Commission Regulation (EC) No 761/2009Show full title

PERFORMANCE STANDARDS

The Performance Standards comprise the following three elements I) Essential Test Method Components, II) Reference Substances and III) Defined Accuracy and Reliability Values (2). These Performance Standards are based on the Performance Standards defined after the completion of the ECVAM skin irritation validation study (3).

(I) Essential Test Method Components

General model conditions

Normal human keratinocytes should be used to construct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) should be present under a functional stratum corneum. Stratum corneum should be multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic marker substances, e.g. SDS or Triton X-100. The barrier function may be assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50 % (IC₅₀) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50 % (ET₅₀) upon application of the marker substance at a specified, fixed concentration. The containment properties of the model should prevent the passage of material around the stratum corneum to the viable tissue, which would lead to poor modelling of skin exposure. The skin model should be free of contamination by bacteria, viruses, mycoplasma, or fungi.

Functional model conditions

Viability

The preferred assay for determining the magnitude of viability is the MTT (4). The OD of the extracted (solubilised) dye from the tissue treated with NC should be at least 20 fold greater than the OD of the extraction solvent alone. It should be documented that the tissue treated with NC is stable in culture (provide similar viability measurements) for the duration of the test exposure period.

Barrier function

The stratum corneum and its lipid composition should be sufficient to resist the rapid penetration of cytotoxic marker substances, e.g. SDS or Triton X-100, as estimated by IC₅₀ or ET₅₀.

Morphology

Histological examination of the reconstructed skin/epidermis should be performed by appropriately qualified personnel demonstrating human skin/epidermis-like structure (including multilayered stratum corneum).

Reproducibility

The results of the method using a specific model should demonstrate reproducibility over time, preferably by an appropriate batch control (benchmark) substance (see definitions in section 1.2).

Quality controls (QC) of the model

Each batch of the epidermal model used should meet defined production release criteria, among which those for viability and for barrier function are the most relevant. An acceptability range (upper and lower limit) for the IC₅₀ or the ET₅₀ should be established by the skin model supplier (or investigator when using an in-house model). The barrier properties of the tissues should be verified by the laboratory after receipt of the tissues. Only results produced with qualified tissues can be accepted for reliable prediction of irritation effects. As an example, the acceptability ranges for the validated reference methods are given below.

Table 1

Examples of QC batch release criteria

	lower acceptance limit	mean of acceptance range	upper acceptance limit
Validated reference method 1 (18 hours treatment with SDS)	IC50 = 1,0 mg/ml	IC50 = 2,32 mg/ml	IC50 = 3,0 mg/ml
Validated reference method 2 (1 % Triton X-100)	ET50 = 4,8 hr	ET50 = 6,7 hr	ET50 = 8,7 hr

(II) Reference Substances

Reference substances are used to determine if the reliability and accuracy of a proposed novel in vitro reconstructed human epidermis test method, proven to be structurally and functionally sufficiently similar to the validated reference methods, or representing a minor modification of a validated reference method, shows comparable performance to that of the validated reference method 1 (1). The 20 reference substances listed in Table 2 include substances representing different chemical classes of interest, as well as substances in UN GHS category 2. The substances included in this list comprise 10 UN GHS category 2 substances, 3 UN GHS optional category 3 substances and 7 non-categorised substances. Under this Test Method, the optional category 3 is considered as no category. These reference substances represent the minimum number of substances that should be used to evaluate the accuracy and reliability of a proposed reconstructed human epidermis test method for skin irritation. In situations where a listed substance is unavailable, other substances for which adequate in vivo reference data are available could be used. If desired, additional substances representing other chemical classes and for which adequate in vivo reference data are available may be added to the minimum list of reference substances to further evaluate the accuracy of the proposed test method.

Table 2

Reference Substances for Determination of Accuracy and Reliability Values for Reconstructed Human Epidermis Skin Irritation Models

