[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 June 2008 to 16 June 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EPISKIN-SMTM Model supplied by SkinEthic Laboratories, Nice, France

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Pre-dates guideline inception

Deviations:

yes

Remarks:

Longer incubation time

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Signature: 15/10/2007; Date of Inspection: 21/08/2007

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of disodium N-(1-oxooctadecyl)-L-glutamate and disodium N-(1-oxohexadecyl)-L-glutamate

EC Number:

700-014-3

Molecular formula:

See remarks

IUPAC Name:

Reaction mass of disodium N-(1-oxooctadecyl)-L-glutamate and disodium N-(1-oxohexadecyl)-L-glutamate

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg of the test material was applied to the epidermis surface.

VEHICLE
No vehicle was used.

Duration of treatment / exposure:

The EPISKINTM reconstituted human epidermis model was subjected to treatment for a period of 15 minutes.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: OD540 Value

Value:

0.78

Remarks on result:

other:

Remarks:

Basis: mean Negative Control. Time point: 15 minutes. Max. score: 0.8. Remarks: Standard Deviation 0.014. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: OD540 value

Value:

0.09

Remarks on result:

other:

Remarks:

Basis: mean Positive control. Time point: 15 minutes. Max. score: 0.11. Remarks: Standard deviation 0.026. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: OD540 value

Value:

0.81

Remarks on result:

other:

Remarks:

Basis: mean Test Material. Time point: 15 minutes. Max. score: 0.84. Remarks: Standard deviation 0.03. (migrated information)

Results 4

Irritation / corrosion parameter:

other: other: Relative mean tissue viability (%)

Value:

100

Remarks on result:

other:

Remarks:

Basis: mean Negative control. Time point: 15 minutes. Max. score: 102.0. Remarks: Standard deviation 1.81. (migrated information)

Results 5

Irritation / corrosion parameter:

other: other: Relative mean Tissue viability (%)

Value:

11.9

Remarks on result:

other:

Remarks:

Basis: mean Positive control. Time point: 15 minutes. Max. score: 13.9. Remarks: Standard deviation 3.35. (migrated information)

Results 6

Irritation / corrosion parameter:

other: other: Relative mean tissue viability (%)

Value:

103.2

Remarks on result:

other:

Remarks:

Basis: mean Test material. Time point: 15 minutes. Max. score: 107.3. Remarks: Standard deviation 3.87. (migrated information)

Any other information on results incl. tables

RESULTS

Direct MTT Reduction

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

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD₅₄₀ values, standard deviations and tissue viabilities for the negative control, test material and positive control 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 103.2 % after a 15‑minute exposure.

The qualitative evaluation of tissue viability is given in Table 2.

Following the 15-minute exposure the test material treated tissues appeared blue which was considered indicative of viable tissue.

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 (SD) value of the % viability was ≤20 %. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₄₀ for the negative control treated tissues was ≥0.6 and the SD value of the % viability was ≤20 %. The negative control acceptance criterion was therefore satisfied.

Table 1:  Mean OD₅₄₀ Values and % Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material	OD540 of tissues	Mean OD540 of triplicate tissues	± SD of OD540	Relative individual tissue viability	Relative mean % viability	± SD of % viability
Negative Control Material Ä	0.799	0.783	0.014	102.0	100*	1.81
	0.772			98.6
	0.777			99.2
Positive Control Material Ä	0.107	0.093	0.026	13.7	11.9	3.35
	0.109			13.9
	0.063			8.0
Test Material	0.780	0.808	0.030	99.6	103.2	3.87
	0.805			102.8
	0.840			107.3

 

Table 2:  Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Tissue 1	Tissue 2	Tissue 3
Negative Control Material Ä	-	-	-
Positive Control Material Ä	+	+	+
Test Material	-	-	-

MTT visual scoring scheme
- = blue tissue (viable)
+ = blue/white tissue (semi-viable)
++ = tissue is completely white (dead)

* = The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the test, the relative mean viability of the test material treated tissues was 103.2 % after a 15 minute exposure. Since this was > 50 % the test material is considered to be a non-irritant, in accordance with the criteria for classification set out in the study.

Executive summary:

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. The concentration of the inflammatory mediator IL-1α 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.

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 % viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Under the conditions of the test, the relative mean viability of the test material treated tissues was 103.2 % after a 15 minute exposure. Since this was > 50 % the test material is considered to be a non-irritant.