2016: Types and Incidence of Neoplasms Wistar vs Spragues

2015: Evaluation of Medical Device Studies

2015: Dextran-Sulfate-Sodium-induced Colitis

2014: Tox Studies with Nanoparticles and Detection Methods

2014: Methyl Methanesulfonate Induced Sperm Lesions

2013: Animal Tumor with Questionable Relevance to Human Risk

2013: Lesions in the Reproductive System of Lab. Animals

2013: Histopathology in Animals receiving Nanomaterials

    Histopathology in Animals receiving Nanomaterials - General aspects & Points to consider

    Yoshimasa Okazaki, AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland


    Original source:

    6th Harlan Global Seminar/Symposium. October 31, 2012; Tokyo, Japan

    Speaker: Dr. Yoshimasa Okazaki (Presentation and Talk in Japanese)




    Very small materials, so-called nanomaterials, have the ability to enter, translocate within, and damage the living organisms. 

    Not only the size effects, nano scale-specific characteristics in surface area, surface chemistry, electromagnetic properties, composition, solubility, crystal structure, and agglomeration state are bringing about unknown toxicity concerns that differ from the hazardous effects caused by the "bulk" materials.

    However, the misapprehension of nanotoxicity may create a general fear that all nanomaterials are toxic.

    Meanwhile it is the fact that nanomaterials have certainly beneficial, fascinating dimension and possibilities leading to growth of several industries and advances in medicine.

    It is therefore important to realize and evaluate properly the biologic events caused by nanomaterials.

    As a first step for understanding the biologic events, this lecture will mainly talk about the representative morphological changes that could be observed in respiratory, cardiovascular and nervous systems of experimental animals treated with some kinds of nanomaterials.

2013: 8-Methyloxypsoralen, ESTP Seminar

2013: Hexachlorophene, ESTP Seminar

2013: Classification of Neural Tumors in Laboratory Animals

    Classification of Neural Tumors in Laboratory Animals

    Klaus Weber, AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland


    Neoplasms of the nervous system are by numerous pathologists and toxicologists considered to be a difficult topic.  Such neoplasms are not often recorded as spontaneous lesion and hence, there may be little experience. However, as lesions of other organ system, nervous system neoplasia implies rules consisting of sex preference, location, age at tumour growth onset and diagnostic features.

    The most common nervous system neoplasia in rats is the granular cell tumour, a neoplasm of the brain leptomeninx. It is the only tumour that may be recorded at a mean incidence of higher than 1% (RCC Historical Data: males – 1.54%, females – 0.9%). Regarding sex preferences, in Wistar rats, granular cell tumours and glial tumours were found at higher incidences in males than in females.

    Regarding the CNS, the spinal cord was found to be affected at 2.14% only (11705 Wistar rats). By mapping, preferred locations could be established for most tumour types of the brain. In the peripheral nervous system, there are generally not a lot of tumours, but the most commonly recorded neoplasm is the schwannoma. Other neural crest tumours may be recorded in the adrenal medulla, eye, skin and less common in ganglia etc..

    The onset of spontaneous tumour development cannot be established in routine oncogenicity studies. However, the time point of diagnose (day of death) revealed significant differences between tumour types. By increasing age, the following statistical differences were recorded: oligodendroglial tumours > astroglial tumours > granular cell tumours > other meningiomas).

    The most important diagnostic feature on nervous system neoplasms depends on the expressed architecture within the neoplastic tissue.  Immunhistochemical analysis is usually to confirm the diagnosis. The aforementioned architectures are usually structures of evidence.


2013: Fluensulfone



    Klaus Weber1), Christian Strupp2), Deborah A. Banas3),  Samuel M. Cohen4),  Elliot Gordon5),  Martina Jaeger6),

    1) AnaPath GmbH,  Oberbuchsiten, Switzerland

    2) Makhteshim Agan Holding B.V. Schaffhausen Branch, Schaffhausen, Switzerland

    3) Experimental Pathology Laboratories Inc., Sterling, VA 20167-0169, United States of America

    4) University of Nebraska, Medical Center, Omaha, NE 68198-3135, United States of America

    5) Elliot Gordon Consulting LLC, Princeton Junction, NJ 08550, United States of America

    6) Harlan Laboratories Ltd., Rossdorf, Germany


    The fluoroalkenyle(-thioether) Fluensulfone  (CAS No.: 318290-98-1) is a new systemic, non-fumigant nematicide for the control of nematodes in agricultural and horticultural crops. It has an activity  on multiple physiological processes which suggests a new MOA. Therefore, it is believed to be unique and different from currently known nematicides and insecticides.

