Regulatory forum opinion piece*: supporting the need for international harmonization of safety assessments for food flavoring substances.

The advancement of technology and the growth of international commerce underscore the need for global harmonization of regulatory safety requirements and their assessment pertaining to consumer products such as drugs, medical devices, and food. This need is particularly relevant when safety requirements involve time-intensive and costly animal safety studies. Here we present the current regulatory requirements in Europe, the United States, and Japan for flavoring substances (FSs) used in foods and point out significant differences relevant to the international standardization for safety assessments that in our opinion need to be addressed and overcome. The safety assessments that are carried out for FSs in various countries are influenced by divergent definitions of FS, by the information required and available for regulatory submission, and by different regulatory procedures, including the use of decision tree approaches. The European Food Safety Authority (EFSA), the Expert Panel of the U.S. Flavor and Extract Manufacturers Association (FEMA), and the Joint Food and Agriculture Organization (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) are making efforts to improve and harmonize the safety assessment of FSs. The application of in silico methods such as quantitative structure-activity relationships and read-across strategies relying on expert input are useful as a first-step screening of the assessment. Application of the Threshold of Toxicological Concern (TTC) approach permits conclusions that are compatible with the risk assessment approaches currently used by international advisory committees. The Japanese Regulatory Authority, on the other hand, does not yet consider in silico methods but still requires in vivo and in vitro genotoxicity test data as well as repeat-dose 90-day toxicity data in at least one species, to be submitted as the first step in the safety assessment of FSs. With this article, we echo requests that have been made for xenobiotics by the pharmaceutical industry worldwide, extending them to food-related products, especially FSs. We encourage regulatory agencies to adopt globally harmonized safety assessment procedures, regulatory guidelines, and review practices for FSs to foster global trade and to reduce costs and laboratory animal use.

International recommendations for training future toxicologic pathologists participating in regulatory-type, nonclinical toxicity studies.

The International Federation of Societies of Toxicologic Pathologists (IFSTP) proposes a common global framework for training future toxicologic pathologists who will support regulatory-type, nonclinical toxicology studies. Optimally, trainees should undertake a scientific curriculum of at least five years at an accredited institution leading to a clinical degree (veterinary medicine or medicine). Trainees should then obtain four or more years of intensive pathology practice during a residency and/or on-the-job "apprenticeship," at least two years of which must be focused on regulatory-type toxicologic pathology topics. Possession of a recognized pathology qualification (i.e., certification) is highly recommended. A nonclinical pathway (e.g., a graduate degree in medical biology or pathology) may be possible if medically trained pathologists are scarce, but this option is not optimal. Regular, lifelong continuing education (peer review of nonclinical studies, professional meetings, reading, short courses) will be necessary to maintain and enhance one's understanding of current toxicologic pathology knowledge, skills, and tools. This framework should provide a rigorous yet flexible way to reliably train future toxicologic pathologists to generate, interpret, integrate, and communicate data in regulatory-type, nonclinical toxicology studies.

Global recognition of qualified toxicologic pathologists: credential review as a potential route for recognizing the proficiency of pathologists involved in regulatory-type nonclinical studies.

Recent international summits of the International Federation of Societies of Toxicologic Pathologists have debated the desirability and potential means by which the proficiency of an individual toxicologic pathologist might be recognized and communicated throughout the world. The present article describes the advantages and disadvantages of implementing such a global recognition system by any means and provides a proposal whereby recognition might be accorded via rigorous credential review of a practitioner's education and experience.

Global Recognition of Qualified Toxicologic Pathologists: Credential Review as a Potential Route for Recognizing the Proficiency of Pathologists Involved in Regulatory-type Nonclinical Studies.

Recent international summits of the International Federation of Societies of Toxicologic Pathologists (IFSTP) have debated the desirability and potential means by which the proficiency of an individual toxicologic pathologist might be recognized and communicated throughout the world. The present document describes the advantages and disadvantages of implementing such a global recognition system by any means, and provides a proposal whereby recognition might be accorded via rigorous credential review of a practitioner's education and experience.

Global recognition of qualified toxicologic pathologists: where we are now and where we need to go.

Although there are a few national schemes for accreditation/certification of toxicologic pathologists (e.g., in Japan and the United Kingdom), a global recognition system for bench toxicologic pathologists is missing, as are universal standards defining their core competencies. This paper summarizes basic means regarding how proficiency in toxicologic pathology is acquired, provides an overview over examinations of interest to toxicologic pathologists, and emphasizes the value of practical experience in the field. The paper then discusses basic approaches to evaluate the proficiency of toxicologic pathologists and examines potential means to recognize qualified toxicologic pathologists. With progressive globalization, it is important that the toxicologic pathology community deepens the discussion regarding a global recognition mechanism for their discipline.

