In a 13-week inhalation toxicity study with three recovery periods (3, 6, and 12 months), Crl: WI rats were allocated to nine groups, each containing 25 animals per sex. Eight groups were treated daily by inhalation with the test items at concentrations of 0.5, 1.0, 2.5, or 5.0mg/m3 (SAS 1 groups 2, 3, 4, or 5, respectively; SAS 2 groups 6, 7, 8, or 9, respectively). Controls (group 1) were treated with air only.In nasal cavities, the major lesions consisted of increased eosinophilic globules and chitinase-3-like-protein-positive crystalloids* in the nasal mucosa, mainly in nasal cavity levels 2-4 up to week 26 of recovery without any further injury in olfactory mucosa, mainly in SAS 1-treated animals. Eosinophilic globules in the rodent nasal cavity are common and increase with age; they represent a particular finding of the rodent nasal mucosa. The relevance of chitinase-3-like protein (Ym1 + Ym2) expression in the rodent nasal mucosa is unknown but is normal in control animals. Both findings developed without any indicator for inflammatory processes. The increase of these unspecific background findings is considered an indicator of minor irritative effects.Due to the clear lack of nasal tissue injury or concurrent changes (degeneration, necrosis, inflammatory infiltrate, dysplasia, and/or neoplasia) following repeated inhalation exposure to SAS, it is deemed that the eosinophilic globules (hyaline inclusions) combined with the formation of eosinophilic protein crystalloids in this study represent an adaptive response.

Ecotoxicology studies were performed in the earthworm Eisenia fetida with four different synthetic amorphous silica (SAS) (SYLOID® AL-1 FP, SYLOID® MX 107, LUDOX® P T-40F, and HDK® N20) mixed into artificial soil to determine a NOEC/LOEC for effects on reproduction (56 days after application), mortality and biomass development (28 days after application) using a standardized artificial soil with 10% peat. The LC50 for test-item effects on adult mortality, and an EC10 and EC50 for reproduction were also determined. Furthermore, earthworms underwent histopathology evaluation, and the amount of silica in different organs from these organisms was evaluated using EDX (Energy Dispersive X-ray Spectroscopy). Histopathology revealed no findings in any organ of the earthworms, except for desiccated dissepiments in evaluated decedents at extremely high SAS doses. To measure SAS uptake into the organs, a fully quantitative method for silica was established and validated using standards containing known concentrations of silica to ensure the accuracy of the analyses undertaken. Results from EDX analysis demonstrated the negligible presence of silicon within the brain ganglia and gonads of adult earthworms comparable to controls. Therefore, any deposition of the test items within these two organs was excluded. In contrast, traces of silicon higher than in controls were found in the intestinal lumina of the earthworms due to ingestion of SAS with soil and feed, but not in other organs.

Bisphenol A (BPA), an endocrine-disrupting chemical and environmental pollutant, has been reported by many researchers to induce male reproductive toxicity in different experimental models. In this study, we investigated whether long-term exposure for two months to 25 µg/kg body weight (low dose) of BPA affects spermatogenesis or sperm quality in young Istrian Pramenka rams exposed via diet. We evaluated body and testicular weights, histopathology of testes and epididymides, and sperm analyses, and compared these parameters between the group of treated rams and the control group of rams. Although there were some differences between the two groups, these differences were not large or statistically significant. The only statistically significant difference was the lower epithelial height of seminiferous tubules in treated rams, compared to control rams. In addition to assessing toxicity, BPA concentrations in the blood plasma of treated rams were determined after the first administration, and the toxicokinetic parameters of total BPA were calculated. In this study, no major signs of altered reproduction in rams were detected.

In-vivo toxicological studies are characterized by multiple primary endpoints with quite different scales. Whereas guidelines and publications provide various statistical tests for normally distributed endpoints (such as organ weights) and proportions (such as tumor rates), few approaches are available for graded histopathological findings, such as 0, +, ++, +++. This represents a basic contradiction of the statistical analysis because these graded findings sometimes show a high predictive value for potential toxic effects. Here we discuss different methods comparatively, especially from the viewpoints of i) designs for very small sample sizes and ii) interpretability by toxicologists. A new approach is recommended where a simultaneous test is performed over all class combinations of score levels, such as (0, +) vs (++, +++). Corresponding R code is provided by way of a data example.