    Regulatory oncogenicity studies were performed in CD-1 mice.  Bronchiolar epithelial hyperplasia increased at 200 and 1200 mg/kg in  incidence and severity. In addition, bronchiolar adenomas increased statistically significant in females at these doses.  The basophilic and multifocal hypertrophic, mainly non-ciliated appearance of the hyperplastic epithelium was deemed  to be of Clara cell origin that was proven by electron microcopy.

    In a cell proliferation study, under treatement with fluensulfone and the positive control isoniazid, there was an approximately four-fold increase of cell proliferation after 3 days. After 7 days, the findings recovered. A comparative in vitro metabolism in mouse and human lung microsomes revealed  in the presence of inhibitors of CYP2E1 and Cyp 2f2 that CYP 2E1 played no important role in this particular metabolism.  In contrast, Cyp 2f2-inhibitor partly inhibited the metabolism. The results revealed that after 120 minutes, the fluensulfone was almost completely metabolized in mouse lung microsomes whereas in the human lung microsomes there was no indicator for metabolic transformation.

    Metabolic activation in mouse lung of xenobiotics by Cyp 2f2 is a well-described phenomenon and its specificity to the mouse has been well-documented.

    The induction of an  increase in cell proliferation leading ultimately to hyperplasia, adenomas and carcinomas is also a well-documented sequence of events for lung tumorigenesis in the mouse. With data already available regarding fluensulfone, there is considerable confidence that the mode of action is as described and that alternative modes of action, especially genotoxicity, can be excluded. Given the information that is available one can confidently conclude that the mode of action for fluensulfone induced lung tumors in mice is known and not relevant for humans.

2013: Induced lesions in the nervous system of lab animals

2013: Bone Marrow Differentiation in Toxicity Studies

    Bone Marrow Differentiation in Toxicity Studies: Sense and Non-Sense


    Klaus Weber, AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland


    Bone marrow smears should be collected, stained and archived in every study. Bone marrow differentiation (BMD) should never be performed as an alone-standing evaluation.. The smear may reveal no changes in Myeloid:Erythroid (M:E) ratios, although a section could reveal hypocellularity. Furthermore, changes in peripheral blood parameters are not necessarily associated with changes in the bone marrow. In contrast, if changes are obvious in bone marrow sections, there are also changes in BMD. BMD is work-intensive and therefore expensive. It could be used to establish the NOEL/NOAEL. In such case  it should be performed in controls and test item-treated groups not showing changes in bone marrow sections and hematology. Often, BMD is helpful in mechanistically considerations. It need also  to be  considered, that changes of a single cell populations will often not lead to changes in the bone marrow section or peripheral blood or vice versa, that the change of only a single subpopulation may be responsible for the changes detected elsewhere. For the latter reason, BMD should be performed in any case of precursor or abnormal cells in the peripheral blood. 

    Hematology parameters are often affected in toxicity studies. Indicators for anemia are often the trigger to perform bone marrow differentiation. Bone marrow differentiation (BMD) however is time-consuming and expensive. Well discussed routine histological sections  may be helpful to avoid unnecessary work to be performed.

    Changes in the bone marrow recorded in routine pathology sections include increased or decreased granulopoiesis or erythropoiesis, fatty replacement, myelofibrosis and other changes. If there are changes in bone marrow section, than there are changes in smears. The value of BMD in groups that are affected by those changes is doubtful and  the question arises on the additional value of such investigation. In contrast, examples should be examined from groups where sections did not indicate induced lesions. In this case, BMD is an interesting tool to establish threshold effects (e.g. PPAR agonists)

    In most cases of anemia, there are no changes in the bone marrow. Otherwise, evidence of changes in  single cell populations in the peripheral blood does not imply changes in bone marrow

    Generally, the whole spectrum of lesions recorded in the dose group of interest need to be considered to judge BMD. Every inflammatory or degenerative change may affect the results. Therefore, side effects by the route of application need to be considered as well, e.g. inflammatory lesions due to emboli induced by liposomes.

