24, 48 and 72 h) were as follow: 0.00 for cornea score; 0.00 for iris score; 0.17 for conjunctivae score and 0.00 for chemosis score, respectively. In this study, γ-Nonalactone is not an eye irritant on female rabbits.

Under the test conditions, γ-Nonalactone is not classified as irritating to eyes according to the Directive 67/548/EEC and the Regulation (EC) No 1272-2008 (CLP).

This study is considered as acceptable and satisfies the requirement for eye irritation endpoint.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation:

No study were located on ɣ-Decalactone, therefore ɣ-Nonalactone and ɣ-Undecalactone data were used to evaluate the skin irritation potential of ɣ-Decalactone. This approach is based on the similarity between aliphatic γ-lactones (see §"Toxicokinetics" for read-across justification).

The study conducted on ɣ-Nonalactone is considered as the key study (Maillet, 2012, rel.2). The skin irritation potential of ɣ-Nonalactone was evaluated using the EPISKIN® reconstructed human epidermis model. The study was conducted according to the OECD Guideline 437 and in compliance with GLP. The relative mean viability of the test item treated tissues was 74.6 ± 2.8 % after the 15‑minute exposure period. With a viability > 50 %,γ-Nonalactone is not a skin irritant.

This result is supported by the absence of skin irritation up to the limit dose of 2000 mg/kg bw in an acute toxicity study by dermal route using γ-Undecalactone (Sanders, 1999, rel.2).

Based on the read-across data, γ-Decalactone is not considered as a skin irritant.

Eye irritation:

No study was available to evaluate the eye irritation potential of γ-Decalactone. However reliable studies were conducted on surrounding lactones, i.e. γ-Nonalactone and γ-Undecalactone. Therefore, a weight of evidence approach, based on the similarity between aliphatic γ-lactones (see §"Toxicokinetics" for read-across justification), was used to evaluate the eye irritation potential of γ-Decalactone.

Both studies were conducted according to the OECD test guideline No.405 and in compliance were GLP (Schreiter, 1999, rel.2). In these eye irritation studies, rabbits were exposed to 0.1 mL of γ-Nonalactone or γ-Undecalactone in their left eye, while the right eye remained untreated and served as control. The calculated mean scores for γ-Decalactone and γ-Undecalactone for each individual lesion for all animals within three scoring times (24, 48 and 72 h) were as follows: 0.00 for cornea score; 0.00 for iris score; 0.00 for conjunctivae score and 0.00 for chemosis score, respectively. In these studies, neither of the two substances was an eye irritant.

Based on the read-across data, γ-Decalactone is not considered as an eye irritant.

Justification for selection of skin irritation / corrosion endpoint:
No study were located on ɣ-Decalactone, therefore ɣ-Nonalactone and ɣ-Undecalactone data were used to evaluate the skin irritation potential of ɣ-Decalactone. This approach is based on the similarity between aliphatic γ-lactones (see §"Toxicokinetics" for read-across justification).

Justification for selection of eye irritation endpoint:
No study was available to evaluate the eye irritation potential of γ-Decalactone. However reliable studies were conducted on surrounding lactones, i.e. γ-Nonalactone and γ-Undecalactone. Therefore, a weight of evidence approach, based on the similarity between aliphatic γ-lactones (see §"Toxicokinetics" for read-across justification), was used to evaluate the eye irritation potential of γ-Decalactone.

Justification for classification or non-classification

Harmonized classification:

γ-Decalactone has no harmonized classification according to the Regulation (EC) No. 1272/2008 including ATP2.

Self classification:

Based on the available data no additional self-classification is proposed regarding both skin and eye irritation according to the Regulation (EC) No. 1272/2008 (CLP) and of the Directive 67/548/EEC.