Alternatives to animal testing in the safety evaluation of products

The conventional method for assessing the safety of products, ranging from pharmaceuticals to agrochemicals, biocides and industrial and household chemicals - including cosmetics - involves determining their toxicological properties by using experimental animals. The aim is to identify any possible adverse effects in humans by using these animal models. Providing safe products is undoubtedly of the utmost importance but, over the last decade or so, this aim has come into conflict with strong public opinion, especially in Europe, against animal testing. Industry, academia and the regulators have worked in partnership to find other ways of evaluating the safety of products, by non-animal testing, or at least by reducing the numbers of animals required and the severity of the tests in which they are used. There is a long way to go before products can be evaluated without any animal studies, and it may be that this laudable aim is an impossible dream. Nevertheless, considerable progress has been made by using a combination of in vitro tests and the prediction of properties based on chemical structure. The aim of this review is to describe these important and worthwhile developments in various areas of toxicological testing, with a focus on the European regulatory framework for general industrial and household chemicals.     

生物制品原辅料及其质量控制

生物制品原辅料的质量控制是保证产品质量的重要因素,就生物制品原辅料分类及质量控制进行了阐述和归纳,并就国家对生物制品原辅料的质量控制要求进行了探讨。     

Heavy metals in wetland soils along a wetland-forming chronosequence in the Yellow River Delta of China: Levels,sources and toxic risks

Surface soil (0–20 cm) samples were collected from four chronological sequences of wetlands (i.e., >50-yr-old wetlands, 40-yr-old wetlands, 30-yr-old wetlands and 10-yr-old wetlands) in the Yellow River Delta of China in May and June of 2007. Total contents of Al, As, Cd, Cr, Cu, Ni, Pb and Zn were determined using inductively coupled plasma atomic absorption spectrometry (ICP-AAS) to investigate the levels, sources and toxic risks of heavy metals in these wetlands. Our results showed an increasing trend for Pb, Cu and Zn along the wetland-forming chronosequence although their pollution levels were low. Both As and Cd exhibited significant enrichment due to their high enrichment factor (EF) values (EF > 5), especially in older wetlands (i.e., >50-yr-old and 40-yr-old wetlands), whereas other heavy metals were minimally or moderately enriched in this region. The results of principal component analysis showed that 83.09% of total variance based on eigenvalues (eigenvalue > 1) could be explained by three principal components (PCs) in four wetlands. The source of Al, Cu, Pb and Zn was different from Cd, Cr and Ni. According to the sediment quality guidelines (SQGs) of China, soil samples in the younger wetlands, especially the 10-yr-old wetlands, were moderately polluted by As, Cd and Ni. According to the SQGs of US EPA, all soil samples were heavily polluted by As and moderately polluted by Ni and soil samples in the older wetlands were moderately polluted by Cr. However, with the exception of As and Ni, the contents of other heavy metals in the four wetlands did not exceed the probable effect level (PEL) values. As, Cd and Ni were identified as heavy metals of primary concerns in four wetlands, Cr were of moderate concern in older wetlands, and Pb, Cu and Zn should be paid more attention in younger wetland (i.e., 10-yr-old and 30-yr-old wetlands). A new and sensitive toxic risk index (TRI) is developed for the accurate assessment of toxic risk for heavy metals in wetland soils compared with the sum of the toxic units (∑TUs), and As, Cr, Ni and Cd showed higher contributions to TRI.     

A comparative evaluation with respect to the bacterial cleanability of a range of wall and floor surface materials used in the food industry

J. H. TAYLOR AND J. T. HOLAH. 1996. A range of floor and wall materials suitable for use in the food industry were selected for comparative bacterial cleanability tests. A standard cleaning regime was carried out on samples after biofilm development of Acinetobacter calcoaceticus (CRA 296). Cleanability was measured as the log reduction in bacterial numbers. The results for the wall materials showed that there were no statistical differences, whilst those for the floor materials were more variable. The grouted joints and texture did not compromise the cleanabilities of tiled surfaces.     

Probability models to assess the safety of foods with respect toClostridium botulinum

Summary The effectiveness of a preservative system to prevent the growth ofClostridium botulinum can be expressed as the probability (P) that not even a single spore will be able to grow and produce toxin. Commerical canning processes for foods have been based upon this principle since the early 1920s. The safety of many current food marketing concepts depends on product formulation, processing, packaging and distribution variables. Direct measurement ofC. botulinum growth in a food system is difficult. Researchers have relied upon bioassay for botulinum toxin detection and Most Probable Number (MPN) techniques to quantifyC. botulinum growth in experimental food systems. The methods used to estimateP for a single spore to initiate growth will lead to a discussion on the use ofP as a dependent variable in predictive models. Modeling the effects of intrinsic and extrinsic processing variables on food safety will be presented.     