Intravenously administered iron-carbohydrate preparations are a structurally heterogenous class of nanomedicines. Iron biodistribution to target tissues is greatly affected by the physicochemical characteristics of these nanoparticles. Some regulatory agencies have recommended performing studies in animal models for biodistribution characterization and bioequivalence evaluation. In the present work, a systematic comparison of iron exposure, tissue biodistribution and pharmacodynamics of four intravenous iron-carbohydrates in anemic CD rats was conducted. A pilot study was performed to establish the anemic rat model, followed by a control study to evaluate the pharmacokinetics (serum iron, biodistribution) and pharmacodynamics (hematological parameters) in healthy and anemic controls and anemic rats receiving ferric carboxymaltose (FCM). The same parameters were then evaluated in a comparative study in anemic rats receiving FCM, iron sucrose (IS), iron isomaltoside 1000 (IIM), and iron dextran (ID). Despite similar serum iron profiles observed across the investigated nanomedicines, tissue iron biodistribution varied markedly between the individual intravenous iron-carbohydrate complexes. Tissue iron repletion differences were also confirmed by histopathology. These results suggest that employing serum iron profiles as a surrogate for tissue biodistribution may be erroneous. The variability observed in tissue biodistribution may indicate different pharmacodynamic profiles and warrants further study.

In 2016, one subject died and four were hospitalized with neurological symptoms during a clinical trial with the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474. The present paper reviews the regulatory toxicology studies that were carried out to support the clinical trial application for BIA 10-2474. Animal studies complied with national and international standards including European regulatory guidelines (e.g. EEC Council Directive 75/318/EEC and subsequent amendments). The CNS effects seen in the rat and mouse appear to be common in rodents in such studies and do not in principle seem to be of the type to generate a signal. In the same way in non-human primates, insignificant alterations in the mesencephalon, and especially of the autonomic nervous system (Meissner's plexus in the bowel) in rodents and monkeys were observed in some animals treated with a high dose. Overall, these data, as well as the extensive additional data generated since the accident, support the conclusion that the tragic fatality that occurred during the clinical trial with BIA 10-2474 was unpredictable and that the mechanism responsible remains unknown, from a non-clinical toxicological perspective.

Inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, are multifactorial inflammatory disorders of the gastrointestinal tract, characterised by abdominal cramps, bloody diarrhoea, and anaemia. Standard therapies, including corticosteroids or biologicals, often induce severe side effects, or patients may develop resistance to those therapies. Thus, new therapeutic options for IBD are urgently needed. This study investigates the therapeutic efficacy and safety of two plant-derived ligands of the aryl hydrocarbon receptor (AhR), quercetin (Q), and indol-3-carbinol (I3C), using a translationally relevant mouse model of IBD. Q and I3C are administered by gavage to C57BL/6 wild-type or C57BL/6 Ahr(-/-) mice suffering from chronic colitis, induced by dextran sulphate sodium (DSS). The course of the disease, intestinal histopathological changes, and in-situ immunological phenotype are scored over 25 days. Our results show that both Q and I3C improved significantly clinical symptoms in moderate DSS colitis, which coincides with a significantly reduced histopathological score. Even in severe DSS colitis I3C, neither Q nor the therapy control 6-thioguanine (6-TG) can prevent a fatal outcome. Moreover, treatment with Q or I3C restored in part DSS-induced loss of epithelial integrity by induction of tight-junction proteins and reduced significantly gut inflammation, as demonstrated by colonoscopy, as well as by immunohistochemistry revealing lower numbers of neutrophils and macrophages. Moreover, the number of Th17 cells is significantly reduced, while the number of Treg cells is significantly increased by treatment with Q or I3C, as well as 6-TG. Q- or I3C-induced amelioration of colitis is not observed in Ahr(-/-) mice suggesting the requirement of AhR ligation and signalling. Based on the results of this study, plant-derived non-toxic AhR agonists can be considered promising therapeutics in IBD therapy in humans. However, they may differ in terms of efficacy; therefore, it is indispensable to study the dose-response relationship of each individual AhR agonist also with regard to potential adverse effects, since they may also exert AhR-independent effects.

