Internal and external validation of the long-term QSARs for neutral organics to fish from ECOSAR™.

This study concentrates on the external validation of an existing Quantitative Structure-Activity Relationship (QSAR) model widely used for long-term aquatic toxicity to fish. In the context of the REACH legislation, QSARs are used as an alternative for experimental data to achieve a complete environmental assessment without the need for animal testing. The predictivity of the model was evaluated in order to increase the reliability of the model. We assessed whether the model met all of the OECD principles. The model was adapted to become more robust, and predictions were made with an external validation set collected from several databases. For the internal validation of the QSAR, the r², Q²(Loo) and Q²(LMO) were used as validation criteria, and for the external validation r², Q²(ext), h and the validation ratio were used. A few substances were classified as outliers and therefore the applicability domain of the QSAR had to be adjusted. The QSAR passed all validation criteria and met all the OECD principles for QSAR validation, and the long-term toxicity QSAR for fish can be applied with high certainty of a correct prediction within the limits of the inherent uncertainty of the model in cases where the substance falls within the applicability domain.

Use of read-across and tiered exposure assessment in risk assessment under REACH--a case study on a phase-in substance.

REACH requests the exploration of alternative strategies for hazard identification before resorting to (in vivo) testing. Here, we combined read-across as non-testing strategy with a tiered exposure assessment for the risk characterisation of 1-methoxypropan-2-ol (PGME) as a representative for phase-in substances to be registered under REACH. Read-across from the selected source substances provided data which were comparable with experimental data available for target substance PGME, resulting in a realistic starting point for both qualitative and quantitative risk assessment. Greater variability was observed in the exposure estimates from a first Tier model (ECETOC TRA) or less conservative further Tier models (Stoffenmanager; RISKOFDERM), when these results were compared with results from a data-rich approach using measured data. When safe use of chemicals cannot be demonstrated with these approaches, refinement can be introduced in the estimation of hazard and exposure, or both. In view of the variability associated with exposure modeling, it may often add more value to invest in realistic exposure data than in toxicity studies, apart from animal welfare considerations.

Improving the applicability of (Q)SARs for percutaneous penetration in regulatory risk assessment.

The new regulatory framework REACH (Registration, Evaluation, and Authorisation of Chemicals) foresees the use of non-testing approaches, such as read-across, chemical categories, structure-activity relationships (SARs) and quantitative structure-activity relationships (QSARs). Although information on skin absorption data are not a formal requirement under REACH, data on dermal absorption are an integral part of risk assessment of substances/products to which man is predominantly exposed via the dermal route. In this study, we assess the present applicability of publicly available QSARs on skin absorption for risk assessment purposes. We explicitly did not aim to give scientific judgments on individual QSARs. A total of 33 QSARs selected from the public domain were evaluated using the OECD (Organisation for Economic Co-operation and Development) Principles for the Validation of (Q)SAR Models. Additionally, several pragmatic criteria were formulated to select QSARs that are most suitable for their use in regulatory risk assessment. Based on these criteria, four QSARs were selected. The predictivity of these QSARs was evaluated by comparing their outcomes with experimentally derived skin absorption data (for 62 compounds). The predictivity was low for three of four QSARs, whereas one model gave reasonable predictions. Several suggestions are made to increase the applicability of QSARs for skin absorption for risk assessment purposes.

4-Substituted 1-chloro-2-nitrobenzenes: structure-activity relationships and extension of the substrate model of rat glutathione S-transferase 4-4.

