On the use of historical control data for trend test in carcinogenicity studies

Historical control data are often available in carcinogenicity studies and are included for testing dose effects in current studies. A new method is developed for incorporating the historical control information into a dose effect test. The method generalizes the test procedures proposed by Tarone (1982, Biometrics 38, 215-220) and Ibrahim and Ryan (1996, Biometrics 52, 1478-1485) by taking into account the variation resulting from parameter estimation based on historical data. Two examples are discussed for illustrating the proposed method.     

Museum genomics: low-cost and high-accuracy genetic data from historical specimens

Natural history collections are unparalleled repositories of geographical and temporal variation in faunal conditions. Molecular studies offer an opportunity to uncover much of this variation; however, genetic studies of historical museum specimens typically rely on extracting highly degraded and chemically modified DNA samples from skins, skulls or other dried samples. Despite this limitation, obtaining short fragments of DNA sequences using traditional PCR amplification of DNA has been the primary method for genetic study of historical specimens. Few laboratories have succeeded in obtaining genome-scale sequences from historical specimens and then only with considerable effort and cost. Here, we describe a low-cost approach using high-throughput next-generation sequencing to obtain reliable genome-scale sequence data from a traditionally preserved mammal skin and skull using a simple extraction protocol. We show that single-nucleotide polymorphisms (SNPs) from the genome sequences obtained independently from the skin and from the skull are highly repeatable compared to a reference genome.     

On the use of historical control data to estimate dose response trends in quantal bioassay.

New tests for trend in proportions, in the presence of historical control data, are proposed. One such test is a simple score statistic based on a binomial likelihood for the "current" study and beta-binomial likelihoods for each historical control series. A closely related trend statistic based on estimating equations is also proposed. Trend statistics that allow overdispersed proportions in the current study are also developed, including a version of Tarone's (1982, Biometrics 38, 215-220) test that acknowledges sampling variation in the beta distribution parameters, and a trend statistic based on estimating equations. Each such trend test is evaluated with respect to size and power under both binomial and beta-binomial sampling conditions for the current study, and illustrations are provided.     

Use of in vivo genetic toxicity data for risk assessment

Mutagenicity studies have been used to identify specific agents as potential carconogens or other human health hazards; however, they have been used minimally for risk assessment or in determining permissible levels of human exposure. The poor predictive value of in vitro mutagenesis tests for carcinogenic activity and a lack of mechanistic understanding of the roles of mutagens in the induction of specific cancers have made these tests unattractive for the purpose of risk assessment. However, the limited resources available for carcinogen testing and large number of chemicals which need to be evaluated necessitate the incorporation of more efficient methods into the evaluation process. In vivo genetic toxicity testing can be recommended for this purpose because in vivo assays incorporate the metabolic activation pathways that are relevant to humans. We propose the use of a multiple end-point in vivo comprehensive testing protocol (CTP) using rodents. Studies using sub-acute exposure to low levels of test agents by routes consistent with human exposure can be a useful adjunct to methods currently used to provide data for risk assessment. Evaluations can include metabolic and pharmacokinetic endpoints, in addition to genetic toxicity studies, in order to provide a comprehensive examination of the mechanism of toxicity of the agent. A parallelogram approach can be used to estimate effects in non-accessible human tissues by using data from accessible human tissues and analogous tissues in animals. A categorical risk assessment procedure can be used which would consider, in order of priority, genetic damage in man, genetic damage in animals that is highly relevant to disease outcome (mutation, chromosome damage), and data from animals that is of less certain relevance to disease. Action levels of environmental exposure would be determined based on the lowest observed effect levels or the highest observed no effect levels, using sub-acute low level exposure studies in rodents. As an example, the known genotoxic effects of benzene exposure at low levels in man and animals are discussed. The lowest observed genotoxic effects were observed at about 1–10 parts per million for man and 0.04–0.1 parts per million in subacute animal studies. If genetic toxicity is to achieve a prominent role in evaluating carcinogens and characterizing germ-cell mutagens, minimal testing requirements must be established to ascertain the risk associated with environmental mutagen exposure. The use of the in vivo approach described here should provide the information needed to meet this goal. In addition, it should allow truly epigenetic or non-genotoxic carcinogens to be distinguished from the genotoxic carcinogens that are not detected by in vitro methods.     

