Prevalidation study of the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0 for assessment of carcinogenic potential of chemicals.

The European Centre for the Validation of Alternative Methods (ECVAM) has organised an interlaboratory prevalidation study on the Syrian hamster embryo (SHE) cell transformation assay (CTA) at pH 7.0 for the detection of rodent carcinogens. The SHE CTA at pH 7.0 has been evaluated for its within-laboratory reproducibility, transferability and between-laboratory reproducibility. Four laboratories using the same basic protocol with minor modifications participated in this study and tested a series of six coded-chemicals: four rodent carcinogens (benzo(a)pyrene, 3-methylcholanthrene, 2,4-diaminotoluene and o-toluidine HCl) and two non-carcinogens (anthracene and phthalic anhydride). All the laboratories found the expected results with coded chemicals except for phthalic anhydride which resulted in a different call in only one laboratory. Based on the outcome of this study, it can be concluded that a standardised protocol is available that should be the basis for future use. This protocol and the assay system itself are transferable between laboratories and the SHE CTA at pH 7.0 is reproducible within- and between-laboratories.

Recommended protocol for the Syrian hamster embryo (SHE) cell transformation assay.

The Syrian hamster embryo (SHE) cell transformation assay (CTA) is a short-term in vitro assay recommended as an alternative method for testing the carcinogenic potential of chemicals. SHE cells are "normal" cells since they are diploid, genetically stable, non-tumourigenic, and have metabolic capabilities for the activation of some classes of carcinogens. The CTA, first developed in the 1960s by Berwald and Sachs (1963,1964) [3,4], is based on the change of the phenotypic feature of cell colonies expressing the first steps of the conversion of normal to neoplastic-like cells with oncogenic properties. Pienta et al. (1977) [22] developed a protocol using cryopreserved cells to enhance practicality of the assay and limit sources of variability. Several variants of the assay are currently in use, which mainly differ by the pH at which the assay is performed. We present here the common version of the SHE pH 6.7 CTA and SHE pH 7.0 CTA protocols used in the ECVAM (European Centre for the Validation of Alternative Methods) prevalidation study on CTA reported in this issue. It is recommended that this protocol, in combination with the photo catalogues presented in this issue, should be used in the future and serve as a basis for the development of the OECD test guideline.

Photo catalogue for the classification of cell colonies in the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0.

This catalogue is a display of Syrian hamster embryo (SHE) cell colony photos representative of the cell transformation assay (CTA) carried out at pH 7.0. It is intended as a visual aid for the identification and the scoring of cell colonies in the conduct of the assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the assay results.

Ecotoxicity of cyanide complexes in industrially contaminated soils.

This study deals with acute and chronic ecotoxicity of leachates from industrially contaminated soils. Analyses focused on cyanides (complex and free forms) to study their possible involvement in leachates toxicity. No acute toxicity on the Microtox and 48 h-Daphnia magna tests was found in leachates collected over 18 months, but a high chronic toxicity was recorded on the reproduction of Ceriodaphnia dubia (EC50-7d=0.31±0.07%) and on the algal growth of Pseudokirchneriella subcapitata (EC50-72 h=0.27±0.09%). Ceriodaphnids were as sensitive to free cyanide as to complex forms (EC50-7d as CN(-)=98 µg/L, 194 µg/L and 216 µg/L for KCN, Fe(CN)(6)K(3) and Fe(CN)(6)K(4), respectively). The EC50-72 h of KCN to P. subcapitata (116 µg/L) as CN(-) was also of the same level as the EC50-72 h of potassium ferricyanide (127 µg/L) and ferrocyanide (267 µg/L). Complex cyanides explained a major part of the toxicity of leachates of the soil. On the other hand, cyanide complexes had no effect on survival of the earthworm Eisenia fetida up to 131 mg CN(-)/kg, while potassium cyanide was highly toxic [EC50-14 d as CN(-)=74 µg/kg soil]. Thermodesorption treatment eliminated a majority of cyanides from the soil and generated much less toxic leachates. Complex cyanides must be integrated into environmental studies to assess the impact of multi-contaminated soils.

Ecotoxicological assessment of PAHs and their dead-end metabolites after degradation by Mycobacterium sp. strain SNP11.

Mycobacterium sp. SNP11 has a high PAH biodegradation potential. In this paper, the toxicity of pyrene, fluoranthene, phenanthrene, and their dead-end metabolites, accumulated in the media after biodegradation by Mycobacterium sp. SNP11, were evaluated by a screening battery of acute, chronic, and genotoxic tests. According to the bioassays, performed on bacteria (Vibrio fischeri, Salmonella typhimurium strains TA1535/pSK1002, TA97a, TA98, TA100), algae (Pseudokirchneriella subcapitata), and crustaceans (Daphnia magna, Ceriodaphnia dubia), total disappearance or a very significant reduction of the (geno)toxic potential was observed after PAH degradation by Mycobacterium sp. SNP11.