Tissue cross-reactivity (TCR) studies for the development of therapeutic antibodies are conducted to estimate any possible binding sites within the human body that can be affected by the antibody when assessing safety in humans. Any possible binding sites include specific binding sites of the antibody to its target antigen and nonspecific or off-target binding sites. In TCR studies the therapeutic antibodies and immunohistochemistry (IHC) of frozen tissues must be applied in assays. However, there are technical issues with applying a therapeutic antibody or test article to IHC, such as human-on-human staining, difficulty in applying the test article to IHC, and retention of the target antigen in frozen sections. In the current review, we introduce three case studies in which these technical issues were addressed, and propose a practical scheme for points to consider when conducting a TCR study. Information on the target antigen distribution obtained through robust assays and case-by-case strategies were found to be useful for understanding and assessing the relevance of toxic effects between animals and humans. Thus, we anticipate that by considering the points discussed in the current review and combining the data with information on the biological features of the target antigens and therapeutic antibodies, it will be possible to predict safety risks in humans with higher accuracy.

The historical control database of a multinational laboratory services provider was queried for all histopathologic findings in New Zealand White rabbits which were used as control animals during a ten-year period (2011–2020). The query included all evaluated tissues, with or without microscopic findings, in studies conducted for safety testing for regulatory approval by the U.S. Food and Drug Agency (FDA) or the U.S. Environmental Protection Agency. A second query included studies conducted in the United Kingdom for control rabbits used in studies compliant with the Healthcare Products Regulatory Agency (MHRA) and/or the European Medicines Agency (EMA), which provide regulatory oversight in the United Kingdom and European Union, respectively. Infiltrates of inflammatory (mixed or mononuclear) cells were commonly noted in various organs including heart, digestive tract, muscle, thyroid, kidney, urinary bladder, eyelid, ocular structures, harderian gland, lacrimal gland, and lung. Mineralization was noted in aorta, kidney, urinary bladder, and ovary. Also noted were degeneration/necrosis in the myocardium, and intramuscular injection sites of the skin, degeneration/regeneration of muscle and diaphragm, ectopic tissue in the pancreas and thyroid, basophilic foci in salivary gland, increased/decreased vacuolation in adrenal gland, increased/decreased lymphocytic cellularity of lymph nodes, intrasinusoidal erythrocytes in lymph nodes, thymic atrophy, increased adipocytes in bone marrow, inflammatory cell foci in the liver and gall bladder, lacrimal gland atrophy, renal tubule basophilia, degeneration/regeneration, and dilatation; oviduct cyst; in the testis, degeneration/atrophy, cellular debris, dilatation, decreased sperm and segmental hypoplasia of seminiferous tubules; and squamous metaplasia of the testis and seminal vesicle.

Duplicate testes lined in series were observed in the right scrotum of a 6-week-old Sprague-Dawley rat in a single-dose toxicity study. Of the two right testicles, one was spherical and less than half the size of a normal testis. The other was oval-shaped, slightly smaller than a normal testis, and possessed clear, tortuous blood vessels similar to those of a normal testis. Each right testis was grossly separated but faced the intertesticular adipose tissue and was sparsely joined by thin cord-like structures. Only one epididymis covered or encompassed the two right testes. The caput epididymis was attached to the smaller spherical testis, whereas the cauda epididymis was attached to the oval testis. Histopathological examination revealed that the smaller spherical testis on the right side and the testis on the left side were normal. The oval-shaped testis on the right exhibited markedly dilated degenerative seminiferous tubules with one to two layers of Sertoli or germ cells, and almost no spermatogenesis was observed. Multinucleated germ cells were observed in the lumen of the degenerated seminiferous tubules. The right epididymis was morphologically normal and contained few sperm in the epididymal duct of the tail. The cord-like structures between duplicate testes comprised fibrous and adipose tissues. Single efferent ductules, ectopic cartilage, and skeletal muscle tissues were buried in the adipose tissue. To our knowledge, this is the first report of spontaneous polyorchidism in a rodent.

We report a histiocytic sarcoma originating from the epididymis observed in a 110-week-old male CD-1 mouse in a carcinogenicity study. At necropsy, no lesions were observed in the epididymis. Histologically, a neoplastic lesion was observed in the cauda of the epididymis that was well demarcated from the surrounding tissues. The lesion mainly consisted of spindle-shaped tumor cells with oval to elongated nuclei and abundant eosinophilic or foamy cytoplasm. The tumor cells were arranged in a fascicular pattern, interlacing bundles, or a whorl pattern. The nuclei showed mild atypia with irregular shapes and varied sizes, whereas few mitotic figures and no typical multinucleated cells were observed. The epididymal ducts remained within the neoplastic lesion, and the tumor cells invaded between the epithelium and the smooth muscle layer of the epididymal duct. Immunohistochemically, the tumor cells were positive for vimentin and macrophage markers (Iba1, CD204, F4/80, and Mac-2) but negative for cytokeratin and other mesenchymal cell (α-smooth muscle actin, desmin, CD31, and platelet-derived growth factor receptor-β), neural cell (S-100 and nestin), or Leydig cell markers (calretinin). Proliferating cell nuclear antigen-positive tumor cells were sporadically observed in the lesion. Based on these results, the tumor was diagnosed as a histiocytic sarcoma originating from the epididymis. This report provides additional histopathological evidence of spontaneous histiocytic sarcomas originating from the epididymis of aged mice.