运用基因芯片技术检测牛、山羊、猪和鸡源性成分

本研究通过对脊椎动物分子标记基因进行序列分析,最终选择线粒体DNA(mtDNA)16S rRNA基因为目标基因,利用一对通用引物,在该引物扩增区间设计了4条特异性基因芯片检测探针及2条质控探针用于对牛、山羊、猪、鸡等4种动物源性成分进行检测。通过对PCR扩增体系及杂交体系的优化,该检测方法能实现对上述4种动物源性成分同时进行快速、准确地检测,具有很好的特异性,灵敏度均达到1pg,最终建立了这4种动物源性基因芯片检测方法。该基因芯片检测技术将为我国进出口饲料中的动物源性成分的鉴别提供新的检测方法和技术支持。     

Phylogenetic analysis of the Cardini group of Drosophila with respect to changes in pigmentation

Phenotypic variability is the engine that drives future diversification with the expectation that polymorphic ancestors give rise to descendants harboring a subset of the ancestral variation. Here we examine evolutionary transitions from polymorphism to monomorphism in a visually striking New World radiation of fruit flies, the Drosophila cardini group. This group is distributed across the Americas and the Caribbean islands and exhibits a wide spectrum of abdominal pigmentation variation. Specifically, the D. dunni subgroup consists of Caribbean island endemics, each of which is monomorphic for its pigmentation pattern, with an interspecific cline of pigmentation across the islands. The D. cardini subgroup consists of American continental species with wide-ranging distributions and intraspecifically variable abdominal pigmentation. We determined the phylogeny of 18 species and subspecies using three nuclear and three mitochondrial regions analyzed with maximum parsimony, maximum likelihood, and Bayesian methods. The topology produced from a combined dataset exhibited high support values at all nodes, and differed from earlier phylogenetic hypotheses based on polytene chromosome inversion patterns and isozyme data. We find that the D. dunni subgroup species, with the exception of D. belladunni, are derived from a single source not of direct South American origin and their dispersal across the islands of the Caribbean does not follow a simple stepping-stone model. Morphological changes in pigmentation across the island species are incongruent with the colonization history of the group indicating that natural selection may have played a role in the determination of this character. Finally, we demonstrate that monomorphic species have arisen independently from polymorphic ancestors two to three times.     

Effects of selenium, iron and cobalt addition to growth and yessotoxin production of the toxic marine dinoflagellate Protoceratium reticulatum in culture

The marine dinoflagellate Protoceratium reticulatum has been recently identified as a source for the disulfated polyether toxin, yessotoxin (YTX), and may pose a risk to human health, aquaculture development and coastal environments. The requirements of P. reticulatum for selenium, iron and cobalt were assessed in culture. P. reticulatum was grown in nutrient enriched seawater (1/10 GP medium) without selenium or with 0.003 and 0.0003 μM selenium added; without iron or with 0.076 and 0.0076 μM iron added; and without cobalt or with 0.008 μM cobalt added. Test flasks were monitored for growth rate, cell yield and YTX production. P. reticulatum was found to exhibit a strong requirement for both selenium and iron. Growth rate and cell yield in treatments without added selenium were significantly (P<0.05) reduced to 60.2% (μ=0.15 day⁻¹) and 20.2% (4942 cell ml⁻¹), respectively, of those with selenium added (μ=0.23 day⁻¹ and 24, 387 cell ml⁻¹). YTX production was significantly increased by addition of selenium in two of three transfers tested. Cells of P. reticulatum subjected to medium without selenium added showed morphological changes observable at the light microscope level which included enlarged cell size. The diameter of cells in medium without selenium added were significantly (P<0.05) enlarged to 36.7±0.90 μm compared to cells in the medium with selenium added, 27.5±1.25 μm. Growth rate and cell yield in treatments without added iron were also significantly reduced to 70.1% (μ=0.16 day⁻¹) and 34.2% (8003 cells ml⁻¹), respectively, of those with iron added (μ=0.23 day⁻¹ and 23,416 cells ml⁻¹). No significant effect on YTX production was measured. In contrast to selenium and iron, no limitation of growth or cell yield or differences in YTX production were observed for flasks without cobalt as compared to those with cobalt added. The possibility that harmful algal events of P. reticulatum may be influenced by selenium or iron in neritic waters is discussed.     

Dynamic structures of globular proteins with respect to correlative movements of residues calculated in the normal mode analysis

Dynamic structures of globular proteins are studied on the basis of correlative movements of residues around their native conformations, which are computed by means of the normal mode analysis. To describe the dynamic structures of a protein, the core regions moving with strong positive or negative correlations to other regions of the polypeptide chain are detected from the correlation maps of the movements of residues. Such core regions are different, according to the definition, from the regions defined from a geometrical point of view, such as secondary structures, domains, modules, and so on. The core regions are actually detected for four proteins, myoglobin, Bence-Jones protein, flavodoxin, and hen egg-white lysozyme, with different folding types from each other. The results show that some of them coincide with the secondary structures, domains, or modules, but others do not. Then, the dynamic structure of each protein is discussed in terms of the dynamic cores detected, as compared with the secondary structures, domains, and modules.     

