N-methyl-N-[C18-(unsaturated)alkanoyl]glycine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Dec 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-methyl-N-[C18-(unsaturated)alkanoyl]glycine

EC Number:

701-177-3

Molecular formula:

C21H39NO3

IUPAC Name:

N-methyl-N-[C18-(unsaturated)alkanoyl]glycine

Test animals

Species:

human

Strain:

other: reconstructed human epidermis EST-200

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: MatTek Corporation, Ashland, USA
- Before the start of the test, the skin models were pre-incubated for 1 h under culture conditions (5% CO2, 37 °C) with assay medium. The medium was changed and the test and reference substances were applicated (50 µL/insert) onto the skin model.

ENVIRONMENTAL CONDITIONS (Incubator)
- Temperature (°C): 37
- CO2 gas concentration (%): 5

Test system

Type of coverage:

open

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent negative control tissue treated with distilled water

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.05 mL (ca. 83 µL/cm²)

Duration of treatment / exposure:

3 min and 1 h

Observation period:

Not applicable

Number of animals:

Not applicable. Tests were performed in triplets for each treatment duration.

Details on study design:

TEST SITE
- Area of exposure: 0.6 cm²

REMOVAL OF TEST SUBSTANCE
- Washing (if done): after the incubation periods, the inserts were washed carefully
- Time after start of exposure: 3 min and 1 h

CELL VIABILITY MEASUREMENTS
- Method: MTT assay
- Details on method used: after exposure to the test substance, the skin model was incubated with MTT solution for 3 h. Thereafter, the inserts were washed 3 times with PBS. For viability testing, the inserts were placed in new 24-well plates and the extraction of blue formazan was performed in the extraction solution on a shaker for 120 min (120 U/min) at room temperature. For determination of cell viability, the solution was homogenized and 3 x 200 µL were inserted into a 96 well plate. The absorption was measured in duplicates at 570 nm in a plate reader.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

72

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

25

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table 1. Results of the MTT assay.

Test substance	Tissue	OD					Cell viability (% of negative control)
		Value 1	Value 2	Value 3	Mean	Mean (tissue 1+2)
Incubation time 3 min
Negative control	1	2.007	1.993	2.010	2.003	2.031	100
	2	2.067	2.032	2.074	2.058
Test substance	1	1.263	1.250	1.292	1.268	1.454	72
	2	1.638	1.635	1.648	1.640
Positive control	1	0.545	0.527	0.533	0.535	0.485	24
	2	0.438	0.431	0.436	0.435
Incubation time 60 min
Negative control	1	1.841	1.848	1.853	1.847	1.755	100
	2	1.687	1.640	1.658	1.662
Test substance	1	0.481	0.457	0.461	0.466	0.445	25
	2	0.426	0.422	0.421	0.423
Positive control	1	0.168	0.163	0.191	0.174	0.213	12
	2	0.251	0.250	0.252	0.251

OD: optical density

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin cor. 1, 1A, 1B/C) based on a positive result in the Reconstructed human epidermis test method (in vitro skin corrosion). A negative in vitro corrosivity response is not conclusive with respect to non-classification or classification as a skin irritant and therefore requires further evaluation and/or data generation.