Controlling cleaning agent residues in pharmaceutical manufacturing: A harmonized scientific strategy.

This paper proposes a scientifically justified and harmonized strategy to control cleaning agent ingredients' (CAIs) residues in pharmaceutical manufacturing. Firstly, we demonstrate that worst-case cleaning validation calculations on CAI residues with representative GMP standard cleaning limits (SCLs) are enough to control CAI residues of low concern to safe levels. Secondly, a new harmonized strategy for the toxicological assessment of CAI residues is presented and validated. The results establish a framework applicable to cleaning agent mixtures based on hazard and exposure considerations. This framework is primarily based on the hierarchy of a single CAI's critical effect, where the lowest resulting limit may become the driver of the cleaning validation process. The six critical effect groups are: (1) CAIs of low concern based on safe exposure reasoning; (2) CAIs of low concern based on the mode of action reasoning; (3) CAIs with local concentration-dependent critical effects; (4) CAIs with dose-dependent systemic critical effects for which a route-specific PDE should be calculated; (5) poorly characterized CAIs with unknown critical effect for which a default value of 100 µg/day is proposed; (6) poorly characterized CAIs which should be avoided because of potential mutagenicity and/or potency.

PDE concept for controlling cleaning agent residues in pharmaceuticals- A critical analysis.

Cleaning agents (CAs) are used in multipurpose facilities to control carryover contamination of active pharmaceutical ingredients (APIs) to scientifically justified limits. While this is often done with the PDE methodology used for API impurities, it is unclear if it is justifiable and necessary for cleaning agents, which generally represent a comparatively lower health risk. Comparing calculated oral PDE values for CA ingredients (CAIs) from four companies with PDEs of a selected number of small-molecule APIs showed that the toxicity of CAIs is several orders of magnitude lower. Furthermore, a critical review of the toxicity and everyday exposure to the general population of the main CAIs functional groups showed that the expected health risks are generally negligible. This is particularly true if the associated mode of actions cause local toxicity that is usually irrelevant at the concentration of potential residue carryover. This work points towards alternative approaches to the PDE concept to control CAIs' contamination and provides some guidance on grouping and identifying compounds with lower health risks based on exposure and mode of action reasoning. In addition, this work supports the concept that limit values should only be set for CAIs of toxicological concern.

Geographic variation in hybridization and ecological differentiation between three syntopic, morphologically similar species of montane lizards.

To understand factors shaping species boundaries in closely related taxa, a powerful approach is to compare levels of genetic admixture at multiple points of contact and determine how this relates to intrinsic and extrinsic factors, such as genetic, morphological and ecological differentiation. In the Australian Alps, the threatened alpine bog skink Pseudemoia cryodroma co-occurs with two morphologically and ecologically similar congeners, P. entrecasteauxii and P. pagenstecheri, and all three species are suspected to hybridize. We predicted that the frequency of hybridization should be negatively correlated with genetic divergence, morphological differentiation and microhabitat separation. We tested this hypothesis using a mitochondrial locus, 13 microsatellite loci, morphological and microhabitat data and compared results across three geographically isolated sites. Despite strong genetic structure between species, we detected hybridization between all species pairs, including evidence of backcrossed individuals at the two sites where all three species are syntopic. Hybridization frequencies were not consistently associated with genetic, morphological or ecological differentiation. Furthermore, P. entrecasteauxii and P. pagenstecheri only hybridized at the two sites where they are syntopic with P. cryodroma, but not at the largest site where P. cryodroma was not recorded, suggesting that P. cryodroma may serve as a bridging species. This study reveals the complex dynamics within a three species hybrid zone and provides a baseline for assessing the impact of climate change and anthropogenic habitat modification on future hybridization frequencies.

Optimization of a novel class of benzimidazole-based farnesoid X receptor (FXR) agonists to improve physicochemical and ADME properties.

Structure-guided lead optimization of recently described benzimidazolyl acetamides addressed the key liabilities of the previous lead compound 1. These efforts culminated in the discovery of 4-{(S)-2-[2-(4-chloro-phenyl)-5,6-difluoro-benzoimidazol-1-yl]-2-cyclohexyl-acetylamino}-3-fluoro-benzoic acid 7g, a highly potent and selective FXR agonist with excellent physicochemical and ADME properties and potent lipid lowering activity after oral administration to LDL receptor deficient mice.

Development of prediction models for three in vitro embryotoxicity tests in an ECVAM validation study.