    BMD is not a routine tool in oncogenicity studies due to the effects on bone marrow by chronic diseases and neoplasia, however BMD may be helpful in understanding induced lesions or to determine types of systemic neoplasia.

2013: Histopath. Approaches for Medical Device Evaluation

    Histopathology Approaches for Medical Device Evaluation

    Weber, K.

    AnaPath GmbH, Oberbuchsiten, Switzerland


    Medical device (MD) studies are usually non-routine studies, except for batch controls. Special designed protocols are under discussion before the study starts.  The studies may become even more complex during performance, e.g.  testing strategies may change during the evaluation of interim sacrifices. Different classes of test items (i.e. stents, bone replacement, dental materials or replacement, engineered tissues, etc.) need different approaches for histological evaluation. The armamentarium is not necessarily restricted on the use of different techniques (i.e. staining methods, immunohistochemistry, hard material embedding, image analysis, electron microscopy, etc.) but also needs a change in thinking on what constitutes the most adequate evaluation. The variety of MDs results in diverse methods of evaluation. It is not difficult to evaluate a subcutaneous implantation site or the mucosa in a cheek pouch test using a scoring system. However, MD studies are not only  undertaken to proof tolerance but also efficacy.  In some cases, the study design, including the method of evaluation, is well described by publications or guides, e.g. for coronary stents.  However, well-described methods may need modification when using stents to cover aortic aneurysms. An evaluation scheme is therefore often prepared specifically before study start and may change during evaluation. In most cases, the local tolerance and the implant-tissue surface is of main interest. Nevertheless, there are circumstances causing degeneration in adjacent tissues that were not predicted in study protocols. Furthermore, implantation techniques may alter the integration of the MD into the tissue or even their functionality. For some MDs, the selection of the test species may affect the outcome of the study, e.g. for dental implants, often the dog is used although it should be considered a compromised model. Otherwise, the size of the test species may be an important factor, e.g. using engineered tissues as vascular grafts or even as replacement for heart valves. For the latter type of implants, special strategies for testing are necessary prior to implantation, e.g. the proof of cell population presence used during the engineering process.  


2013: Role of Toxicologic Pathologist

    Role of Toxicologic Pathologist

    Weber, K.

    AnaPath GmbH, Oberbuchsiten, Switzerland


    Is there is only one main difference between  diagnostic and toxicologic pathology? Whereas in diagnostic pathology all available data obtained from a single individual have to be considered, in toxicologic pathology a group of animals is evaluated.  Pathology is an integrative discipline. The way to think about chemical or biological structures, molecular biology, physiology and morphology as it is necessary for candidate selection, early development or preclinical/toxicology studies need training and experience.

    Nevertheless, the majority of human and veterinary pathologists are by training not focused on pre-clinical toxicologic pathology. Therefore, special courses for further education are organized on national and international levels. Experience does matter. A major fact to reach a high level of experience is due to cooperation with other pathologists, training periods in other institutions, slide seminars, but also by participation in pathology working groups etc. Learning success is determined by personal behavior and not at least driven by the acceptance of more experienced opinions.

    To understand the underlaying mechanisms in induced lesions, the cooperation with the study director is essential in obtaining background information. Before study start, the pathologist should be involved in the design of the protocol. Detailed discussions with the sponsor on test item structures or the composition of a medical device, intended indications (medicinal, chemical, agrochemical), results of previously performed studies with the test item under investigation or similar compounds etc. will allow best possible interpretations of study results. The credibility of the results increases by peer review.

    A toxicologic pathologist should have profound knowledge in basic sciences including molecular biology, immunology, chemistry, physiology, morphology etc. but need to be also trained in general pathology. The understanding of special pathology is obligatory to understand possibly background lesions. Professional ethics cannot be neglected taking into consideration the possible harm by wrong interpretations for both the usage of  a test item in the future or the losses for the sponsor. 

    It is questionable if a board certified pathologist is the better pathologist considering the limited education in toxicologic pathology. There is a lack in specific training in the latter field. 


2013: Evaluation of environmental toxicants using sentinels

    Evaluation of environmental toxicants using local sentinels

     Weber, K.