Retrospective optimization of time-dependent fermentation control strategies using time-independent historical data

We have previously shown the usefulness of historical data for fermentation process optimization. The methodology developed includes identification of important process inputs, training of an artificial neural network (ANN) process model, and ultimately use of the ANN model with a genetic algorithm to find the optimal values of each critical process input. However, this approach ignores the time-dependent nature of the system, and therefore, does not fully utilize the available information within a database. In this work, we propose a method for incorporating time-dependent optimization into our previously developed three-step optimization routine. This is achieved by an additional step that uses a fermentation model (consisting of coupled ordinary differential equations (ODE)) to interpret important time-course features of the collected data through adjustments in model parameters. Important process variables not explicitly included in the model were then identified for each model parameter using automatic relevance determination (ARD) with Gaussian process (GP) models. The developed GP models were then combined with the fermentation model to form a hybrid neural network model that predicted the time-course activity of the cell and protein concentrations of novel fermentation conditions. A hybrid-genetic algorithm was then used in conjunction with the hybrid model to suggest optimal time-dependent control strategies. The presented method was implemented upon an E. coli fermentation database generated in our laboratory. Optimization of two different criteria (final protein yield and a simplified economic criteria) was attempted. While the overall protein yield was not increased using this methodology, we were successful in increasing a simplified economic criterion by 15% compared to what had been previously observed. These process conditions included using 35% less arabinose (the inducer) and 33% less typtone in the media and reducing the time required to reach the maximum protein concentration by 10% while producing approximately the same level of protein as the previous optimum.     

Analysis of historical control litter parameters of reproduction toxicity studies in Sprague-Dawley derived rats

Control litter data from reproduction toxicity studies in SD derived rats bred in our closed colony were investigated for historical changes, differences due to study design or generations, seasonal variations and effects of vehicle-treatment. The litter size did not change visibly during the entire 16-year period, but the number of live fetuses differed significantly between study designs or generations. The fetal weight gradually increased during these years. The malformation rate decreased, while the rate of 14 th ribs remained stable. There were no seasonal variations and no effects of vehicle-treatment.     

On the use of historical control information for trend test in carcinogenesis

In this article a new non-model-based significance test for detecting dose-response relationship with the incorporation of historical control data is proposed. This non-model-based test is considered simpler from a regulatory perspective because it does not require validating any modeling assumptions. Moreover, our test is especially appropriate to those studies in which the intravenous doses for the investigational chemical are labeled as, e.g., low, medium and high or the dose labels do not suggest any obvious choices of dose scores. This test can be easily adopted for detecting general dose-response shape, such as an umbrella pattern. Simple adjustments will be proposed for better control of the actual Type I error. Data sets from two carcinogenesis studies will be used to illustrate our method. We also evaluate the performance of the proposed test and the famous model-based Tarone's trend test with respect to size and power.     

Approaches to genetic control for the use of pesticides

Analysis of data available in literature has shown that 65% of 400 pesticides studied for their mutagenicity exert a mutagenic effect on any test-object. The number of revealed mutagens approaches 100% when four or more test-objects are used. Recommendations for quantitative estimation of genetic risk worked out under conditions of model experiments with standard mutagens are not available for pesticides since they are slight mutagens. The necessity for genetic stage-by-stage monitoring of pesticide use is substantiated. This monitoring should be carried out at the stage of experimental studies by means of classifications by the degree of potential mutagenic danger (the method is described) and at the stage of ecological and genetic investigations--by means of the regulation for application with due regard for the summary mutagenic background.     

A decade of rabbit fertility data: study of historical control animals.

Reproductive data for 1795 artificially inseminated Hra: (NZW)SPF control rabbits in 93 developmental toxicity studies conducted from 1980 through 1989 were summarized. Data were obtained during terminal Caesarean-sectioning procedures performed on animals which survived to day 29 of gestation and during postmortem evaluation of does which aborted, delivered prematurely, or were found dead. Significant seasonal variation was not observed. Average pregnancy rate, percentage of rabbits that aborted, and percentage of rabbits that delivered prematurely throughout the decade were 86, 2.0, and 1.6%, respectively. Average numbers of corpora lutea, implantations, live fetuses, dead fetuses, early resorptions, and late resorptions for each doe that survived to scheduled termination were 10.8, 7.8, 7.1, 0.02, 0.49, and 0.15, respectively. The various vehicles used, routes of administration, and a variety of maternal and paternal factors were compared with the fertility data, and no correlations were observed. Rabbits that aborted earlier in gestation had fewer implantations than does which aborted late or delivered prematurely. Does which resorbed 100% of their conceptuses had fewer corpora lutea and implantations when compared with rabbits in the remainder of the population. Rabbits pregnant at scheduled termination which had a low number of corpora lutea or implantation sites had higher than expected pre- and postimplantation losses relative to the population as a whole. Does with a high number of corpora lutea had significantly higher preimplantation loss relative to the general population. This may indicate the presence of a "ceiling value" for the number of ova that can become fertilized and/or implant when the ovulation rate is high and which probably varies according to strain as well as a number of factors related to the individual rabbit. Based upon the results of this study and the work of a previous author, a minimum of four corpora lutea may be necessary for the successful maintenance of pregnancy in the New Zealand white rabbit. The minimum number of corpora lutea required may be strain dependent and may bear a relationship to the normal litter size of the strain.     

