The EpiDerm Test Protocol for the Upcoming ECVAM Validation Study on In Vitro Skin Irritation Tests — An Assessment of the Performance of the Optimised Test

Abstract

During the past decade, several validation studies have been conducted on in vitro methods for discriminating between skin irritating and non-irritating chemicals. The reconstructed human skin models, EpiDerm and EPISKIN, provided the most promising results. Based on experience of the similar performance of the two skin models, it was suggested that a common test protocol and prediction model should be developed for the prediction of skin irritation potential with the two models. When the EPISKIN protocol was applied with the EpiDerm model, an acceptable specificity (80%) was achieved, whereas the sensitivity (60%) was low. In 2003, the EPISKIN protocol was further refined by extending the post-incubation period following exposure to test chemicals. This extension and additional technical improvements to the EpiDerm protocol were evaluated with 19 chemicals from the prevalidation study. With the new test design, high sensitivity (80%) and specificity (78%) were obtained. The statistical probability for correct classifications was high, so the test was considered to be ready for formal validation. However, since test optimisation had been conducted with the same test chemicals as were used in the ECVAM prevalidation study, it was decided that the optimisation of the protocol had to be verified with a new set of chemicals. Thus, in the current study, 26 additional chemicals (10 rabbit irritants and 16 non-irritants), which had previously been selected and tested by L'ORΈAL with EPISKIN, were evaluated in three independent experiments with EpiDerm. With this unbalanced testing set, a specificity of 94%, and a sensitivity of 60% were obtained, while the positive and negative predictivity and accuracy remained almost unchanged (around 80%) in comparison to the in vivo rabbit data. Overall, 45 chemicals (20 irritants and 25 non-irritants) were tested according to the final protocol. The resulting high positive (82%) and negative predictive values (79%) confirmed the reliability (accuracy of 80%) of the improved test protocol of the EpiDerm model.

Keywords

alternative methods common protocol ECVAM EpiDerm prevalidation skin irritation validation

Get full access to this article

View all access options for this article.

References

Botham

P.A.

, Earl

L.K.

, Fentem

J.H.

, Roguet

, & van de Sandt

J.J.M.

(1998). Alternative methods for skin irritation testing: the current status. ECVAM Skin Irritation Task Force report 1. ATLA 26, 195–211.

OECD (2004). Guidelines for the Testing of Chemicals, No. 431: In Vitro Skin Corrosion: Human Skin Model Test. 8 pp. Paris, France: OECD.

Zuang

, Balls

, Botham

, Coquette

, Corsini

, Curren

R.D.

, Elliott

G.R.

, Fentem

J.H.

, Heylings

J.R.

, Liebsch

, Medina

, Roguet

, van de Sandt

J.J.M.

, Wiemann

, & Worth

A.P.

(2002). Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. ECVAM Skin Irritation Task Force Report 2. ATLA 30, 109–129.

Liebsch

, Barrabas

, Traue

, & Spielmann

(1997). Entwicklung eines neuen in vitro Tests auf dermale Phototoxizitat mit einem Modell menschlicher Epidermis (EpiDerm™). ALTEX 14, 165–174.

Liebsch

, Traue

, Barrabas

, Spielmann

, Upholl

, Wilkins

, McPherson

J.P.

, Wiemann

, Kaufmann

, Remmele

, & Holzhütter

H.-G.

(2000). The ECVAM prevalidation study on the use of EpiDerm for skin corrosivity testing. ATLA 28, 371–401.

Portes

, Grandidier

M.H.

, Cohen

, & Roguet

(2002). Refinement of the Episkin protocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidation study. Toxicology in Vitro 16, 765–770.

Kandárová

, Liebsch

, Genschow

, Gerner

, Traue

, Slawik

, & Spielmann

(2004). Optimisation of the EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation tests. ALTEX 21, 107–114.

Cotovio

, Grandidier

M.-H.

, Portes

, Rouguet

, & Rubinsteen

(2005). In vitro assessment of the acute skin irritation of chemicals: Optimisation of the EPISKIN prediction model within the framework of the ECVAM validation process. ATLA 33, 329–349.

OECD (2002). Guideline for the Testing of Chemicals, No. 404: Acute Dermal Irritation/Corrosion. 13 pp. Paris, France: OECD.

10.

European Commission (1967). Council Directive 67/548/EEC of 27 June 1967 on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances. Official Journal of the European Communities 196, 1–98.

11.

United Nations Economic Commission for Europe (2003). The Globally Harmonized System of Classification and Labelling of Chemicals (GHS). UN ST/SG/AC.10/30, United Nations, New York, NY, USA/Geneva, Switzerland: United Nations Organisation. Website http://www.unece.org/trans/danger/publi/ghs (Accessed 15.8.03).

12.

Cannon

C.L.

, Neal

P.J.

, Southee

J.A.

, Kubilus

, & Klausner

(1994). New epidermal model for dermal irritancy testing. Toxicology in Vitro 8, 889–891.

13.

Ponec

, Boelsma

, Weerheim

, Mulder

, Bouwstra

, & Mommaas

(2000). Lipid and ultrastructural characterization of reconstructed skin models. International Journal of Pharmaceutics 203, 211–225.

14.

Fentem

J.H.

, Briggs

, Chesné

, Elliott

G.R.

, Harbell

J.W.