    AnaPath GmbH, Oberbuchsiten, Switzerland


    Environmental disasters raise public attention. They may be of local interest, or affecting national or even international ecological and economical interests. The list of such ‘accidents’ is long.

    Well known examples of international significance are oil spills (e.g., 2003 Galicia (Costa de Muerte), 2010 Golf of Mexico).

    National disasters were by toxic floods due to mine accidents (e.g., 1998 Aznalcóllar, Sevilla), or eutrophication (e.g. 2005/2010 blue algal bloom in the Baltic See). Mass growing  of toxic algae like Pfiesteria piscicida in the 1980s and 1990s on the coast of North Carolina and Maryland caused widespread death of fish and affected human health. However, also introduced pathogens (e.g. Batrachochytrium dendrobatidis related to global amphibian decline) cause tremendous damage in ecosystems.

    Most investigations are performed by non-profit making public institutions and universities in cooperation with facultative participants. There are national and regional monitoring programs like:


    Mussel Watch

    introduced in 1975, that uses bivalves widely chosen in the aaquatic environments as an ideal bio-monitors. Mussel Watch Program is the longest continuous, nationwide contaminant monitoring program in U.S. coastal waters. The program analyzes sediment and bivalve tissue chemistry for a suite of organic contaminants and trace metals to identify trends at over 300 selected coastal sites from 1986 to present. 


    Bioeffects Assessment Program

    identifies and assesses biological effects associated with contaminant exposure. The investigational data include: sediment chemistry, toxicity, and species diversity and quantity for the same suite of organic contaminants and trace metals as the Mussel Watch Program


    Where are we involved as pathologists?


2013: Evaluation of Environmental Toxins: Fish and Frog

    Evaluation of Environmental Toxins: Fish and Frog used as Models

    Weber, K.

    AnaPath GmbH, Oberbuchsiten, Switzerland


    Aquatic toxicology includes the development, application, and validation of methods to assess the effects of contaminants, degraded water quality, and other factors on aquatic organisms.

    The first observations on estrogenic effects of DDT were made in the fifties. Thereafter, reports on environmental pollutants with estrogenic properties increased. In the nineties, concerns raised about influences on ecosystems including fish populations. 

    If a species is not considered endangered, any ecological risk assessment with toxic chemicals focus on population levels. Therefore, end points need to be interpreted for a whole population (all species within this ecosystem).  These endpoints include survival, growth and reproductive success (gonadal development, egg production, fertility, hatching success). Biomarkers of exposure, as vitellogenin induction in males are considered to be powerful tools in tracking single substances and mixtures of concern. Such biomarkes link field and laboratory data.

    To assess possible endocrine disruptors, after prioritization of chemicals,  would be a test in fish to identify chemicals that affect reproductive function through alterations in the HPG axis, followed by more extensive tests (full life cycle and multigenerational tests).  Different laboratory fish species, the Japanese medaka (Oryzias latipes), the zebrafish (Danio rerio), and the fathead minnow (Pimephales promelas) were suggested.

    Fish are used in the laboratory however not only for endocrine disruptor testing. Toxic pollutants, including   metals, pesticides, and other organics pose serious risks to many aquatic organisms. 

    Evaluation of environmental toxins. Frog and snail used as animal models.

    Ecotoxicology deals with the impact of chemical compounds on the animate environment.

    Main issues that may be considered for testing on endocrine activity on aquatic organisms includes:

    -           product safety for humans

    -           product safety in environment

    -           economical considerations

    A high incidence of developmentally deformed frogs was observed in North America. This is associated with a worldwide decline of amphibian species. It was suggested that environmental changes were causative factors. As a consequence, namely endocrine disruptors are under test. The latter is reflected in the Amphibian Metamorphosis screening assay (Endocrine Disruptor Screening Program) that  is intended to identify chemicals which may interfere with thyroid-dependent processes the currently only existing assay that detects thyroid activity in an animal undergoing morphological development.