Serous borderline tumor of the paratestis.

Reported herein is a case of serous borderline tumor (SBT, ovarian epithelial type tumor) of the paratestis, involving the tunica vaginalis, in a 64-year-old man. The patient complained of right hydrocele; puncture cytology of the turbid fluid pointed to an adenocarcinoma. Right orchiectomy was performed and multiple micronodules were grossly observed in the paratestis. On microscopy small papillary epithelial lesions were found with psammoma bodies and intraglandular papillary lesions were irregularly recognized in the stroma of the paratestis, similar to SBT of the ovary. The tumor cells had often short microvilli. Mucin production was evident on PAS and colloid iron staining. Both papillary and glandular epithelial cells were positive on immunohistochemistry for Ber-EP4/epithelial antigen, low-molecular-weight cytokeratin (CAM5.2), cytokeratin 7 and estrogen and progesterone hormone receptors, but negative for CEA, cytokeratin 20 and calretinin. The average proliferative index was approximately 10.5% as assessed on Ki-67 (MIB-1) staining. Ultrastructurally, the cells did not demonstrate any well-developed microvilli or secretory granules and immunohistochemical findings supported SBT of Müllerian type (ovarian epithelial type tumor), while excluding a papillary type of malignant mesothelioma. The lesion in the present case was concluded to be a testicular serous tumor of Müllerian type, similar to SBT of the ovary.

Global recognition of qualified toxicologic pathologists: where we are now and where we need to go.

While there are a few national schemes for accreditation/certification of toxicologic pathologists (e.g. in Japan and UK), a global recognition system for bench toxicologic pathologists is missing, as are universal standards defining their core competencies. This paper summarizes the basic means, how proficiency in toxicologic pathology is acquired, provides an overview over examinations of interest to toxicologic pathologists and emphasizes the value of practical experience in the field. The paper then discusses basic approaches to evaluate the proficiency of toxicologic pathologists and examines potential means to recognize qualified toxicologic pathologists. With progressive globalization it is important that the toxicologic pathology community intensifies the discussion regarding a global recognition of their discipline and seeks to agree on the way forward.

Inhibitory effects of lemon grass (Cymbopogon citratus, Stapf) extract on the early phase of hepatocarcinogenesis after initiation with diethylnitrosamine in male Fischer 344 rats.

Effects of lemon grass extract (LGE) on hepatocarcinogenesis were examined in male Fischer 344 rats, administered diethylnitrosamine (DEN) at three weekly intraperitoneal doses of 100 mg/kg body weight and partially hepatectomized at the end of week 5. LGE was given at dietary concentrations of 0, 0.2, 0.6 or 1.8% from the end of week 4 for 10 weeks. All rats were sacrificed at the end of week 14. LGE reduced the number of putatively preneoplastic, glutathione S-transferase placental form-positive lesions and the level of oxidative hepatocyte nuclear DNA injury, as assessed in terms of 8-hydroxydeoxyguanosine production. In contrast, LGE did not affect the size of the preneoplastic lesions, hepatocyte proliferative activity, activities of phase II enzymes or hepatocyte extra-nuclear oxidative injury. These results suggest inhibitory effects of LGE on the early phase hepatocarcinogenesis in rats after initiation with DEN.

Lack of initiation activity in rat liver of low doses of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline.

It has been generally accepted that genotoxic carcinogens have no threshold in exerting their potential for cancer induction. However, the non-threshold theory can be challenged for cancer risk assessment in humans. Here we examined low dose carcinogenicity of a food-derived, genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), using an in vivo medium-term bioassay to detect initiating activity for rat hepatocarcinogenesis. With MeIQx initiation at various doses followed by administration of phenobarbital, a well known hepatopromoter, no induction of glutathione S-transferase placental form-positive foci, assessed as preneoplastic lesions, was noted at doses of 0.001-1 ppm. The results imply a no-observed effect level for hepatocarcinogenicity with this genotoxic agent.

Inhibition of the development of hepatocellular carcinomas by phenyl N-tert-butyl nitrone in rats fed with a choline-deficient, L-amino acid-defined diet.

Effects of phenyl N-tert-butyl nitrone (PBN), a spin-trapping agent, on the development of frank cancers were examined in male Wistar rats fed with a choline-deficient, l-amino acid-defined (CDAA) diet for 70 weeks. PBN (0.065% in the drinking water) reduced incidences, multiplicities and possibly sizes of both hepatocellular adenomas and carcinomas when administered for all 70 weeks or only for the first 26 weeks, and those of carcinomas but not adenomas, when administered only for the last 44 weeks. These results indicate that PBN can prevent the development of frank HCCs in the CDAA diet model. The anti-carcinogenic effect of PBN may be ascribed to the prevention of both the development of HCAs and their malignant conversion to HCCs. If such findings can be generalized, PBN may be able to serve as a good tool to investigate molecular mechanisms underlying carcinogenic processes.