Scaling of long bones in ruminants with respect to the scapula

The significance of the scapula for locomotion is becoming more and more established. Studies of locomotion in small and medium‐sized mammals show a considerable amplitude of the scapula and a large contribution to step length. Taking this into account, long bone studies of forelimb movement restricted to the ‘arm’ miss one important segment. A regression model (reduced major axis) was used for analysis of a sample of 77 species of ruminants. This sample was divided according to (1) phylogenetic relationships and (2) habitat. The proximal elements of the limbs, scapula and humerus in the anterior extremity, femur in the hindlimb, show a similar scaling in the different analyses. The changes to limb proportions in the different subsamples are caused by the variability of the distal segments. The anterior extremity scales with a higher coefficient than the hindlimb in all analyses. Concepts like elastic or geometric similarity are inadequate for long bone scaling when the full range of body size in the sample is used. Taking all analyses into account, the differences in limb proportions are due more to phylogenetic relationships than to habitat.     

Chemical and microbiological characterization of artisan inoculants used for the fermentation of traditional dairy products in Epirus area (Greece)

The artisan production of a variety of dairy products is a historical tradition since antiquity, which is still practiced nowadays, in the area of Epirus, (northwestern Greece). The common denominator of these products, regardless of the final form of the end product, is the fermentation of raw milk by the use of dried artisan inoculants made of the dehydrated rumen of small ruminants, mainly lambs. The aim of this research project is to study the microbiological parameters as well as certain technological parameters (chymosin activity and lipolytic activity) of these inoculants. The results revealed a wide biodiversity of microorganisms such as Lactobacilli, Lactococci, Leuconostoc, Pediococci, Streptococci, Bifidobacteria, Enterococci, Clostridia and coliforms. Chymosin activity and lipolytic activity were found to be higher in artisan inoculants than in the commercial ones.     

太阳能灭虫器对吐鲁番葡萄产区害虫的诱捕效果及对天敌安全性评价

本文研究了FWS-DBL-1新型太阳能灭虫器在320～580、360、400、460和520 nm 5种不同波长下对吐鲁番葡萄产区害虫的诱捕效果及对天敌安全性。结果表明:在5种波长下,灭虫器诱捕昆虫的种类相似,以鞘翅目、鳞翅目、半翅目、脉翅目、双翅目、直翅目和膜翅目等为主。诱捕的主要昆虫是蜉金龟Aphodius sp.、白云斑鳃金龟Polyphylla alba vicaria Semenov、额喙丽金龟Adoretus nigriforns Steven、毛喙丽金龟Adoretus hirsutus Ohaus、绣罗夜蛾Leucanitis picta Christoph和淘赏夜蛾Catocala puerpera Giorna等。其中:灭虫器在400 nm波长下的诱捕量最多,其次是320～580 nm和360 nm波长。综合分析比较:400 nm波长对鞘翅目害虫的诱捕效果最好,360 nm波长对鳞翅目和双翅目害虫的诱捕能力最强,520 nm波长对半翅目害虫的诱捕效果最好。对天敌的安全性研究表明:FWS-DBL-1太阳能灭虫器对天敌有一定的诱捕作用,益害比为1∶4～1∶5。诱捕量较多的是步甲科和草蛉科的天敌,占天敌总诱捕量的85.82%。在这5个波长中,360 nm波长下,灭虫器对天敌的诱捕作用最小,对天敌的安全性相对较高。     

Evaluation of the effectiveness of the FWS-DBL-1 solar insect killer and its safety with respect to predatory insects in the Turpan grape growing region

本文研究了FWS-DBL-1新型太阳能灭虫器在320～580、360、400、460和520nm 5种不同波长下对吐鲁番葡萄产区害虫的诱捕效果及对天敌安全性.结果表明:在5种波长下,灭虫器诱捕昆虫的种类相似,以鞘翅目、鳞翅目、半翅目、脉翅目、双翅目、直翅目和膜翅目等为主.诱捕的主要昆虫是蜉金龟Aphodius sp.、白云斑鳃金龟Polyphyyla alba vicaria Semenov、额喙丽金龟Adoretus nigriforns Steven、毛喙丽金龟Adoretus hirsutus Ohaus、绣罗夜蛾Leucanitis picta Christoph和淘赏夜蛾Catocala puerpera Gioma等.其中:灭虫器在400nm波长下的诱捕量最多,其次是320～580nm和360nm波长.综合分析比较:400nm波长对鞘翅目害虫的诱捕效果最好,360nm波长对鳞翅目和双翅目害虫的诱捕能力最强,520nm波长对半翅目害虫的诱捕效果最好.对天敌的安全性研究表明:FWS-DBL-1太阳能灭虫器对天敌有一定的诱捕作用,益害比为1:4～1:5.诱捕量较多的是步甲科和草蛉科的天敌,占天敌总诱捕量的85.82％.在这5个波长中,360nm波长下,灭虫器对天敌的诱捕作用最小,对天敌的安全性相对较高.     

Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase

Oxygen and other molecules of similar size take part in a variety of protein reactions. Therefore, it is critical to understand how these small molecules penetrate the protein matrix. The protein system studied in this case is horseradish peroxidase (HRP). We have converted the native HRP into a phosphorescent analog by replacing the heme prosthetic group by Pd-mesoporphyrin. Oxygen readily quenches the phosphorescence of Pd porphyrins, and this can be used to characterize oxygen diffusion through the protein matrix. Our measurements indicate that solvent viscosity and pH modulate the accessibility of the heme pocket relative to small molecules. The binding of the substrate benzohydroxamic acid (BHA) to the protein drastically impedes oxygen access to the heme pocket. These results indicate that, first, the penetration of small molecules through the protein matrix is a function of protein dynamics, and second, there are specific pathways for the diffusion of these molecules. The effect of substrate and pH on protein dynamics has been investigated with the use of molecular dynamics calculations. We demonstrate that the model of a "fluctuating entry point," as suggested by Zwanzig (J Chem Phys 1992;97:3587-3589), properly describes the diffusion of oxygen through the protein matrix.     

Improvement of methane biofiltration by the addition of non-ionic surfactants to biofilters packed with inert materials

The effect of non-ionic surfactants on the biofiltration of methane (CH₄) was analyzed. Two biofilters (BF) treating CH₄ were operated for one year at fixed CH₄ concentration of 4.8 g m⁻³ and air flow rate of 0.25 m⁻³ h⁻¹. Three polyoxyethylenes (Brijs), and 3 mono polyoxyethylenesorbitans (Tweens) were added to the nutrient solution at a concentration of 0.5% (w/w). Without surfactant, CH₄ conversion had an average level of 35%, with Brijs the CH₄ conversion varied between 38% and 46%, and with Tweens between 43% and 48%. The non-ionic surfactants decreased the biomass accumulation in the packed bed due to their detergent character. Biofilters were operated in a range of nitrogen concentration in the nutrient solution from 0.5 to 2 gN L⁻¹ using Tween 20 at a concentration of 0.5% (w/w). The EC_max observed in this study, 45 g m⁻³ h⁻¹, occurred when the nitrogen concentration was 1 gN L⁻¹.     

Application of near‐infrared (NIR) spectroscopy for screening of raw materials used in the cell culture medium for the production of a recombinant therapeutic protein

Control of raw materials based on an understanding of their impact on product attributes has been identified as a key aspect of developing a control strategy in the Quality by Design (QbD) paradigm. This article presents a case study involving use of a combined approach of Near‐infrared (NIR) spectroscopy and Multivariate Data Analysis (MVDA) for screening of lots of basal medium powders based on their impact on process performance and product attributes. These lots had identical composition as per the supplier and were manufactured at different scales using an identical process. The NIR/MVDA analysis, combined with further investigation at the supplier site, concluded that grouping of medium components during the milling and blending process varied with the scale of production and media type. As a result, uniformity of blending, impurity levels, chemical compatibility, and/or heat sensitivity during the milling process for batches of large‐scale media powder were deemed to be the source of variation as detected by NIR spectra. This variability in the raw materials was enough to cause unacceptably large variability in the performance of the cell culture step and impact the attributes of the resulting product. A combined NIR/MVDA approach made it possible to finger print the raw materials and distinguish between good and poor performing media lots. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Differentiating the effects of origin and frequency in reciprocal transplant experiments used to test negative frequency-dependent selection hypotheses

Reciprocal transplant experiments have been used to estimate the probability that negative frequency-dependent selection by natural enemies has occurred in host populations by determining whether pest populations are less adapted to “foreign” (rare) hosts, which originate from a population with which the pests have not coevolved. However, these experiments usually confound the effects of frequency and origin: the rare genotypes are also genotypes that did not originate at a site. When clonal organisms are used, and the clones occur in more than one population, it is possible to separate the effects of origin and frequency. Here I present the results of an experiment in which Arabis clones of known frequency were reciprocally transplanted among sites. Contrary to expectations, clones at their site of origin had less disease, less herbivory, and higher fitness than foreign clones. However, variation within and among sites in herbivory and infection was large, suggesting that the number of sites and clones needed to thoroughly test the hypothesis of negative frequency-dependent selection in this system is very large: thus, these results are suggestive but not conclusive. Received: 20 October 1997 / Accepted: 8 February 1998