Testicular histopathology is considered the most sensitive and reliable method to detect the effects of chemicals on sperm production. To carry out a sensitive examination of testicular histopathology and interpret the changes require knowledge of spermatogenic stages. Spermatogenic staging based on acrosome development during spermiogenesis is conventionally performed in animal species routinely used for research and toxicity testing. In contrast, small ruminants, such as sheep and goats, are rarely used as animal models to evaluate toxicity in male reproductive organs. To the best of our knowledge, a comparable spermatogenic staging system in rams has not yet been fully characterised. Hence, this study aimed to adapt the existing spermatogenic staging based on acrosome development in bull testes to fit the seminiferous epithelium cycle of ram testes. The results show that spermatogenic staging based on acrosome development in bull testes can, with slight modifications, be efficiently used for the staging of ram testes.

The inter-laboratory performance of Isolated Chicken Eye (ICE) histopathology scoring was assessed for predicting EU CLP/UN GHS Cat. 1 surfactants. Furthermore, the predictive capacity of ICE histopathology was evaluated for the combined dataset of surfactants and existing data for non-extreme pH (2 < pH < 11.5) detergents. Use of ICE histopathology led to increased sensitivity compared to the ICE test method alone for surfactants. When combined with the existing dataset of detergents, use of histopathology in addition to the standard ICE test method decreased the false negative rates from 64% (14/22) to 27% (6/22); increased accuracy from 53% (16/30) to 77% (23/30); and led to acceptable level of false positives (from 0/8 to 1/8 (12.5%). Moreover, good reproducibility of ICE histopathology predictions conducted on the same slides was found between pathologists and peer-reviewers from three independent laboratories (10/12 or 83%) and over time. Use of ICE histopathology was therefore found suitable to predict EU CLP/UN GHS Cat. 1 surfactants and non-extreme pH detergents. In addition, appropriate reproducibility of ICE histopathology was found, provided that i) an internal peer-review system was in place; ii) original slides were assessed to enable evaluation of three dimensional effects; and iii) appropriate training and proficiency appraisal were conducted.

We examined a 110-week-old RccHan (TM): WIST Wistar male rat from a carcinogenicity study. No clinical signs were observed, and the rat was sacrificed at the end of the study. Macroscopically, within the midline of the sphenoid bone, was a 10 mm, non-infiltrative, soft, heterogeneous mass. Microscopic evaluation showed an expansile, cystic proliferation, consisting of two patterns of epithelial lining: well- differentiated areas lined by a single layer to a pseudostratified, ciliated-cuboidal epithelia with Goblet cells compatible with Rathke's cleft cyst; and poorly differentiated ones that formed irregular papillary projections, covered by atypical epithelia with squamous differentiation and hyperkeratosis compatible with areas of craniopharyngioma. Pleomorphisms were high in atypical areas with up to 2-3 mitotic figures per high power field. Within the cystic cavities, there was abrupt keratinization, mucus, cholesterol clefts, and foci of foamy macrophages. Immunohistochemistry revealed strong pancytokeratin immunolabelling of neoplastic cells confirming the epithelial origin. Well-differentiated epithelial lining showed cytokeratin-20 and cytokeratin-8 immunoreactivity, whereas the atypical squamous epithelium presented with a loss of cytokeratin-20 positive signal and weak to moderate positivity with cytokeratin-8. Areas compatible with a Rathke's cleft cyst and craniopharyngioma were considered to co-exist in the same mass.

In 2016 one person died and others had neurological sequelae during a clinical trial with BIA 10-2474 (3-(1-(cyclohexyl(methyl)carbamoyl)-lH-imidazol-4-yl)pyridine 1-oxide), a novel fatty acid amide hydrolase (FAAH) inhibitor being developed for the treatment of medical conditions such as pain. Prior to the clinical trial a full battery of regulatory toxicology tests were carried out and this paper describes the genotoxicity/mutagenicity tests undertaken with BIA 10-2474 using the Ames (Salmonella typhimurium) reverse mutation test, the Escherichia coli WP2uvrA forward mutation test, an in vitro chromosome damage assay in human lymphocytes, and an in vivo micronucleus test in mice. All tests were conducted with and without a rat liver S9 metabolic activation system. None of the test results were judged to be positive with regards to the mutagenicity/genotoxicity of BIA 10-2474 making it unlikely that any such effect was involved in the toxicity observed in the clinic.