, Heylings

J.R.

, Portes

, Roguet

, van de Sandt

J.J.M.

, & Botham

P.A.

(2001). A prevalidation study on in vitro tests for acute skin irritation: results and evaluation by the Management Team. Toxicology in Vitro 15, 57–93.

15.

Balls

, Blaauboer

, Brusick

, Fraizer

, Lamb

, Pemberton

, Reinhardt

, Roberfroid

, Rosenkranz

, Schmid

, Spielmann

, Stanmmati

A-L.

, & Waulm

(1990). Report and recommendations of the CAAT/ERGATT workshop on the validation of toxicity test procedures. ATLA 18, 313–337.

16.

Worth

A.P.

, & Balls

(2001). The importance of the prediction model in the validation of alternative tests. ATLA 29, 135–143.

17.

Combes

R.D.

, & Balls

(2003). How much flexibility is possible when validating new in vivo and in vitro test methods? ATLA 31, 225–232.

18.

ECETOC (1995). Skin Irritation and Corrosion: Reference Chemicals Data Bank. ECETOC Technical Report No. 66. 247 pp. Brussels, Belgium: European Centre for Ecotoxicology and Toxicology of Chemicals.

19.

Mossman

(1983). Rapid colorimetric assay for cell growth and survival-application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55–63.

20.

Norušis

M.J.

(1994). SPSS Professional Statistics 6.1. Chicago, IL, USA: SPSS Inc.

21.

Backhaus

, Erichson

, Plinke

, & Weiber

(1996). Multivariate Analyse-methoden. 8th edn, 591 pp. Berlin & Heidelberg, Germany: Springer Verlag.

22.

Bortz

(1993). Statistik. 3rd edn, 753 pp. Berlin & Heidelberg, German: Springer Verlag.

23.

Landau

, & Everitt

S.B.

(2003). A Handbook of Statistical Analyses using SPSS. 354 pp. London, UK & Boca Raton, FL, USA: Chapmann & Hall/CRC Press.

24.

Basketter

D.A.

(1999). Contact irritation models. In Toxicology of Contact Dermatitis (ed. Basketter

D.A.

, Gerberick

, Kimber

, & Willis

), pp. 39–56. Chichester, UK: John Wiley & Sons.

25.

OECD (1999). Series on Testing and Assessment, No.16. Detailed Review Document on Classification Systems for Skin Irritation/Corrosion in OECD Member Countries. 28 pp. ENV/JM/MONO(99)6. Paris, France: OECD.

26.

Heylings

J.R.

, Diot

, Esdaile

D.J.

, Fasano

W.J.

, Manning

L.A.

, & Owen

H.M.

(2003). A prevalidation study on the in vitro skin irritation function test (SIFT) for prediction of acute skin irritation in vivo: results and evaluation of ECVAM Phase III. Toxicology in Vitro 17, 123–138.

27.

ECETOC (2002). Use of Human Data in Hazard Classification for Irritation and Sensitisation. ECETOC Monograph No. 32, 54 pp. Brussels, Belgium: European Centre for Ecotoxicology and Toxicology of Chemicals.

28.

Basketter

D.A.

, York

, McFadden

J.P.

, & Robinson

M.K.

(2004). Determination of skin irritation potential in the human 4-hour patch test. Contact Dermatitis 51, 1–4.

29.

Basketter

D.A.

, Chamberlain

, Griffiths

H.A.

, Rowson

, Whittle

, & York

(1997). The classification of skin irritants by human patch test. Food and Chemical Toxicology 35, 845–852.

30.

Bickers

, Calow

, Greim

, Hanfin

J.M.

, Rogers

A.E.

, Saurat

J.H.

, Sipes

I.G.

, Smith

R.L.

, & Tagami

(2003). A toxicologic and dermatologic assessment of linalool and related esters when used as fragrance ingredients. Food and Chemical Toxicology 41, 919–942.

31.

Letizia

S.C.

, Cocchiara

, Lalko

, & Api

A.M.

(2003). Fragrance material review on linalool. Food and Chemical Toxicology 41, 943–964.

32.

Letizia

S.C.

, Cocchiara

, Lalko

, & Api

A.M.

(2003). Fragrance material review on linalyl acetate. Food and Chemical Toxicology 41, 965–976.

33.

Fentem

J.H.

, Archer

G.E.B.

, Balls

, Botham

P.A.

, Curren

R.D.

, Earl

L.K.

, Esdaile

D.J.

, Holzhütter

H.-G.

, & Liebsch

(1998). The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxicology in Vitro 12, 483–524.

34.

Phillips

, Steinberg

, Maibach

H.I.

, & Akers

W.A.

(1972). A comparison of rabbit and human skin response to certain irritants. Toxicology and Applied Pharmacology 21, 369–382.

35.

Robinson

M.K.

, McFadden

J.P.

, & Basketter

D.A.

(2001). Validity and ethics of the human 4 hour patch test as an alternative method to assess skin irritation potential. Contact Dermatitis 45, 1–12.

36.

Genschow

, Spielmann

, Scholz

, Seiler

, Brown

, Piersma

, Brady

, Huuskonen

, Paillard

, Bremer

, Clemann

, & Becker

(2002). The ECVAM international validation study on in vitro embryotoxicity tests. Results of the definitive phase and evaluation of prediction models. ATLA 30, 151–176.