Effect of reducing the top concentration used in the in vitro chromosomal aberration test in CHL cells on the evaluation of industrial chemical genotoxicity.

A current concern with in vitro mammalian cell genotoxicity testing is the high frequency of false or misleading positive results caused in part by the past use of excessively high test concentrations. A dataset of 249 industrial chemicals used in Japan and tested for genotoxicity was analyzed. Of these, 116 (46.6%) were positive in the in vitro chromosomal aberration (CA) test, including 6 that were positive only at test concentrations >10mM. There were 59 CA-positive chemicals at test concentrations ≤ 1mM. At >1mM, 51 chemicals were CA-positive, including 13 Ames-positive chemicals, which were therefore not "missed" by the test battery. Thus, 38 potentially positive chemicals would not have been detected in the test battery if the top test concentration was limited to 1mM in CA test. Analysis of the relevance of CA results on the 38 missed chemicals was conducted based on a weight of evidence approach, including evaluations of effects of extreme culture conditions (low pH, high toxicity, or precipitation), in silico structural alert analysis, in vivo genotoxicity and carcinogenicity test data (where available), mode of action, or information from closely related chemicals. After an exhaustive review, there were four chemicals with some concern for human health risk assessment, nine with minimal concern, and the remaining 25 with negligible concern. We apply different top concentrations to the 38 missed chemicals to identify the most accurate approach for predicting the genotoxicity of industrial chemicals. Of these 2mM or 1mg/mL, whichever is higher, was the most effective in detecting these chemicals, i.e., relatively higher (8/13) or lower (17/25) detection among 13 chemicals with some or minimal concern, or 25 with negligible concern, respectively. Lower top concentration limits, 1mM or 0.5mg/mL, whichever is higher, are not as effective (2/13) for detecting these chemicals with concern. Therefore, we conclude 2mM or 1mg/mL, whichever is higher, would be an appropriate top concentration limit for testing industrial chemicals for chromosome damage.

[Analysis of drug safety information using large-scale adverse drug reactions database].

The worldwide situations of drug safety have changed dramatically. Drugs are used based on the evaluation of safety data collected in clinical practice worldwide. US Food Drug Administration collects spontaneous reports and requires manufacturers to report adverse drug reactions (ADRs) of US marketed drugs occurring worldwide. These worldwide data are available through the Adverse Event Reporting System (AERS) (about 4.1 million reports on about 3,073,340 patients, for 13 years: 1997.4th qr-2010.4th qr.). The current issues are how to analyze and utilize such large-scale safety data. Potential biases should always be kept in mind, because AERS is based on spontaneous reports. However, its huge volumes and exhaustiveness allow for sufficient scientific evaluation with the aid of current IT technology. Therefore, analysis of large-scale ADR database becomes a new research area not only from the medical science but also from the statistical viewpoint. In this report, I introduce some case studies in which we analyzed the AERS data on psychotropics including antipsychotics, antiepileptics, and antidepressants. Antipsychotics caused ADRs specific to each drug, and, in combination therapy, increased the incidences of diabetes mellitus, pancreatitis, and neuroleptic malignant syndrome; antiepileptics caused AEs (adverse events) including serious skin reactions such as Stevens-Johnson syndrome (SJS), congenital anomaly, and closed-angle glaucoma; and antidepressants caused AEs including serotonin syndrome, suicidal events, and congenital anomaly, and AEs occurring at a higher incidence for other indications, drugs often used in the elderly and AEs in combination therapy. We have analyzed ADRs associated with concomitant drug therapies using Bayesian approach. In the analysis we faced difficulties of overdispersion and we have to estimate a number of parameters, given a large number of target drugs as well as ADRs. In addition, ADR reports are not collected from uniform populations, we also have to consider the variations in the target populations. So, we use Bayesian statistics. Bayesian analysis has become feasible with advances in computer technologies and the Markov chain Monte Carlo (MCMC) methods. It allows us to analyze ADRs associated with concomitant drug therapies and estimate the ADR signals for each drug. Therefore, the analysis and evaluation of large-scale ADR database can provide important safety information in clinical practice and the studies on ADR database are the most important issues in ensuring the postmark safety of pharmaceutical products.