a The 20 reference substances comprise a representative selection from the 58 substances which were originally used to validate reference method 1 (EpiSkin™). A complete list of test substances and the criteria for their selection are available (5).
Substancea	CAS No	EINECS No	Physical state	In vivo score	GHS in vitro cat.	GHS in vivo cat.
1-bromo-4-chlorobutane	6940-78-9	230-089-3	L	0	Cat. 2	No Cat.
Diethyl phthalate	84-66-2	201-550-6	L	0	No Cat.	No Cat.
Naphthalene acetic acid	86-87-3	201-705-8	S	0	No Cat.	No Cat.
Allyl phenoxy-acetate	7493-74-5	231-335-2	L	0,3	No Cat.	No Cat.
Isopropanol	67-63-0	200-661-7	L	0,3	No Cat.	No Cat.
4-methyl-thio-benzaldehyde	3446-89-7	222-365-7	L	1	Cat. 2	No Cat.
Methyl stearate	112-61-8	203-990-4	S	1	No Cat.	No Cat.
Heptyl butyrate	5870-93-9	227-526-5	L	1,7	No Cat.	Optional Cat. 3
Hexyl salicylate	6259-76-3	228-408-6	L	2	No Cat.	Optional Cat. 3
Tri-isobutyl phosphate	126-71-6	204-798-3	L	2	Cat. 2	Optional Cat. 3
1-decanol	112-30-1	203-956-9	L	2,3	Cat. 2	Cat. 2
Cyclamen aldehyde	103-95-7	203-161-7	L	2,3	Cat. 2	Cat. 2
1-bromohexane	111-25-1	203-850-2	L	2,7	Cat. 2	Cat. 2
2-chloromethyl-3,5-dimethyl-4-methoxypyridine hydrochloride	86604-75-3	434-680-9	S	2,7	Cat. 2	Cat. 2
a-terpineol	98-55-5	202-680-6	L	2,7	Cat. 2	Cat. 2
di-n-propyl disulphide	629-19-6	211-079-8	L	3	No Cat.	Cat. 2
Butyl methacrylate	97-88-1	202-615-1	L	3	Cat. 2	Cat. 2
Benzenethiol, 5-(1,1-dimethylethyl)-2-methyl	7340-90-1	438-520-9	L	3,3	Cat. 2	Cat. 2
1-methyl-3-phenyl-1-piperazine	5271-27-2	431-180-2	S	3,3	Cat. 2	Cat. 2
Heptanal	111-71-7	203-898-4	L	4	Cat. 2	Cat. 2

The substances listed in Table 2 provide a representative distribution of the 58 substances used in the ECVAM international skin irritation validation study (1). Their selection is based on the following criteria:

• the substances are commercially available,

• they are representative of the full range of Draize irritancy scores (from non-irritant to strong irritant),

• they have a well-defined chemical structure,

• they are representative of the validated method’s reproducibility and predictive capacity as determined in the ECVAM validation study,

• they are representative of the chemical functionality used in the validation process,

• they are not associated with an extremely toxic profile (e.g. carcinogenic or toxic to the reproductive system) and they are not associated with prohibitive disposal costs.

(III) Defined Accuracy and Reliability Values

The performance (sensitivity, specificity, false negative rate, false positive rate and accuracy) of the proposed test method should be comparable to that of the validated reference method 1 (Table 3), i.e. sensitivity should be equal or higher (≥) than 80 %, specificity should be equal or higher (≥) than 70 %, and accuracy should be equal or higher (≥) than 75 %. The calculation of the performance should be done using all classifications obtained for the 20 substances in the different participating laboratories. The classification for each substance in each laboratory should be obtained by using the mean value of viability over the different runs performed (minimum three valid runs).

Table 3

Performance of the Validated Reference Method 1^a

a Table 3 provides the performance of the validated reference method 1, with regard to its ability to correctly identify irritant substances (UN GHS category 2) and non-classified substances (no category including optional category 3) for the 58 and 20 Reference Substances (Table 2), respectively.
b EpiSkin™
c Based on 13 GHS cat. 2 irritants.
d Based on 45 GHS cat. 3 irritants or GHS no category chemicals.
Test method	No. of Substances	Sensitivity	Specificity	False Negative Rate	False Positive rate	Accuracy
Validated Reference Method 1b	58	87,2 %c	71,1 %d	12,8 %	29,9 %	74,7 %
Validated Reference Method 1b	20	90 %	73,3 %	10 %	26,7 %	81,7 %

The reliability of the proposed test method should be comparable to that of the validated reference methods.

Within-laboratory reproducibility

An assessment of within-laboratory variability should show a concordance of classifications (category 2/no category) obtained in different, independent test runs of the 20 Reference Substances within one single laboratory equal or higher (≥) than 90 %.

Between-laboratory reproducibility

An assessment of between-laboratory reproducibility is not essential if the proposed test method is to be used in one laboratory only. For methods to be transferred between laboratories, the concordance of classifications (category 2/no category) obtained in different, independent test runs of the 20 Reference Substances between preferentially a minimum of three laboratories should be equal or higher (≥) than 80 %.