Reconstruction of a windborne insect invasion using a particle dispersal model,historical wind data,and Bayesian analysis of genetic data

Understanding how invasive species establish and spread is vital for developing effective management strategies for invaded areas and identifying new areas where the risk of invasion is highest. We investigated the explanatory power of dispersal histories reconstructed based on local‐scale wind data and a regional‐scale wind‐dispersed particle trajectory model for the invasive seed chalcid wasp Megastigmus schimitscheki (Hymenoptera: Torymidae) in France. The explanatory power was tested by: (1) survival analysis of empirical data on M. schimitscheki presence, absence and year of arrival at 52 stands of the wasp's obligate hosts, Cedrus (true cedar trees); and (2) Approximate Bayesian analysis of M. schimitscheki genetic data using a coalescence model. The Bayesian demographic modeling and traditional population genetic analysis suggested that initial invasion across the range was the result of long‐distance dispersal from the longest established sites. The survival analyses of the windborne expansion patterns derived from a particle dispersal model indicated that there was an informative correlation between the M. schimitscheki presence/absence data from the annual surveys and the scenarios based on regional‐scale wind data. These three very different analyses produced highly congruent results supporting our proposal that wind is the most probable vector for passive long‐distance dispersal of this invasive seed wasp. This result confirms that long‐distance dispersal from introduction areas is a likely driver of secondary expansion of alien invasive species. Based on our results, management programs for this and other windborne invasive species may consider (1) focusing effort at the longest established sites and (2) monitoring outlying populations remains critically important due to their influence on rates of spread. We also suggest that there is a distinct need for new analysis methods that have the capacity to combine empirical spatiotemporal field data, genetic data, and environmental data to investigate dispersal and invasion.     

Coupling historical prospection data and a remotely-sensed vegetation index for the preventative control of Desert locusts

Locusts are grasshopper species that exhibit phase polyphenism resulting in the expression of gregarious behaviors that favor the development of large devastating bands and swarms. Desert locust preventative management aims to prevent crop damage by controlling populations before they can reach high densities and form mass migrating swarms. The areas of potential gregarization for Desert locust are large and need to be physically assessed by survey teams for efficient preventative management. An ongoing challenge is to be able to guide where prospection surveys should occur depending on local meteorological and vegetation conditions. In this study, we analyzed the relationship between historical prospection data of Desert locust observations from 2005 to 2009 and spatio-temporal statistics of a vegetation index gathered by remote-sensing with the help of multiple models of logistic regression. The vegetation index was a composite Normalized Difference Vegetation Index (NDVI) given every 16 days and at 250 m spatial resolution (MOD13Q1 from MODIS satellite). The statistics extracted from this index were: (1) spatial means at different scales around the prospection point, (2) relative differences of NDVI variation through time before the prospection, and (3) large-scale summary of vegetation quantity. The multi-model framework showed that vegetation development a month and a half before the survey was amongst the best predictors of locust presence. Also, the local vegetation quantity was not enough to predict locust presence. Vegetation quantity on a scale of a few kilometers was a better predictor but varied non-linearly, reflecting specific biotope types that support Desert locust development. Using one of the best logistic regression models and NDVI data, we were able to derive a predictive model of probability of finding locusts in specific areas. This methodology should help in more efficiently focusing survey efforts on specific parts of the gregarization areas based on the predicted probability of locusts being present.     

Inferring contemporary and historical genetic connectivity from juveniles

Measuring population connectivity is a critical task in conservation biology. While genetic markers can provide reliable long‐term historical estimates of population connectivity, scientists are still limited in their ability to determine contemporary patterns of gene flow, the most practical time frame for management. Here, we tackled this issue by developing a new approach that only requires juvenile sampling at a single time period. To demonstrate the usefulness of our method, we used the Speartooth shark (Glyphis glyphis), a critically endangered species of river shark found only in tropical northern Australia and southern Papua New Guinea. Contemporary adult and juvenile shark movements, estimated with the spatial distribution of kin pairs across and within three river systems, was contrasted with historical long‐term connectivity patterns, estimated from mitogenomes and genome‐wide SNP data. We found strong support for river fidelity in juveniles with the within‐cohort relationship analysis. Male breeding movements were highlighted with the cross‐cohort relationship analysis, and female reproductive philopatry to the river systems was revealed by the mitogenomic analysis. We show that accounting for juvenile river fidelity and female philopatry is important in population structure analysis and that targeted sampling in nurseries and juvenile aggregations should be included in the genomic toolbox of threatened species management.     