Establishment of a transgenic hairy root system in wild and domesticated watermelon (Citrullus lanatus) for studying root vigor under drought.

Root vigor is an important trait for the growth of terrestrial plants, especially in water-deficit environments. Although deserts plants are known for their highly developed root architecture, the molecular mechanism responsible for this trait has not been determined. Here we established an efficient protocol for the genetic manipulation of two varieties of watermelon plants: a desert-grown wild watermelon that shows vigorous root growth under drought, and a domesticated cultivar showing retardation of root growth under drought stress. Agrobacterium rhizogenes-mediated transgenic hairy roots were efficiently induced and selected from the hypocotyls of these plants. Transgenic GUS expression was detected in the roots by RT-PCR and histochemical GUS staining. Moreover, a liquid culture system for evaluating their root growth was also established. Interestingly, growth of the hairy roots derived from domesticated variety of watermelon strongly inhibited under high osmotic condition, whereas the hairy roots derived from wild variety of watermelon retained substantial growth rates under the stress condition. The new protocol presented here offers a powerful tool for the comparative study of the molecular mechanism underlying drought-induced root growth in desert plants.

Practical issues on the application of the GHS classification criteria for germ cell mutagens.

The Globally Harmonized System of Classification and Labeling of Chemicals (GHS) requires classification of chemicals on germ cell mutagenicity. The Japanese government has conducted GHS classification on about 1400 chemicals in a 2-year project (J-GHS) for implementing GHS domestically. Prior to the classification work, the technical guidance for classification of germ cell mutagens was prepared. This guidance introduces the concept of heritable mutagenicity, and presents detailed criteria for germ cell mutagens, test data to be used, and a practical decision tree for classification. These practical guidance and supporting explanations are useful for non-expert Classifiers (scientists applying the classification criteria). Several issues, however, were identified during the course of J-GHS and in re-evaluating the classification results. These include: (1) the information sources when available data are limited; (2) lack of understanding GHS classification criteria or insufficient review of the information by Classifiers; (3) varying opinions of experts on data quality and weight of evidence, and; (4) decision tree approaches, e.g., inadequacy for use in overall evaluation in some cases. Ideally, classification should be performed by Classifiers with high expertise using high quality information sources. Genetic toxicologists as experts should consider data quality and reliability, and give a critical review of all available information for support of classification. A weight of evidence approach is also required to assess mutagenic potential of chemicals. Critical points for suitable classification for GHS are discussed.

[Histamine food poisonings in Japan and other countries].

Histamine food poisonings are allergy-like food poisonings caused by the ingestion of spoiled fish containing markedly elevated histamine levels. We examined histamine food poisonings in Japan from 1998 to 2008. In average 8 food poisonings and 150 cases were reported annually and there was no fatality case. In more than 80% of remaining food samples, histamine content exceeded 20 mg/100 g. These poisonings were caused by tuna, billfish (marlin) and mackerel, which contained higher level of histamine than other fishes in histamine food poisonings in Japan. Cooking methods of these fishes were mainly "broiled". We also studied histamine food poisonings in other countries. Tuna was the main fish in histamine food poisonings reported to Centers for the Disease Control and Prevention (CDC) in the US and Ozfoodnet in Australia from 2000 to 2006. In the US, histamine food poisonings were also caused by mahimahi and escolar fish. Our review will be useful for in taking measures to reduce risk of histamine food poisonings.

[Studies on the results of monitoring of veterinary drug residues in food products of animal origin in Japan and other countries].