Since 1997 the National Center for Documentation and Evaluation of Alternative Methods to Animal Experiments, ZEBET, in Berlin, has been coordinating a validation study aimed at prevalidation and validation of three in vitro embryotoxicity tests, funded by the European Center for the Validation of Alternative Methods (ECVAM) at the Joint Research Center (JRC, Ispra, Italy). The tests use the cultivation of postimplantation rat whole embryos (WEC test), cultures of primary limb bud cells of rat embryos (micromass or, MM, test), and cultures of a pluripotent mouse embryonic stem cell line (embryonic stem cell test or EST). Each of the tests was performed in four laboratories under blind conditions. In the preliminary phase of the validation study 6 out of 20 test chemicals comprising different embryotoxic potential (non, weakly, and strongly embryotoxic) were tested. The results were used to define biostatistically based prediction models (PMs) to identify the embryotoxic potential of test chemicals for the WEC test and the MM test. The PMs developed with the results of the preliminary phase of the validation study (training set) will be evaluated with the results of the remaining 14 test chemicals (definitive phase) by the end of the study. In addition, the existing, improved PM (iPM) for the EST, which had been defined previously, was evaluated using the results of the preliminary phase of this study. Applying the iPM of the EST to the results of this study, in 79% of the experiments, chemicals were classified correctly according to the embryotoxic potential defined by in vivo testing. For the MM and the WEC test, the PMs developed during the preliminary phase of this validation study provided 81% (MM test) and 72% (WEC test) correct classifications. Because the PM of the WEC test took into account only parameters of growth and development, but not cytotoxicity data, a second PM (PM2) was developed for the WEC test by incorporating cytotoxicity data of the differentiated mouse fibroblast cell line 3T3, which was derived from the EST. This approach, which has previously never been used, resulted in an increase to 84% correct classifications in the WEC test.

[Development of prediction models for three in vitro embryotoxicity tests which are evaluated in an ECVAM validation study] / Die Entwicklung von Prädiktionsmodellen für drei in vitro Embryotoxizitätstests im Rahmen einer ECVAM Validierungsstudie.

In 1997 ZEBET started the co-ordination of a study funded by the European Centre for the Validation of Alternative Methods (ECVAM) with the aim of pre-validation and validation of three in vitro embryotoxicity tests. These tests employ the cultivation of post-implantation whole rat embryos (WEC test), cultures of primary limb bud cells of rat embryos (micromass test, MM-Test), and cultures of a pluripotent mouse embryonic stem cell line (embryonic stem cell test, EST). In the current Validation Study each of the tests is evaluated in four laboratories under blind conditions. In an initial phase of the validation study six out of 20 test chemicals comprising different embryotoxic potential (non, weak and strong embryotoxic) were tested. The results were used to improve the prediction models (PM) for the WEC test and the MM-Test in order to identify the embryotoxic potential of test chemicals. In addition, the existing PM for the EST was evaluated using the results from testing of the initial six chemicals. The PM for the EST was developed using the results of a previous pre-validation study (Scholz et al.,1999), in which 94% of the learning sample were classified correctly. Applying this PM to the results of the initial phase of the current validation study, 80% of the experiments were classified correctly according to the embryotoxic potential of the tested chemicals in vivo. Applying the PM for the MM-Test and the WEC test that were developed during the current validation study in both tests, 79% correct classifications were achieved. Since the PM of the WEC-Test took into account only parameters of growth and development, but not cytotoxicity data, a second PM (PM2) for the WEC was developed that was improved by incorporating cytotoxicity data of the differentiated mouse fibroblast cell line 3T3 derived from the EST. This approach, which has previously never been used as an adjunct to the WEC test, resulted in an increase of correct classifications to 96%.

New and traditional approaches for the assessment of testicular toxicity.

In this study, the suitability of several methods for the assessment of testicular damage, including histopathology, flow cytometry (FCM), testicular sperm head counts, and secretion of androgen binding protein (ABP), has been evaluated. Testicular toxicity after acute exposure of adult rats to different doses of the known toxicant 1,3-dinitrobenzene (DNB) was analyzed. The effects showed dose dependence, in spite of the large variability within each dose group. Histopathology and FCM showed germ cell depletion, particularly of round spermatids; testicular sperm head counts were reduced and ABP production was increased. All evaluated methods showed similar sensitivities. The increased testicular ABP levels support the theory that the Sertoli cell is the likely target of DNB induced testicular toxicity, producing subsequent germ cell depletion. The presented results show the suitability of FCM for the analysis of testicular damage and also support the usefulness of including a metabolic marker for Sertoli cell function.