Aberrant transcription of FHIT gene in intrahepatic cholangiocellular carcinomas induced by N-nitrosobis(2-oxopropyl)amine in hamsters.

Aberrant transcription of the fragile histidine triad (FHIT) gene was investigated in intrahepatic cholangiocellular carcinomas (ICCs) induced by N-nitrosobis(2-oxopropyl)amine (BOP) in female Syrian golden hamsters. The animals received 70 mg/kg of BOP followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with DL-ethionine and then L-methionine and administration of 20 mg/kg BOP. A total of 14 ICCs were obtained 10 weeks after the beginning of the experiment and total RNAs were extracted from each for assessment of aberrant transcription of the FHIT gene by reverse transcription-polymerase chain reaction analysis. Aberrant transcripts were detected in four out of 14 ICCs (28.6%), as absence in the regions of nucleotides (nt) -75 to 279, nt -75 to 348 and nt -75 to 447. These results suggest that alteration of the FHIT gene may play a role in a small fraction of ICCs induced by BOP in the hamster.

Multi-step lung carcinogenesis model induced by oral administration of N-nitrosobis(2-hydroxypropyl)amine in rats.

N-Nitrosobis(2-hydroxypropyl)amine (BHP) was first synthesized by Krüger et al. (1974), and has been shown to primarily induce pancreatic duct adenocarcinomas by a subcutaneous injection in Syrian hamsters. By contrast, the carcinogenic effect of BHP has been indicated at the different target organs in rats, namely the lung. When rats are received by an oral administration of BHP in drinking water for 25 weeks, a high incidence of lung carcinomas are induced, which include adenocarcinomas, squamous cell carcinomas and combined squamous cell and adenocarcinomas. So many similarities are observed in terms of not only histological appearances but also gene alterations between human and BHP-induced rat lung cancers. Moreover, the step by step development of lung lesions, from preneoplastic lesions to cancers in rat lung carcinogenesis by BHP offers a good model to investigate the mechanisms underlying the pathogenesis of lung cancers. Because data for genetic and epigenetic alterations have indeed been accumulated during the BHP-induced rat lung carcinogenesis, we will introduce them in this review and hence demonstrate that this lung carcinogenesis model provides a useful opportunity for the research on the pathogenesis of lung cancers of both humans and rats.

Foundation and progress of Japanese society of toxicologic pathology*.

The Japanese Society of Toxicologic Pathology (JSTP) has a differing conceptual framework from the Japanese Society of Pathology (JSP) and Japanese Society of Toxicology (JST) and was founded in 1985 by the leadership of late Dr. Yasukazu Nishiyama with the cooperation of several founding members and the support of JSP. The aim of the JSTP is to improve the human and animal health using an interdisciplinary scientific approach based on pathology and toxicology. In its development as a professional society, the JSTP has established society rules and activities. The JSTP has grown in terms of membership and financial aspects and is now recognized not only domestically but also internationally as a well-organized scientific society. To maintain the high professional standard and visibility of JSTP, we here provide the historical background of the society as a basis for current members to contribute to the continued improvement of our scientific organization.

International recommendations for training future toxicologic pathologists participating in regulatory-type, nonclinical toxicity studies.

The International Federation of Societies of Toxicologic Pathologists (IFSTP) proposes a common global framework for training future toxicologic pathologists who will support regulatory-type - nonclinical toxicology studies. Trainees optimally should undertake a scientific curriculum of at least 5 years at an accredited institution leading to a clinical degree (veterinary medicine or medicine). Trainees should then obtain 4 or more years of intensive pathology practice during a residency and/or on-the-job "apprenticeship," at least 2 years of which must be focused on regulatory-type toxicologic pathology topics. Possession of a recognized pathology qualification (i.e., certification) is highly recommended. A nonclinical pathway (e.g., a graduate degree in medical biology or pathology) may be possible if medically trained pathologists are scarce, but this option is not optimal. Regular, lifelong continuing education (peer review of nonclinical studies, professional meetings, reading, short courses) will be necessary to maintain and enhance one's understanding of current toxicologic pathology knowledge, skills, and tools. This framework should provide a rigorous yet flexible way to reliably train future toxicologic pathologists to generate, interpret, integrate, and communicate data in regulatory-type, nonclinical toxicology studies.