In the present study, eleven 4-substituted 1-chloro-2-nitrobenzenes were tested for their GSH conjugation capacity when catalyzed by base or rat glutathione S-transferase (GST) 4-4. Kinetic parameters (ks and K(m), kcat, and kcat/K(m)) were determined and subsequently used for the description of structure-activity relationships (SAR's). For this purpose, eight physicochemical parameters (electronic, steric, and lipophilic) of the substituents and five computer-calculated parameters of the substrates (charge distributions and several energy values) were used in regression analyses with the kinetic parameters. The obtained SAR's are compared with corresponding SAR's for the GSH conjugation of 2-substituted 1-chloro-4-nitrobenzenes, previously determined [Van der Aar et al. (1996) Chem. Res. Toxicol. 9, 527-534]. The kinetic parameters of the 4-substituted 1-chloro-2-nitrobenzenes correlated well with the Hammett sigma p- constant; the Hammett sigma p constant corrected for "through resonance", while the corresponding kinetic parameters of the 2-substituted 1-chloro-4-nitrobenzenes did not. The base- and GST 4-4-catalyzed GSH conjugation reactions of 2-substituted 1-chloro-4-nitrobenzenes depend to a different extent on the electronic properties of the ortho substituents, suggesting the involvement of different rate-limiting transition states. The base- and GST 4-4-catalyzed conjugation of 4-substituted 1-chloro-2-nitrobenzenes, however, showed a similar dependence on the electronic properties of the para substituents, indicating that these substrates are conjugated to GSH via a similar transition state. Multiple regression analyses revealed that, besides electronic interactions, also steric and lipophilic restrictions appeared to play an important role in the GST 4-4-catalyzed GSH conjugation of 4-substituted 1-chloro-2-nitrobenzenes. Finally, the 4-substituted 1-chloro-2-nitrobenzenes were also used to extend the previously described substrate model for GST 4-4 [De Groot et al. (1995) Chem. Res. Toxicol. 8, 649-658], by which a specific steric restriction of substrates for GST 4-4 became clear.

Structure-activity relationships for chemical and glutathione S-transferase-catalysed glutathione conjugation reactions of a series of 2-substituted 1-chloro-4-nitrobenzenes.

Glutathione S-transferases (GSTs) constitute an important class of phase II (de)toxifying enzymes, catalysing the conjugation of glutathione (GSH) with electrophilic compounds. In the present study, Km, kcat and kcat/Km values for the rat GST 1-1-, 3-3-, 4-4- and 7-7-catalysed conjugation reactions between GSH and a series of 10 different 2-substituted 1-chloro-4-nitrobenzenes, and the second-order rate constants (ks) of the corresponding base-catalysed reactions, were correlated with nine classical physicochemical parameters (electronic, steric and lipophilic) of the substituents and with 16 computer-calculated molecular parameters of the substrates and of the corresponding Meisenheimer complexes with MeS- as a model nucleophile for GS- (charge distributions and several energy values), giving structure-activity relationships. On the basis of an identical dependence of the base-catalysed as well as the GST 1-1- and GST 7-7-catalysed reactions on electronic parameters (among others, Hammett substituent constant sigma p and charge on p-nitro substituents), and the finding that the corresponding reactions catalysed by GSTs 3-3 and 4-4 depend to a significantly lesser extent on these parameters, it was concluded that the Mu-class GST isoenzymes have a rate-determining transition state in the conjugation reaction between 2-substituted 1-chloro-4-nitrobenzenes and GSH which is different from that of the other two GSTs. Several alternative rate-limiting transition states for GST 3-3 and 4-4 are discussed. Furthermore, based on the obtained structure-activity relationships, it was possible to predict the kcat/Km values of the four GST isoenzymes and the ks of the base-catalysed GSH conjugation of 1-chloro-4-nitrobenzene.

A single injection of a biodegradable microsphere formulation of the ACTH-(4-9) analogue ORG 2766 accelerates functional recovery after brain damage.

The functional recovery from impaired motor activity induced by 6-hydroxydopamine lesions in rat nucleus accumbens was accelerated by subcutaneous treatment with the ACTH-(4-9) analogue Met/O2/-Glu-His-Phe-D-Lys-Phe (ORG 2766). Treatment was effective after daily injections of ORG 2766 dissolved in saline during the first 6 days following the lesion (ED50: 28.5 ng kg-1 day-1) or after a single injection of the peptide in a biodegradable microsphere formulation administered after the lesion (ED50: 8.9 ng kg-1 day-1). This study shows that a single injection of a microsphere preparation can replace multiple injections with ORG 2766 in order to facilitate functional recovery after brain damage.