2013: Endocrine Compounds affecting Snails

    Endocrine Compounds affecting Snails


    K.Weber, D. Mahnke

    Invertebrates play an important role in ecosystems as herbivores, carnivores, and destruents, thus contributing to the maintenance of energy flow and cycles of matter. With a number of approximately up to 130’000 living species, the diverse molluscan phylum can be found  in almost all biotopes world-wide. There were many discussions about the use of molluscs to control effects of chemicals to the environment. Consequently, there is a number of publications available dealing with the effects of chemicals on wild living bivalves and gastropods.

    The extent of possible effects of chemicals on molluscs came into public regard with the dramatic effects of tributyltin (TBT) compounds, which have broadly been used as antifouling agents for ships. The females of the Dog Whelk (Nucella lapillus) and of at least 160 further species developed male parts in addition to the female genital organs, a syndrome named imposex. Imposex and intersex induced  by TBT are meanwhile described worldwide for more the 160 snail species. Misinterpretation for endocrine effects in testing products on snails need to be elucidated by histopathology. Mainly irritative effects (inflammation, degeneration etc.) and parasitic infections may be primary causes of changed physiological parameters (e.g. fertility, fecundity).

    There are even proposed strategies for the use of molluscs as multidisciplinary models in ecotoxicity studies mainly due the fact that some metabolic pathways shows homologies to vertebrates. Since 1998, there is a guideline for the testing of chemicals in marine bivalves (ASTM E724-98). Currently, there is a guideline for testing on marine and freshwater molluscs under discussion. 


2013: Induced and Spontaneous Lesions in the Larynx

    Induced and Spontaneous Lesions in the Larynx of Laboratory Animals

    Weber, K.

    AnaPath GmbH, Oberbuchsiten, Switzerland


    Little is published on induced and spontaneous lesions in the larynx of laboratory animals, although the hamster, mice, rat, rabbit, dog and monkey are species used in inhalation toxicology.  Even detailed descriptions for the histological evaluation are limited (Renne and Gideon, 2006; Renne et al., 1992, 1993, 2007;  Sagartz et al.,1992).


    In order to collect information on the usefulness of trimming techniques, the influence of different vehicles, the impact of different application routes in toxicity studies, and differences between induced vs. spontaneous lesions, the author compiled and reviewed the data obtained from a high number of studies performed at  RCC Itingen during 1999 – 2007.  

    In inhalation studies, the trimming and cutting of the rat larynx according to Sagartz et al. (1992) revealed most lesions compared to the usually longitudinal section plan applied in non-inhalation studies. In the latter technique, there is a loss of information on lesions in the ventral aspect of the larynx. The larynx of other rodent species is trimmed similarly at RCC Itingen as described for rats.

    An interesting trimming technique was found to be the   sagittolongitudinal sectioning described by Germann et al. (1998) which result in a very satisfying overview on the laryngeal structures in the complete course of the laryngeal length. Furthermore, it was deemed to be very important that this technique provide information on the current contents in the laryngeal lumen that may be influenced by impacted food particles or even the test item. The latter technique was helpful for mechanistic considerations in studies with a high number of decedents by asphyxia. 

    For dogs, in all studies performed at RCC Itingen, two sections per larynx were made, one sagittolongitudinal section through the epiglottis and one transversal section through the corpus crossing the region of the lateral laryngeal pouches. The cutting planes are deemed to be very consistent, however, the histological patterns of epithelia are found to be varying at these locations mainly for the transversal sections.  The same technique is applied for rabbits and minipigs.


    The comparison of lesions encountered in control animals of inhalation studies (rats, mice, dogs) treated with different vehicles (air, lactose, Mg-stearate, saline), did not reveal differences in the type, distribution pattern, incidence and/or severity of spontaneous lesions. Furthermore, the type, incidence and quality of spontaneous lesions recorded in dog studies did not differ between inhalation and non-inhalation studies. In rodents, the type of lesions was not different comparing studies of different application routes in non-inhalation studies compared to inhalation studies, however, the incidence completely depends on the different trimming technique used for these study types.


    The pattern of spontaneous lesions in the rodent larynx was found to be dominated by degenerative and inflammatory lesions starting most often in the submucosal glands by dissected secretions followed by mineralization and local inflammation or were induced by impacted foreign bodies, mainly food remnants but rarely hairs. In level 6 according to Sagartz et al. (1992), spontaneously existing squamous metaplasia was recorded as a spontaneous lesion only in male Wistar rats from Inhalation studies as far (13-Week Studies: 0.5±1.7%, 26-Week Studies: 2.2±5.0%, Oncogenicity study: 20.0%).

