Preclinical Toxicity Studies for Regenerative Medicine in Japan.

PURPOSE: Advances in methods designed to evaluate preclinical toxicity have not kept up with progress in regenerative medicine. Preclinical toxicity studies of regenerative therapies must be designed logically and should be flexible to accurately reflect toxicity of products under development. The purpose of this review is to discuss requirements of preclinical toxicity studies of this type developed in Japan. METHODS: We conducted MEDLINE and PubMed literature searches to identify recent reports relevant to regenerative medicine. Information regarding approved drugs and public announcements, including existing guidelines and guidance in Japan, was collected from the website of Japan's Ministry of Health, Labor and Welfare (https://www.mhlw.go.jp/index.html) and the Pharmaceuticals and Medical Devices Agency (https://www.pmda.go.jp/). FINDINGS: Four cell therapy products have been developed and approved in Japan so far. The principal preclinical toxicity data submitted to regulatory authorities in the Pharmaceuticals and Medical Devices Agency in Japan are summarized here. The potential for tumor formation, a major concern in such clinical applications, is assessed in 3 ways: tumor-forming capacity of the original cell, quantitation of residual pluripotent stem cells in the product, and the possibility that a tumor will form at the product's engraftment site. Although gene therapy and oncolytic virus products are under development, these types of products are not yet approved in Japan. Guidelines relevant to the development of these products are now being created based on existing guidelines and considerations established by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use. IMPLICATIONS: Because of cell tropism and heterologous immunity, animal species or strains useful for preclinical studies of regenerative therapies are often restricted. Nonetheless, preclinical toxicity studies must be designed to predict results relevant to humans.

A repeated 28-day oral dose toxicity study of nonylphenol in rats, based on the 'Enhanced OECD Test Guideline 407' for screening of endocrine-disrupting chemicals.

A 28-day repeated oral dose toxicity study of nonylphenol (NP) was performed for an international validation of the 'Enhanced OECD Test Guideline 407' paying particular attention to the sensitivity of individual endocrine-related parameters. Sprague-Dawley rats, each group consisting of ten males and ten females, were administered NP once daily by gavage at doses of 0 (control), 10, 50, or 250 mg/kg body weight. At 250 mg/kg, three females died or became moribund during the experiment. At this dose, hepatic and renal toxicity was evident in both sexes with increase of relative liver and kidney weights as well as histopathological changes, such as centrilobular liver cell hypertrophy and a variety of renal tubular lesions, and alteration of serum biochemical parameters, some of them being evident from 50 mg/kg in females (glucose and inorganic phosphates). Hematologically, development of anemia was evident at 250 mg/kg in both sexes. Regarding endocrine-related effects, increase of thyroid weight in males was detected from 50 mg/kg. At 250 mg/kg, males exhibited reduction of relative weights of the ventral prostate and seminal vesicles, and females developed irregular estrous cyclicity and vaginal mucosal hyperplasia. Although changes in serum hormone levels were detected in both sexes, magnitude of the changes was small to be regarded as a low toxicological significance. In summary, repeated oral doses of NP to rats for 28 days resulted in hepato-renal toxicity from 50 mg/kg and anemia at 250 mg/kg. Effects on the endocrine system were observed from 50 mg/kg, and assessment of weights and histopathology of endocrine-related organs and estrous cyclicity may be valid in a battery for detecting endocrine effects of NP. The no-observed-adverse-effect level of NP was estimated to be 10 mg/kg per day.

Antibodies that label paraffin-embedded mouse tissues: a collaborative endeavor.

Histology and immunohistochemistry are important tools in the study of human diseases and their respective animal models. The study of mouse models has been hampered by the absence of a large set of mouse-specific antibodies adapted to paraffin-embedded tissues. A total of 196 antibodies were tested on paraffin-embedded mouse tissues preserved in five different fixatives (Fekete's acid-alcohol-formalin, 10% neutral buffered formalin, 4% paraformaldehyde, IHC Zinc Fixative, and Bouin's fixative). The antibodies were targeted to proteins of the cytoplasm (n = 100), plasma membrane (n = 48), nucleus (n = 36), extracellular compartment (n = 5), cytoplasm/cell membrane (n = 4), and viral proteins (n = 3). A total of 83 antibodies provided an adequate signal to noise ratio. Of these, adequate labeling required heat-mediated epitope retrieval or enzymatic digestion for 32 and 8 antibodies, respectively. Epitope recognition was best for tissues fixed with Fekete's acid-alcohol-formalin. However, some proteins could be detected only in IHC Zinc Fixative, confirming that there is no single fixative suitable for the preservation of all epitopes. Four of 13 antibodies that failed to label their cellular targets on tissue sections successfully labeled whole-mount tissues, indicating that tissue processing plays an important role in epitope degradation. Regularly updated information on immunohistochemistry of normal and neoplastic mouse tissues is accessible online at (http://tumor.informatics.jax.org); links to antibody suppliers' web sites are provided.