Two beagle dog strains were used in a 14-day intrathecal infusion study for a small molecule test article. A moderate number of Renaut bodies (RBs) were observed in the sciatic nerves of control and test article–treated adult animals as early as 1 day after test article infusion (ie, 5 days after catheter implantation in the lumbar cistern). In most cases, the sciatic nerve was affected unilaterally, apparently in association with extended lateral recumbency on one side. The lighter beagle strain (Marshall), and especially the females (which weighed less than age-matched Marshall males), developed more RBs. In contrast, neither females nor males of the larger strain (Harlan) developed any nerve lesions. These data support the hypothesis that RBs develop following mechanical stress to sciatic nerves, suggest that this change may develop fairly quickly following an insult, and demonstrate that different dog strains exhibit strain-specific nerve changes.

Boudard et al.1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar in “Chronic Oral Exposure to Synthetic Amorphous Silica (NM-200) Results in Renal and Liver Lesions in Mice” described lesions in the kidneys and livers of mice exposed to NM-200, a synthetic precipitated amorphous silica (SAS). The lesions occurred at a low-dose exposure taken up by drinking water. Three experiments are described in this publication, which together tend to highlight a specific behavior of the tested substance; however, having some scientific deficiencies that jeopardize the overall drawn conclusion. The authors have performed 3 experiments in which small groups of animals were exposed via drinking water starting at an age of 3 months over a long period up to 18 months. Three experiments have been performed according to the described study design. The study design has been described as “[The] experiment was carried out in parallel using 2 different wild-type mouse lines, namely C57BL/6 and C57BL/6S. Besides, a 9-month exposure study was performed in a transgenic mouse line (TgHuA53T) expressing the human mutated (A53T) α-synuclein protein.”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar The identified scientific and study design deficiencies of the study are described in this letter to the editor. Interestingly, the authors mentioned that the mouse lines C57BL/6 (Charles River, France) and C57BL/6S (Harlan, France), were “selected on the criteria of absence of any spontaneous diseases, in particular no kidney or liver tumors that could appear with natural aging.”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar This statement is insofar surprising because especially in males, liver tumors are not unusual at all.2Blackwell B.N. Bucci T.J. Hart R.W. et al.Longevity, body weight, and neoplasia in ad libitum-fed and diet-restricted C57BL6 mice fed NIH-31 open formula diet.Toxicol Pathol. 1995; 23: 570-582Crossref PubMed Scopus (146) Google Scholar,3Frith C.H. Highman B. Burger G. Sheldon W.D. Spontaneous lesions in virgin and retired breeder BALB/c and C57BL/6 mice.Lab Anim Sci. 1983; 33: 273-286PubMed Google Scholar,S1–S3 Liver neoplasms were even considered the second most common tumors in C57BL/6 mice.S1 Furthermore, the animals were placed into this study at an age of 3 months and were maintained for a further 18 months (i.e., the animals are senile individuals at the end of the study). In addition, the statement that C57BL/6 mice do not express spontaneous renal diseases is wrong. Nephropathy was among the most common non-neoplastic findings that contributed to death in long-term survival studies of ad libitum fed and restricted diet feeding in C57BL/6 mice.2Blackwell B.N. Bucci T.J. Hart R.W. et al.Longevity, body weight, and neoplasia in ad libitum-fed and diet-restricted C57BL6 mice fed NIH-31 open formula diet.Toxicol Pathol. 1995; 23: 570-582Crossref PubMed Scopus (146) Google Scholar The most common non-neoplastic findings in another study included glomerulonephritis.S3 Furthermore, the genetic background of C57BL/6 mice was used to establish the renal phenotype of the cystinosis mouse model.S4 Regarding the study design, it is stated that groups of “5 to 8 female mice, 3 months old at the beginning of the experiment (average weight 20 to 25 g), were exposed orally to NPs through their drinking water for 18 months” and “[c]ontrol group of each mouse line (n = 7 and n = 8) received only tap water during the 18 months.”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar In addition, a third experiment was performed “to study the impact of 3-, 6-, and 9-month exposure to silica in drinking water on young (8 weeks old, average weight 20 to 25 g) transgenic mice (n = 15, male and female)” expressing the human mutated (A53T) α-synuclein protein (TgHuA53T) “compared [to] the matched controls (n = 10, unexposed transgenic mice).”