    Nevertheless, the squamous metaplasia of the respiratory epithelium overlaying the ventral gland in level 6 is the most important induced lesion in rodents.  The metaplastic change recorded in the larynx of rodents is a very important issue of discussions on its prospective behaviour. However, this lesion was never seen to turn into neoplasia. It is reversible in nature and an indicator of the especially high sensitivity of the rodent larynx to any mild irritant.

    Other induced lesions in inhalation studies consisted of submucosal edema, necrosis, inflammation and/or formation of granuloma. Induced lesions in non-inhalation studies were found to be exclusively related to reflux laryngitis.

    It is concluded, that in rodents induced lesions of the larynx differ in type, distribution pattern, severity and incidence from spontaneous lesions.

    Neoplasia in the rodent larynx is an extreme rare occasion. An induced spontaneous carcinoma in the hamster and a spontaneous adenomatoid lesion of a control rat will be demonstrated.


    In dogs, the range of spontaneous lesions was found to be even more limited compared to rodents. Inflammatory lesions are rare events and often restricted to the submucosal seromucinous glands or related to the impact of foreign bodies. Occasionally, a minor erosion or inflammations of the epiglottis or the glottis mucosa in control animals were considered to be induced by trauma. In contrast to rodents, however, there is no possibility to differ between induced and spontaneous lesions except the incidence. Again, a significant difference in lesions observed in inhalation and non-inhalation studies could not be demonstrated.


    Germann PG et al., Toxicol Pathol, 26: 283-289. 1998

    Renne RA, Gideon KM. Toxicol Pathol, 34: 281-285. 2006

    Renne RA et al., Toxicol Pathol, 35: 163-169. 2007

    Renne RA et al., Toxicol Pathol, 20: 44-51. 1992

    Renne RA et al., Toxicol Pathol, 21: 542-546. 1993

    Sagartz, J.W. et al.: Toxicol Pathol,  20: 118-121 (1992)


2013: Toxicologic Pathology in Inhalation Studies

    Toxicologic Pathology in Inhalation Studies


    Weber, K.

    AnaPath GmbH, Oberbuchsiten, Switzerland


    Inhalation toxicology is a challenge for pathologists. There are organs and tissues under examination that for other studies may be not regarded, e.g. the nasopharyngeal duct. Furthermore, there are cut-accompanying tissues visible that under other circumstances may not be considered, e.g. Steno’s glands. Some of these tissues are of high metabolic functionality, e.g. P450 contents of olfactory mucosa, or may be even totally different from the study species to the target species, e.g. P450 enzymes in Clara cells.  Therefore, a deep knowledge of the anatomical and  physiological situation of these organs is the basis for a perfect evaluation and the avoidance of misinterpretation. The study pathologists need basic knowledge of inhalation toxicology and technique. Other factors influencing the outcome of an inhalation toxicity study consist of a high quality level technical preparation of the tissues and the most adequate fixation. A considerable experience level is necessary for risk assessment.   

    The presentation gives an overview on the organs and tissues necessary to evaluate in this type of studies in different species. The technical considerations are explained and examples for possible artifact inductions are presented. For the evaluation, mapping procedures, and evaluation techniques are presented. This presentation bases on experiences made during 20 years on different compound classes and includes examples of pharmaceuticals, chemicals, agrochemicals, cosmetics and carriers.


2013: Path. Evaluation involved in the Development of ATMPs

    Pathology Evaluation involved in the Development of ATMPs


    Weber K1),  Füner  J4), Gähler S1), Hofman-Hüter, H2), Janke O4), Lehmann  J3), Riedel W2), Schulz R3), Weidmann K2)


    1) AnaPath GmbH,Oberbuchsiten,Switzerland

    2) BSL Bioservice Scientific Laboratories GmbH,Planegg,Germany

    3) Fraunhofer Institute for Cell Therapy and Immunology,Leipzig,Germany

    4) preclinics GmbH,Potsdam, German


    Advanced Therapy Medicinal Products (ATMPs) consist of three major therapeutical groups including gen therapeutics, somatic cell therapeutics, and engineered tissues. A number of ATMPs is classified and listed under the ‘Summaries of scientific recommendations on classification of advanced-therapy medicinal products’ by the EMA. All ATMPs contain partially or consist fully of living cells or tissues. A further option is the combination of ATMPs with medical devices. Therefore, ATMPs are complex, and the preclinical testing differs in many aspects from ‘classical’ testing strategies. 