The positive list system was introduced in Japan for agricultural chemicals such as pesticides, feed additives and veterinary drugs in May 2006. After this time, food containing residues of these chemicals exceeding MRLs, or the default tolerance (uniform limit: 0.01 ppm) cannot be marketed in Japan. The number of chemicals to be tested for imported food at the port increased remarkably. We studied on the results of monitoring of imported food for the residues of veterinary drugs and change in trend of violation after the enforcement of the system. The number of violation of the veterinary drugs in imported food increased largely, most of them were attributed to chloramphenicol and nitrofurans in seafood from Asian countries. Monitoring of the veterinary drugs in seafood in other countries such as EU, United States, United Kingdom and Australia were also studied.

The human health burden of foodborne infections caused by Campylobacter, Salmonella, and Vibrio parahaemolyticus in Miyagi Prefecture, Japan.

To estimate the human health burden of foodborne infections caused by Campylobacter, Salmonella, and Vibrio parahaemolyticus in Japan, an epidemiological study was conducted in Miyagi Prefecture. Laboratory-confirmed infections among patients with diarrhea caused by the three pathogens were ascertained from two clinical laboratories in the prefecture from April 2005 to March 2006. To estimate the number of ill persons who were not laboratory-confirmed, we estimated physician-consultation rates for patients with acute diarrhea by analyzing foodborne outbreak investigation data for each pathogen and the frequency at which stool specimens were submitted from a physician survey. Each factor was added to a Monte-Carlo simulation model as a probability distribution, and the number of laboratory-confirmed cases was extrapolated to estimate the total number of ill persons. The estimated incidence of foodborne infections per 100,000 per year in this region estimated by this model was 237 cases for Campylobacter, 32 cases for Salmonella, and 15 cases for V. parahaemolyticus. Simulated results indicate a significant difference between our estimated incidence and the reported cases of food poisoning in this region. An enhanced surveillance system is needed to complement the present passive surveillance on foodborne illnesses in Japan to identify food safety issues more precisely, and to monitor the effectiveness of risk management options.

Monitoring minimization of grade B environments based on risk assessment using three-dimensional airflow measurements and computer simulation.

A practical, risk-based monitoring approach using the combined data collected from actual experiments and computer simulations was developed for the qualification of an EU GMP Annex 1 Grade B, ISO Class 7 area. This approach can locate and minimize the representative number of sampling points used for microbial contamination risk assessment. We conducted a case study on an aseptic clean room, newly constructed and specifically designed for the use of a restricted access barrier system (RABS). Hotspots were located using three-dimensional airflow analysis based on a previously published empirical measurement method, the three-dimensional airflow analysis. Local mean age of air (LMAA) values were calculated based on computer simulations. Comparable results were found using actual measurements and simulations, demonstrating the potential usefulness of such tools in estimating contamination risks based on the airflow characteristics of a clean room. Intensive microbial monitoring and particle monitoring at the Grade B environmental qualification stage, as well as three-dimensional airflow analysis, were also conducted to reveal contamination hotspots. We found representative hotspots were located at perforated panels covering the air exhausts where the major piston airflows collect in the Grade B room, as well as at any locations within the room that were identified as having stagnant air. However, we also found that the floor surface air around the exit airway of the RABS EU GMP Annex 1 Grade A, ISO Class 5 area was always remarkably clean, possibly due to the immediate sweep of the piston airflow, which prevents dispersed human microbes from falling in a Stokes-type manner on settling plates placed on the floor around the Grade A exit airway. In addition, this airflow is expected to be clean with a significantly low LMAA. Based on these observed results, we propose a simplified daily monitoring program to monitor microbial contamination in Grade B environments. To locate hotspots we propose using a combination of computer simulation, actual airflow measurements, and intensive environmental monitoring at the qualification stage. Thereafter, instead of particle or microbial air monitoring, we recommend the use of microbial surface monitoring at the main air exhaust. These measures would be sufficient to assure the efficiency of the monitoring program, as well as to minimize the number of surface sampling points used in environments surrounding a RABS.

[Study on pesticides residue monitoring data in some countries of the world].