Compilation of human mtDNA control region sequences.

This paper describes the organisation of a database for human mitochondrial control-region sequences. The data are divided into three ASCII files that contain aligned sequences from the hypervariable region I (HVRI), from the hypervariable region II (HVRII), and the available information about the individuals, from whom the sequences stem. The current collection comprises 4079 HVRI and 969 HVRII sequences. From 728 individuals sequences of both HVRI and HVRII are available. For easy access, the collection is made available to the scientific community via World Wide Web at URL http://www.zi.biologie.uni-muenchen.de/[symbol: see text]meyers/mtdna.html     

Testing historical phylogeographic inferences with contemporary gene flow data: population genetic structure of the Qinghai toad-headed lizard

Life history theory suggests that variation in individual body size is often the result of resource availability and is controlled by trade-offs between growth, reproductive features and subsequently, survival. In the land snail Cornu aspersum , body size and correlated life history traits vary strongly intraspecifically, leading to geographic patterns of life history tactics. This work investigates the potential role of repeated exposure to stress during the growth and reproduction stage on the life history responses of two subspecies. Cornu aspersum aspersa and Cornu aspersum maxima exhibit contrasting life history strategies, as shown morphologically by the giant size of maxima . In this study, we postulated that the two subspecies would respond differently when exposed to a similar, non-specific stress. Thus, snails were regularly challenged with injections of Gram-negative heat-killed bacteria Escherichia coli suspensions. As expected, we found that stress induced by bacterial challenges leads to a significant decrease in fecundity in the two subspecies. We also observed differences between the challenged and the control snails in terms of the (1) growth features for C. a. maxima and (2) timing of the first egg-laying for C. a. aspersa . Moreover, a delayed development of the reproductive organs in the post-challenged animals suggested a trade-off between growth and sexual maturity.     

Overview of the genetic toxicity of caprolactam and benzoin

DNA binding in vitro was measured with 2-amino-3- methylimidazo(4,5-f)[5-3H]quinoline (3H-IQ) in the presence of S9 and microsomes from Wistar rat liver. DNA binding of 3H-IQ was catalyzed by both microsomes and S9 and was increased 5-fold in Aroclor (PCB)-pretreated animals. DNA binding was reduced by the specific inhibitor of sulfotransferase 2,6-dichloro-4-nitrophenol and slightly increased by 3'-phosphoadenosine 5'-phosphosulfate. The incubation of IQ in media with increasing concentrations of sulfate produced a modest dose-dependent increase in DNA binding. Liver S9 obtained from rats which had been administered 1% sulfite in drinking water catalyzed a higher binding of IQ to DNA, and this effect was inhibited by pentachlorophenol. The data demonstrate that sulfotransferase has a role in the activation of IQ and that variations in the activity of this enzyme induced by dietary changes may vary the genotoxic activity of IQ.     

Combining genetic, historical and geographical data to reconstruct the dynamics of bioinvasions: application to the cane toad Bufo marinus

We developed a spatially explicit model of a bioinvasion and used an approximate Bayesian computation (ABC) framework to make various inferences from a combination of genetic (microsatellite genotypes), historical (first observation dates) and geographical (spatial coordinates of introduction and sampled sites) information. Our method aims to discriminate between alternative introduction scenarios and to estimate posterior densities of demographically relevant parameters of the invasive process. The performance of our landscape-ABC method is assessed using simulated data sets differing in their information content (genetic and/or historical data). We apply our methodology to the recent introduction and spatial expansion of the cane toad, Bufo marinus, in northern Australia. We find that, at least in the context of cane toad invasion, historical data are more informative than genetic data for discriminating between introduction scenarios. However, the combination of historical and genetic data provides the most accurate estimates of demographic parameters. For the cane toad, we find some evidence for a strong bottleneck prior to introduction, a small initial number of founder individuals (about 15), a large population growth rate (about 400% per generation), a standard deviation of dispersal distance of 19 km per generation and a high invasion speed at equilibrium (50 km per year). Our approach strengthens the application of the ABC method to the field of bioinvasion by allowing statistical inferences to be made on the introduction and the spatial expansion dynamics of invasive species using a combination of various relevant sources of information.     

