Oils, fish, oxidized, bisulfited, sodium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 13-19, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to well-documented and supported procedures for which no guideline (OECD, etc.) exists

Justification for data waiving:

other:

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material for 15 minutes and incubation for 42 hours by means of the colourmetric MTT reduction assay.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Oils, fish, oxidized, bisulfited, sodium salts

EC Number:

307-037-4

EC Name:

Oils, fish, oxidized, bisulfited, sodium salts

Cas Number:

97488-98-7

Molecular formula:

Not applicable

IUPAC Name:

not available

Details on test material:

- Name of test material (as cited in study report): FLL sample 3
- Chemical name: Oils, fish, oxidized, bisulfited, sodium salts (triglycerides, C12 to C24, even, saturated and unsaturated, bisulfited, sodium salt)
- Physical state: viscous liquid
- Purity: 90%
- Impurities: 10% water content by Karl Fischer
- Lot/batch No.: LS06921
- Expiration date of the lot/batch: 13 October 2010
- Storage condition of test material: room temperature in the dark
- Other: amber color
- All other template details: Not reported

Test animals

Species:

other: reconstituted human epidermis model

Details on test animals or test system and environmental conditions:

TEST ANIMALS - EPISKIN Model Kit
- Source: SkinEthic Laboratories, Nice, France
- Date received: 13 April 2010
- All other template details: Not applicable

ENVIRONMENTAL CONDITIONS: Not applicable

IN-LIFE DATES: Not applicable

Test system

Type of coverage:

other: uniform topical coverage

Preparation of test site:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 µl

VEHICLE: Not applicable

Duration of treatment / exposure:

15 minutes

Observation period:

Not applicable

Number of animals:

Three tissues per test material, positive control, and negative control for a total of 9 skin models analyzed

Details on study design:

TEST SITE
- Area of exposure: entire EPISKIN tissue
- % coverage: 100
- Type of wrap if used: Not applicable

REMOVAL OF TEST SUBSTANCE
- Washing: rinsed with dulbeccos phosphate buffered saline (PBS) with calcium and magnesium
- Time after start of exposure: at the end of the 15 minute exposure period

SCORING SYSTEM: optical density at 540 nm was measured and divided by the negative control optical density and multiplied by 100 to obtain a percentage for the relative mean viability.
- If the relative mean tissue viability was less or equal to 50%, the compound is predicted to be an irritant.
- If the relative mean tissue viability was greater than 50%, the compound is predicted not to be an irritant.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: relative mean viability based on OD540 measurements

Value:

103.9

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes. Max. score: 106.7. Reversibility: other: not applicable. Remarks: scores are percentages of mean negative control OD540 values . (migrated information)

In vivo

Irritant / corrosive response data:

See Table 1 for raw data values

Other effects:

Test material was shown to directly reduce MTT. However, rinsing was effective at removing the test material on or in the tissue following each exposure period and no degree of interference was noted.

Any other information on results incl. tables

Table 1. Individual and Mean OD540 Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material.

Material	OD540of tissues	Mean OD540of triplicate tissues (±SD)	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative control material	0.638	0.629 (0.008)	101.4	100	1.2
	0.623		99.0
	0.627		99.7
Positive control material	0.194	0.175 (0.016)	30.8	27.9	2.6
	0.164		26.1
	0.168		26.7
Test material	0.653	0.653 (0.018)	103.8	103.9	2.8
	0.636		101.1
	0.671		106.7

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material was considered to be a non-irritant to the skin.

Executive summary:

Summary from data report:

Introduction: The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN^TMreconstituted 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 colourmetric MTT reduction assay. Cell viability is measured by enzymatic reduction of 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. The concentration of the inflammatory mediator IL-1alpha in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

 

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 incubation 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 200µl 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 the negative control tissues).

 

Results: The relative mean viability of the test material treated tissues was 103.9% 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.