In May 2006, the positive list system for residues of pesticide and veterinary medicines in food was introduced in Japan. Under this system, the number of regulated pesticides remarkably increased. The information of food items and foods/origins combinations in which pesticides were frequently detected over the detection limits and/or exceeding MRL in the foreign pesticides residue monitoring are useful to achieve the Japanese pesticides residue monitoring programme more efficiently and effectively. We investigated pesticides residue monitoring data published by food safety authorities of North America, Europe and Oceania countries in their web sites. We focused food items and foods/origins combinations which Japan imported in large quantity. Most of pesticides residues detected in twelve food items were mainly fungicides and insecticides, and some residues level of their samples were exceeding the MRLs (maximum residue limits) established in Japan. Most of pesticides residues detected in European countries were also fungicides (e.g. maneb-group and procymidone) and insecticides (e.g. pirimiphos-methyl and malathion), but herbicides were less detected. We provided the collection of Web links to information sites of MRLs and the pesticides residue monitoring reports published by food safety authorities of other countries.

[Study on usage of pesticides in various countries].

Usage of pesticides in food items in export countries was studied, focusing items which Japan imports in large quantity. Japan has imported field crops such as wheat, corn and soy bean, and also grapefruit in large quantity on a weight base, mainly from United States, Australia and Canada. While, Japan has imported various kinds of vegetables in which China had the largest share. We collected usage data of pesticides for 44 food items of 17 countries of 2004. Pesticides which were used frequently (usage rank within top ten in each item/country) were dichlorvos, carbofuran, chlorpyrifos, dimethoate (insecticides), mancozeb, carbendazim, thiophanate-methyl, chlorthalonil (fungicides), glyphosate, 2,4-D, paraquat, acetochlor (herbicides). Carbendazim, thiophanate-methyl, acetochlor and dichlorvos were mainly used in China. Dithiocarbamates are used frequently in various food items in various countries, and also frequently detected in monitoring in foreign countries. Some pesticides such as bisultap, monosultap, etaboxam and triazmate were used only in certain countries, and available information on toxicity or analytical method was very limited. Some of pesticides described above have not been analyzed in the pesticide residue monitoring in Japan before 2005,however, many of them are subjects of analysis for import food after 2006 with the enforcement of positivelist system for residues of pesticide and veterinary medicines in food in Japan.

[Chemical incidents and gathering information on toxicity].

Major cases of chemical incidents and information on chemical agents and chemical terrorist attacks are outlined. Since the late 1990s, major incidents occurred consecutively, such as two cases of sarin attack in 1994 and 1995, an oil spill from a Russian oil tanker in the Japan Sea in 1997, arsenic poisoning in Wakayama in 1998, the criticality incident at Tokai-Mura in 1999 in Japan, and terrorist attacks on September 11, 2001, in New York. The importance of crisis management and cooperation among relevant organizations has been emphasized. To provide information for an appropriate and quick response in emergencies, we prepared a Web portal site for information on chemicals including chemical agents, a chemical incident database, and links to relevant Web sites. In intentional cases of poisoning caused by toxic chemicals in Japan, 111 cases were collected mainly from a newspaper database (1984-1999). Many copy-cat poisonings occurred, especially in 1984-1985 and in 1998 just after an arsenic poisoning incident in Wakayama. Many cases occurred in the laboratories of institutes, universities, and hospitals where various types of chemicals are used.

[Trends in the utilization of internet for facilitating transparent and more active stakeholder participation in the Codex process in several countries].