    For example, the preclinical testing of a recombinant HSV-1 containing the gene encoding the

    human GM-CSF (therapeutic effect is related directly to the product, GM-CSF, from the expression of the added sequence) may include the testing on virus replication, transgene expression (site, duration), biological activity and efficacy vs toxicity, etc.  Considering such aspects, a first question is told about the test model often being different from conventional animal models, e.g., immunocompromized mice, immunocompetent mice, hamsters, tumor models.

    Testing strategies may be totally different when using somatic cell therapeutics or stem cells, e.g. xenogeneic islets will have different safety and efficacy concerns than human stem cell-derived progenitor, beta, or islet cells. Furthermore, while a number of models are useful for various aspects of type-1 and type-2 diabetes, there is no one model serving as the gold standard. Conventional animal models may serve for toxicity studies, e.g. recent studies described the transplantation of microencapsulated neonatal pig islets in an alginate matrix to confirmed their biocompatibility in non-diabetic monkeys. In addition, this group of ATMPs requires tumorigenicity studies. The latter is also true for the testing of engineered tissues.


    The requirements on non-clinical development of cell-based medicinal products (CBMPs) are fixed in the EMA-GUIDELINE ON HUMAN CELL-BASED MEDICINAL PRODUCTS (Doc. Ref. EMEA/CHMP/410869/2006), Draft guideline on the risk-based approach according to Annex I, part IV of Directive 2001/83/EC applied to Advanced Therapy Medicinal Products (Doc. Ref. EMA/CAT/CPWP/686637/2011). The scrutiny applied during non-clinical testing should be proportional to the risk expected to be associated with clinical use. Therefore the approval for clinical application of a certain CBMP requires special procedures with regard to their efficacy and safety testing. Important endpoints are the assessment of toxicity related to de-differentiation or loss of cell function, cell transformation, and tumorigenicity (summarized as “Tumorigenicity”), or ectopic engraftment in non-target tissues (“Biodistribution”). The induction of an immune response against the CBMP itself and/or towards cell-derived pharmacologically active substances might significantly modulate the efficacy. Therefore, the possible immunogenicity of a CBMP should be considered. Moreover, the development of autoimmunity has to be considered when cells are used for immunotherapy purposes, e.g. cancer immunotherapeutic products (summarized as “Immunotoxicity”).

    Finding a relevant animal model may be challenging as, strictly speaking, the only relevant species for testing human cells – when all aspects including receptors, cytokines and micro-environment are considered – is the human being itself, and so any animal model can be expected to have inherent limitations. Therefore, immunotoxicity studies should be done in appropriate in-vitro models using human cells.

    A special safety testing strategy for cartilage/chondrocyte CBMPs has been developed at Fraunhofer IZI. Here, biodistribution of human cells and tumorigenicity are proved using an immunocompromized mouse model (NSG mouse) and immunotoxicity/immunogenicity is tested using special in-vitro assays applying human primary cells or cell lines. Currently, this testing strategy is expanded for myocard CBMPs and combined ATMPs for the treatment of chronic wounds and burn wounds. The entire testing procedure can be realized under GLP conditions in order to guarantee highest quality standards during non-clinical development of ATMPs.

    Whatever the product under test is, the preclinical testing strategies include considerations on the species specificity (molecular, cellular and tissue level), safety predictivity, efficacy/ proof of concept (diseases models), biological relevance (e.g. delivery), animal models reliable in surgical procedures, methods for tracking cells in vivo etc. 

    The use of many different species including diseases models as well as the complex application of molecular biology approaches  and the usage of an extended armamentarium to trace ATMPs in vivo not alone cause changes in the way of classical pathology evaluation. In addition, the pathologist may be involved in the classification of elements from engineered tissue production during the manufacturing process (GMP/QC), in the evaluation of product stability, homogenicity and others. 

    The presentation will show examples on strategies of pathology evaluation during the development of ATMPs.    




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