Global recognition of qualified toxicologic pathologists: credential review as a potential route for recognizing the proficiency of pathologists involved in regulatory-type nonclinical studies.

Recent international summits of the International Federation of Societies of Toxicologic Pathologists (IFSTP) have debated the desirability and potential means by which the proficiency of an individual toxicologic pathologist might be recognized and communicated throughout the world. The present document describes the advantages and disadvantages of implementing such a global recognition system by any means, and provides a proposal whereby recognition might be accorded via rigorous credential review of a practitioner's education and experience.

International recommendations for training future toxicologic pathologists participating in regulatory-type, nonclinical toxicity studies.

The International Federation of Societies of Toxicologic Pathologists (IFSTP) proposes a common global framework for training future toxicologic pathologists who will support regulatory-type nonclinical toxicology studies. Trainees optimally should undertake a scientific curriculum of at least 5 years at an accredited institution leading to a clinical degree (veterinary medicine or medicine). Trainees should then obtain 4 or more years of intensive pathology practice during a residency and/or on-the-job "apprenticeship," at least 2 years of which must be focused on regulatory-type toxicologic pathology topics. Possession of a recognized pathology qualification (i.e., certification) is highly recommended. A non-clinical pathway (e.g., a graduate degree in medical biology or pathology) may be possible if medically trained pathologists are scarce, but this option is not optimal. Regular, lifelong continuing education (peer review of nonclinical studies, professional meetings, reading, short courses) will be necessary to maintain and enhance one's understanding of current toxicologic pathology knowledge, skills, and tools. This framework should provide a rigorous yet flexible way to reliably train future toxicologic pathologists to generate, interpret, integrate, and communicate data in regulatory-type, nonclinical toxicology studies.

Expression of inducible nitric oxide (NO) synthase but not prevention by its gene ablation of hepatocarcinogenesis with fibrosis caused by a choline-deficient, L-amino acid-defined diet in rats and mice.

Expression of inducible nitric oxide synthase (iNOS) and effects of iNOS gene ablation on the hepatocarcinogenesis associated with fibrosis caused by a choline-deficient, L-amino acid-defined (CDAA) diet, were examined in male F344 rats and C57BL/6J wild-type and iNOS-/- mice. Western blot, RT-PCR and immunohistochemical analyses revealed increased expression of iNOS protein and mRNA in the livers of rats and wild-type mice fed a CDAA diet for 12-80 weeks, associated with elevated serum NO(x) and liver nitrotyrosine levels. iNOS-/- mice demonstrated greater liver injury and fibrosis in the early stage than their wild-type counterparts, but this did not significantly affect the incidence and multiplicity of altered foci, adenomas and hepatocellular carcinomas in spite of immunohistochemical iNOS expression in these lesions. Results suggested no major determinant roles of the expressed iNOS in the development of liver tumors caused by the CDAA diet.

Alterations of the M6p/Igf2 receptor gene in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine in rats.

Alterations of the mannose 6-phosphate/insulin-like growth factor II receptor (M6p/lgf2r) gene were investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 wk old, were given 2000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until killed at week 25. A total of 12 lung adenocarcinomas were obtained, and total RNAs were extracted from each for assessment of mutations and levels of aberrant transcripts of the M6p/Igf2r gene by reverse transcription (RT)-polymerase chain reaction (PCR) single-strand conformation polymorphism analysis and RT-PCR analysis, respectively. No mutations were found in exons 27, 28, 31, 33, and 34. Aberrant transcripts bearing deletions of nt 3698 to 4902, 3366 to 4902, and 3817 to 4697 were detected in three of 12 adenocarcinomas (25%). These results suggest that alterations of the M6p/Igf2r gene may be involved in the development of lung adenocarcinomas induced by BHP in rats.

Alterations of the retinoblastoma-related gene RB2/p130 in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine in rats.

Alteration of the retinoblastoma-related gene RB2/p130 was investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in male Wistar rats. At 6 wk of age, 21 animals were given 2000 ppm of BHP in their drinking water for 12 wk and then maintained without further treatment until they were killed at the end of week 25. A total of 21 lung adenocarcinomas were obtained, and total RNAs were extracted from each for mutation analysis of RB2/p130 by the reverse transcription-polymerase chain reaction-single-strand comformation polymorphism approach. No mutations were found in exons 19-22. However, examination of the expression of the RB2/p130 gene by Northern blot analysis showed mRNA levels to be significantly lower than those of normal lung tissues. Western blot analysis showed reduction of the pRb2/p130 protein in all of the adenocarcinomas examined. These results suggest that alteration of the RB2/p130 gene may play important roles in the development of lung adenocarcinomas induced by BHP in rats.