Lithium isooctadecanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 1 March 2017 and 01 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Lithium isooctadecanoate

EC Number:

283-815-6

EC Name:

Lithium isooctadecanoate

Cas Number:

84731-55-5

Molecular formula:

C18H35O2.Li

IUPAC Name:

Lithium isooctadecanoate

Constituent 2

Reference substance name:

Isooctadecanoic acid

EC Number:

250-178-0

EC Name:

Isooctadecanoic acid

Cas Number:

30399-84-9

Molecular formula:

C18H36O2

IUPAC Name:

Isooctadecanoic acid

Constituent 3

Reference substance name:

Lithium laurate

EC Number:

238-663-5

EC Name:

Lithium laurate

Cas Number:

14622-13-0

Molecular formula:

C12H24O2.Li

IUPAC Name:

lithium laurate

Constituent 4

Reference substance name:

Lithium myristate

EC Number:

243-743-8

EC Name:

Lithium myristate

Cas Number:

20336-96-3

Molecular formula:

C14H27O2.Li

IUPAC Name:

lithium myristate

Constituent 5

Reference substance name:

Lithium palmitate

EC Number:

243-842-6

EC Name:

Lithium palmitate

Cas Number:

20466-33-5

Molecular formula:

C16H32O2.Li

IUPAC Name:

lithium palmitate

Constituent 6

Reference substance name:

Lithium nonadecanoate

Molecular formula:

C19H37O2Li

IUPAC Name:

Lithium nonadecanoate

Constituent 7

Reference substance name:

Lithium icosanoate

EC Number:

257-088-0

EC Name:

Lithium icosanoate

Cas Number:

51250-21-6

Molecular formula:

C20H39O2.Li

IUPAC Name:

lithium icosanoate

Test material form:

solid

Details on test material:

- Batch number: A104/99
- Expiration date: 30 November 2018
- Storage condition of test material: At room temperature, protected from light

Specific details on test material used for the study:

- Stability under test conditions: Stable
- Treatment of test material prior to testing: None
- Preliminary purification step: None
- Final preparation of a solid: Applied neat

In vitro test system

Test system:

other: EPISKIN™ Reconstructed Human Epidermis Model

Source species:

other: EPISKIN™ Reconstructed Human Epidermis Model

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN™ Reconstructed Human Epidermis Model

Source strain:

other: EPISKIN™ Reconstructed Human Epidermis Model

Details on animal used as source of test system:

The EPISKINTM 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.

Justification for test system used:

This is the test system described in OECD 439. Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items. The procedure followed is based on the recommended EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 17-EKIN-017
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: 25 April 2017
- Date of initiation of testing: 1 March 2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 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
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: None
- Filter bandwidth: Not applicable
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Not specified
- Barrier function: Not specified
- Morphology: Not specified
- Contamination: Not specified
- Reproducibility: Not specified

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: water-killed tissues
- N. of replicates: Not specified
- Method of calculation used: Not specified

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be an irritant to skin if the relative mean tissue viability after 15-Minute exposure period followed by the 42-Hour post-exposure incubation period is less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15-Minute exposure period followed by the 42-Hour post-exposure incubation period is greater than or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: Appropriately sized discs of the test item were applied
- Concentration: Test material is solid

VEHICLE: Not applicable

NEGATIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration: 0.3 mg/mL in DPBS

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration: 5% w/v aqueous solution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Test animals

Species:

other: reconstituted human epidermis model

Strain:

other: reconstituted human epidermis model

Details on test animals or test system and environmental conditions:

The plates were kept in the biological safety cabinet at room temperature for 15 minutes. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 hours.

Test system

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- The test material was applied neat.
- Amount(s) applied (volume or weight with unit): Appropriately sized discs of the test item were applied
- Application method: Prior to the test item being applied 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Appropriately sized discs of the test item were applied to the epidermis surface ensuring uniform covering.
- Concentration: The test material was used as supplied.

VEHICLE: No vehicle used

Duration of treatment / exposure:

15 Minutes & 42 hour post exposure incubation

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

TEST SITE
- Area of exposure:Appropriately sized discs of the test item were applied
- % coverage: The test material was applied topically to the corresponding tissues ensuring uniform covering.
- Type of wrap if used: None used

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 Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material.
- Time after start of exposure: 15 Minutes post exposure

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
For the test material, the relative mean tissue viabilities obtained after the 15 minute treatment 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:

mean OD570 of test material / mean OD570 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is =50% : Irritant (I) H315 Category 2

Mean tissue viability is >50% : Non-Irritant (NI) Not classified. Category 3 cannot be determined

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Relative mean viability

Value:

89.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal.
The relative mean tissue viability for the positive control treated tissues was 13.8% relative to the negative control treated tissues and the standard deviation value of the viability was 15.4%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.666 and the standard deviation value of the viability was 12.6%. The negative control acceptance criteria were therefore satisfied.

Any other information on results incl. tables

RESULTS

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD₅₇₀ values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test material treated tissues was 89.3% after a 15 -minute exposure.

Quality Criteria

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 was =20%. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₇₀ for the negative control treated tissues was =0.6 and the SD value of the percentage viability was =20%. The negative control acceptance criterion was therefore satisfied.

Table 1 : Mean OD₅₇₀ Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material	OD570 of tissues	Mean OD570 of triplicate tissues	±SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Material	0.761	0.666	0.084	114.3	100*	12.6
	0.636			95.5
	0.602			90.4
Positive Control Material	0.209	0.092	0.102	31.4	13.8	15.4
	0.048			7.2
	0.019			2.9
Test Material	0.605	0.595	0.059	90.8	89.3	8.9
	0.648			97.3
	0.531			79.7

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:

not irritating

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

The test material was considered to be Non-Irritant (NI)

Executive summary:

Introduction:

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN^TM reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was 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. 

Methods:

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 approximately 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 540 nm. Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Results: 

The relative mean viability of the test material treated tissues was 89.3% after a 15-minute exposure.

Quality criteria: 

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

Conclusion:  The test material was considered to be Non-Irritant (NI).