Codex standards have become the benchmarks against which national food safety control measures and regulations are evaluated within the legal parameters of the World Trade Organization (WTO) Agreements. For this reason, high-level representation at meetings of the Codex Alimentarius Commission, it's related committees and ad hoc intergovernmental task forces continue to be a priority for many governments. Opportunities broaden for stakeholder input towards the development of government positions for all Codex work through the national Codex website. Some countries utilize a national Codex website as an effective communication tool between the national Codex Contact Point (CCP) and stakeholders. In this regard, stockholder participation in the national Codex preparation process is insufficient in Japan. One of the reasons for this could be the lack of information on Codex and the insufficient understanding of the work of Codex among Japanese stakeholders. To overcome these problems, more active and effective utilization of the Japanese Codex webpage should be considered. In this paper, we show analyses of recent trends of the information on Codex available from national Codex websites from six countries in order to identify the needs to establish a similar Japanese Codex website and possible contents of the site. The six websites of the national CCPs analyzed are regularly updated and utilized for the means of active information interchange between national CCPs and stakeholders, for example, providing basic general information on Codex, including its purpose, structure and meeting schedule, posting Codex working documents open for comment, and the Terms of Reference, key issues under discussion, and delegation reports of the previous sessions. Consequently, stakeholders interested in the paper could submit their comments to the delegate of the country whose contact details are made available on the website. This is one of the examples of active stakeholder participation. By establishing a similar communication system in Japan between the National Codex Contact Point and stakeholders, a more active stakeholder participation in the national codex process could be achieved. The web site could be used to provide information on the issues under discussion in each Committee and summaries of the Codex working documents circulated for the comments and their potential implications in Japan.

[Outbreaks of Salmonella in infants associated with powdered infant formula].

Historically, outbreaks associated with Salmonella-contaminated milk products were recognized as early as the 1950's in the United Kingdom and Bulgaria. In the 1960's and 1970's there were also a number of outbreaks related to Salmonella in various powdered milk products. As a result, Salmonella criterion was included in the Codex Code of hygienic practice for foods for infants and children. Between 1985 and 2005 at least 6 outbreaks of salmonellosis, involving as many as 250 infants, have been associated with powdered infant formula (PIF). In 2005, in France, an outbreak affecting more than 100 infants was associated with PIF contaminated with Salmonella Agona. These reported outbreaks indicated that problems persisted. Experts from two FAO/WHO Expert Consultations, held in 2004 and 2006, concluded that intrinsic contamination of PIF with Enterobacter sakazakii and Salmonella has been a cause of infection and illness in infants, including severe disease which can lead to serious developmental sequelae and death. Most of the Salmonella outbreaks associated with PIF involved unusual Salmonella serotypes, which likely aided in the recognition of these outbreaks. In many regions of the world where Salmonella serotyping is not routinely performed, identification of geographically or temporarily diffused outbreaks could be difficult. It is therefore important to use the appropriate methodology to detect unusual strains of Salmonella that cause illnesses in infants, such as the lactose-positive strain, and to perform serotyping and/or pulsed-field gel electrophoresis (PFGE) genotyping for rapid identification of Salmonella outbreaks and to establish linkages between the illness and implicated food.

[Development of the databases for ADI (acceptable daily intake) and relevant information on food additives, pesticides and veterinary drugs].

Databases for ADI (Acceptable Daily Intake) and relevant information on food additives, pesticides and veterinary drugs were developed. The databases we developed are easily accessible on the web, and contain ADIs, latest evaluation year, classification and use, as well as synonym and CAS registry number. The databases are designed to be easily updated by researchers as ADI and relevant information are updated or added without delay. The database for food additives has already provided from the homepage of NIHS, and the access log of the web site was 1325/month in December 2005 and 2179/month in March 2006.

Establishment of critical contamination risk locations ("hot spots") in environmental monitoring by means of three-dimensional airflow analysis and particulate evaluation.

A practical approach for the qualification of the surrounding environment of the critical area in aseptic processing has been developed. This method uses three-dimensional air velocity measurements combined with airborne particle monitoring. The analysis of the results obtained using the methods described in this article are beneficial in the selection of sample sites and frequencies and in refining personnel procedures and materials flow in aseptic processing. We propose that this improved qualification method can be widely applicable for both existing and new aseptic processing areas. This paper shows the results of one case study utilizing this method. The particle distribution map of a Grade B environment based upon extensive analysis was found to correspond to room airflow, as visualized by air vector mapping. The actual annual environmental monitoring data, which include airborne particles and microbes, as well as other microbial monitoring data, are also presented with respect to their relationship to the airflow pattern.