Utility of short-term tests for genetic toxicity

By definition, short-term tests (STTs) for genetic toxicity detect genotoxic agents, not carcinogens specifically. However, there is sufficient evidence, based on mechanistic considerations alone, to say that genotoxic agents are potential carcinogens. STTs have high statistical power, are almost always replicated, can be performed rather easily under various sets of experimental conditions, are relatively inexpensive, and detect a variety of endpoints relevant to carcinogenesis. In addition, several STTs have shown considerable utility in evaluating the genotoxic effects of real-world, environmental complex mixtures as well as the antimutagenic effects of various pure compounds and complex mixtures. STTs are likely to continue to be refined, resulting in STTs that are increasingly more relevant to human mutation and disease. Their utility should not be judged solely against the questionable standard of a rodent carcinogenicity assay.     

Evolution of testing strategies for genetic toxicity

Shortly following the inception of genetic toxicology as a distinct discipline within toxicology, questions arose regarding the type and number of tests needed to classify a chemical as a mutagenic hazard or as a potential carcinogen. To some degree the discipline separated into two sub-specialties, (1) genetic risk assessment and (2) cancer prediction since data from experimental oncology also supports the existence of a genotoxic step in tumor initiation. The issue of which and how many tests continued to be debated, but is now focused more tightly around two independent phenomena. Tier or sequential testing was initially proposed as a logical and cost-effective method, but was discarded on the basis that the lower tier tests appeared to have too many false responses to force or exclude further testing of the test agent. Matrix (battery) testing was proposed for screening on the hypothesis that combinations of endpoints and multiple phylogenetic target organisms were needed to achieve satisfactory predictability. As the results from short-term test 'validation' studies for carcinogen prediction and evaluations of EPA's Gene-Tox data accumulated, it became obvious that qualitative differences remained between predictive and definitive tests and by assembling different combinations of short-term assays investigators did not appear to resolve the lack of concordance. Recent trends in genetic toxicology testing have focused on mathematical models for test selection, and standardized systems for multi-test data assessment.     

Refinement and reduction of acute oral toxicity testing: a critical review of the use of cytotoxicity data

Acute oral toxicity testing is still required for the classification and labelling of chemicals, agrochemicals and related formulations. There have been increasing efforts over the last two decades to reduce the number of animals needed for this testing, according to the Three Rs concept. To evaluate the utility of an in vitro cytotoxicity test in our routine testing for acute oral toxicity, we have implemented in our laboratory the neutral red uptake (NRU) method, with Balb/c 3T3 fibroblasts after a 48-hour exposure, which was recommended in ICCVAM Report 07-4519, 2006. Initially, we tested 16 substances that had existing in vivo and in vitro data available, to prove our technical proficiency with the in vitro test. Then, testing was performed with 187 test substances, including a broad variety of chemicals, agrochemicals and formulations. The starting dose for acute oral systemic toxicity assays in rats (LD50) was estimated by using the prediction model presented in the ICCVAM validation study, and subsequently compared to the results obtained by in vivo testing performed according to, or similar to, OECD Test Guideline 423. Comparison of all of the 203 predicted LD50 values that were deduced from the in vitro IC50 values, with the in vivo results from oral toxicity studies in rats, resulted in a low overall concordance of 35%. The in vitro cytotoxicity assay achieved a good concordance of 74%, only for the weakly toxic substances (EU-GHS Cat. 4). However, it must be noted that 71% of the substances tested (i.e. 145/203) were classified as being weakly toxic in vitro. We further analysed the utility of the in vitro test for predicting the starting dose for an in vivo study, and the potential reduction in animal usage that this would engender. In this regard, the prediction by the cytotoxicity test was useful for 59% of the substances. However, the use of a standard starting dose of 300 mg/kg bw by default (without previous cytotoxicity testing) would have been almost as useful (50%). In contrast, the prediction by an experienced toxicologist was correct for 95% of the substances. However, this was only performed for 40% of the substances, mainly those of no to low toxicity. Calculating the theoretical animal numbers needed in several scenarios supported these results. The additional analysis, considering some physicochemical data (solubility, molecular weight, log POW), substance class and mode of action, revealed no specific applicability domains. In summary, the use of the 3T3 NRU cytotoxicity data alone did not sufficiently contribute to refinement and reduction in the acute oral toxicity testing of the substance portfolio tested routinely in our laboratory.