Reaction mass of L-Glutamic acid, N-coco acyl derivs., monosodium salts and sodium hydrogen N-(1-oxooctadecyl)-L-glutamate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 10 June 2008 and 16 June 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Remarks:

Study conducted on read-across material

Justification for type of information:

A RAAF report will shortly be provided.

Cross-reference

Reason / purpose for cross-reference:

other: read across target

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

A RAAF report will shortly be provided.

Reason / purpose for cross-reference:

read-across source

Irritation / corrosion parameter:

other: Assessment of direct test material production of MTT (tissue viability)

Value:

76.3

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes. Max. score: 100.0. Reversibility: no data. (migrated information)

Irritation / corrosion parameter:

other: Absorbance (540 nm)

Value:

0.598

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes. Max. score: 0.783. Reversibility: no data. (migrated information)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2009

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

(pre-dates inception of guideline)

Deviations:

yes

Remarks:

Longer incubation time

Principles of method if other than guideline:

Performed using the protocol supplied with the in vitro model (EpiSkin-SM Model supplied by SkinEthic Laboratories, Nice, France)

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 sodium hydrogen N-(1-oxooctadecyl)-L-glutamate and sodium hydrogen N-(1-oxohexadecyl)-L-glutamate

EC Number:

936-610-7

Molecular formula:

See remarks

IUPAC Name:

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

Constituent 2

Reference substance name:

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

IUPAC Name:

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

Test animals

Details on test animals or test system and environmental conditions:

An in vitro study, therefore no animals used.

Test system

Type of coverage:

other: not applicable for this type of study

Preparation of test site:

other: not applicable for this type of study

Vehicle:

unchanged (no vehicle)

Controls:

other: Triplicate tissues treated with 10 µl of Phosphate Buffered Saline Dulbeccos (PBS) were used to serve as negative controls. Triplicate tissues treated with 10 µl of SDS 5% w/v were used to serve as positive controls.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg of the test material was applied to the epidermis surface.
- Concentration (if solution): not applicable


VEHICLE
- Amount(s) applied (volume or weight with unit): not applicable
- Concentration (if solution): not applicable
- Lot/batch no. (if required): not applicable
- Purity: not applicable

Duration of treatment / exposure:

Exposure period of 15 minutes.

Observation period:

42-hour post-exposure incubation period

Number of animals:

Triplicate tissues were treated with the test material

Details on study design:

TEST SITE
- Area of exposure: total exposure
- % coverage: 100 %
- Type of wrap if used: not applicable


REMOVAL OF TEST SUBSTANCE
- Washing (if done): rinsed with PBS
- Time after start of exposure: 15 minutes


SCORING SYSTEM: The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: Assessment of direct test material production of MTT (tissue viability)

Value:

76.3

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes. Max. score: 100.0. Reversibility: no data. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: Absorbance (540 nm)

Value:

0.598

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes. Max. score: 0.783. Reversibility: no data. (migrated information)

Any other information on results incl. tables

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 76.3% after a 15‑minute exposure.

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

Following the 15-minute exposure two of the test material treated tissues appeared blue which was considered indicative of viable tissue and one of the test material treated tissues appeared blue/white which was considered indicative of semi-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.

Table1. Mean OD₅₄₀ Values and % Viabilities for the Negative Control Material, Positive Control Material and Test Material.

Material	OD540of tissues	Mean OD540of triplicate tissues	± SD of OD540	Relative individual tissue viability	Relative mean % viability	± SD of % viability
Negative Control MaterialÄ	0.799	0.783	0.014	102.0	100*	1.81
	0.772			98.6
	0.777			99.2
Positive Control MaterialÄ	0.107	0.093	0.026	13.7	11.9	3.35
	0.109			13.9
	0.063			8.0
Test Material	0.339	0.598	0.238	43.3	76.3	30.3
	0.648			82.8
	0.806			102.9

Ä= Control group shared with another study

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

Table2.Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation).

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

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

Ä= Control group shared with SPL Project number 1071/0025

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test material was considered to be Non-Irritant.

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. 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.

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 % 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 76.3% 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.