[Child health and chemical safety].

Recently concern over the hazards posed by chemicals to children has become more active. Many chemicals have been introduced into the market within the past several decades. These chemicals are used widely in consumer products and dispersed in the environment. Children are at risk of exposure to such chemicals. Scientific understanding has also improved about the vulnerability of children to chemical hazards. As children represent the future of our societies, protecting their health is an important issue. Thus, many actions are being undertaken by international organizations, e.g., the World Health Organization and the United Nations, and regulatory bodies in Japan, the US and the EU, based on the probable vulnerability of infants and children to chemicals. In this paper, these efforts and state measures are summarized, the characteristics of children at risk assessed, and the list of chemicals concerning child health as well as future actions in Japan are presented.

[Recent trends in evaluating risk associated with acrylamide in foods. --Focus on a new approach (MOE) to risk assessment by JECFA--].

The safety of acrylamide in foods was evaluated in the 64th meeting (2005) of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Acrylamide is classified as "probably carcinogenic to humans (Group 2A)" by the International Agency for Research on Cancer (IARC) from evidence of carcinogenicity in experimental animals and from evidence that acrylamide is metabolized to a genotoxic compound, glycidamide, in both rodents and humans. Acrylamide is also known to have genotoxicity, neurotoxicity, reproductive and developmental toxicity. In this meeting, the Committee used "Margin of Exposure (MOE)" as a new approach to risk assessment for compounds that were both genotoxic and carcinogenic, the lower the MOE the greater the health concern. JECFA calculated MOE values of 300 for the general population and 75 for consumers of large quantities of food containing high acrylamide. These MOEs were considered low and a potential human health concern. Therefore, appropriate efforts to reduce acrylamide concentrations in foodstuffs should continue. This report discusses how JECFA applied the MOE concept to the risk assessment of acrylamide in foods.

[Global trends of food safety information associated with chemicals in food].

Recently, a number of food safety problems have frequently arisen and consumer concerns have drastically increased. In order to meet these concerns, we have been publishing a biweekly bulletin called "Food Safety Information" since April 2003, monitoring the latest information from overseas on food safety. In this paper, we analyze the recent trends of information on food chemicals in the bulletin published between April 2003 and March 2005 in order to clarify the problems that need to be followed up. Among the 1,199 entries on food chemicals included in the bulletin, about 50% were from the EU and European organizations such as the FSA (UK). Approximately 20% of the total information focused on food contaminants such as heavy metals, dioxins, PCBs and mycotoxins. Scientific evidence-based information on dietary supplements and herb products was also suggested to be important to protect public health as well as food contaminants. We monitor the latest information on food safety constantly and continuously, which is important for long-term follow up of food safety issues of concern. We also provide the bulletin to the general public through the website as well as to researchers and risk managers.

[Influence of implants on human body during MRI examinations: fundamental experiment using metal balls].

It is increasingly the case that patients who have implants feel pain during high-field MRI examinations. A probable reason for the pain is the generation by irradiation of RF pulses and changing of the magnetic field gradient. As a fundamental study on the effect of implants on the human body under MRI procedures, temperature measurements were obtained from metal balls incorporated into gel-filled phantoms by using two kinds of measuring instruments, a copper-constantan thermocouple and a fluorescence fiber thermometer. At first we pursued a correlation between a copper-constantan thermocouple (absolute measurement) and fluoroptic thermometer and confirmed the precision and stability of the fluoroptic thermometer under MRI procedures. When a stainless steel ball with or without a loop antenna was used, only in the former case did the temperature rise during RF pulse irradiation. There was no significant difference between the magnetic field gradient ON and OFF. Furthermore, differences in metal (steel, aluminum, brass, stainless steel, copper) and size (5, 10, 20 mmPhi) were affected according to the increase of temperature. In conclusion, both RF pulse irradiation and a loop antenna are necessary for heat generation on the surface of metals.