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar No information has been provided on the silica content in the tap water and diet and thus about the background magnitude already present through normal water and diet consumption (see the section “Silica Content in Different Organs,” later in this article). It is also well known that rodent feed for mice and rats is enriched with certain vitamins that usually use synthetic amorphous silica as a carrier material and thus create a second background, again no data were provided on this aspect neither on the amorphous silica concentration in the used feed nor about the feed consumption of each animal on average. Even, for a special study design, where no standard guidance is used as a basis and besides of performing such an impacting study outside GLP, it is unusual that mice were used that were 3 months of age at study start. It surprises even more that a such low number of animals have been used in such a long experiment, knowing that a huge number of age-related background lesions, and even mortality, will interfere with the study results. It is furthermore unclear how the daily SAS consumption was evaluated considering a water uptake of “average daily water intake is estimated to be around 3–5 mL (1.5 mL/10 g body weight/day) . . . .”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar The authors declared furthermore that “the usual estimated individual intake of 4 mL/day was used to calculate the average daily intake of mice.”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar One might ask the question, was it measured or estimated. But if it was estimated, it is questionable to declare a daily SAS uptake of “4.8 mg SiO2/kg body weight/day.”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Although no mortality was reported (except in a table for TgHuA53T mice [Supplementary Table S4], without evaluation of the cause of death) during the conduct of the study, it remains unclear why obviously several organs have not been evaluated regarding histopathological changes or Si tissue distribution. It was necessary to compile a table (see Table 1) for the C57BL6/S and C57BL6 mice to get an overview on the evaluation of animals. In Supplementary Figure S1 of the publication, the authors describe the experimental setup with animal numbers per group. In Supplementary Table S3 and Table 1, histopathological results are reported for part of exposed animals in C57BL6 and C57BL6/S nanoparticle exposed animals. Information on the selection of animals is missing. It further remains unclear how 2 age-matched animals were included in the experiment in C57BL6 Control only and how they were treated. It gets even more obscure as in the publication Table 1 this changes to 7 old and 9 young adult mice (Table 1).Table 1Summary on study designStrainExposure phase (Supplementary Figure S1)Liver histopathology table (Supplementary Table S3)Histo summary table (Table 1)C57BL6-C7n = 2 age matched, n = 7 young adultn = 7 old and n = 9 young adultC57BL6/S-C88n = 6 oldC57BL6-NP863C57BL6/S-NP555 Open table in a new tab It is unclear, what “age matched” does mean (Table 1). In the supplementary document, it is stated that TgHu53T mice were used to study the impact of 3, 6, and 9 months exposure on young mice at an age of 8 weeks. It may be considered that transgenic mice have entered the study at an age of 8 weeks and the sentence “compared with the matched controls (n = 10, unexposed transgenic mice)” is a statement of using controls at the same age. This is reasonable but is different from the main document statement on mice entering the study at an age of 3 months. A 3-score system has been applied to describe the lesions: “Grade 1, for 0% to 25% of lesions observed in the section (+); Grade 2, for 25% to 50% of lesions observed in the section (++); Grade 3, when there was more than 50% of lesions in the section (+++)”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Such scoring scheme is not considered adequate to differ induced lesions from spontaneous background changes nor to properly classify lesions. The STP Best Practice Paper on pathology report writing recommends: “When severity grading is important to the understanding of major study findings, it may be useful to provide a description of the distinguishing features of each severity grade.”S5 Furthermore, a 5-grade system (beside of 0, where no findings are present) has been recommended by several authors,S6 but also by the INHAND-Nomenclature.S7 The authors stated that “No morphological abnormalities were noted on young adult or age-matched control mice.”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar This sentence astonishes likely every, even little experienced toxicologic pathologist. Renal background alterations are common in every mouse strain, and, especially in aged animals; histological lesions are accumulating in any rodent species and strain. Moreover, changes in the urinary system in C57BL/6 mice are, for example, summarized as mouse urologic syndrome by Szymanska et al.S1 Nephrotic lesions were also reported by Brayton et al.S8 The authors stated that the observation of findings in kidneys as “[v]acuoles within tubules, especially proximal tubules, were seen in all the animals exposed to NM-200 whatever the mouse line, without atrophic lesions or tubular necrosis (Figure 2h)”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar and “unrelated to normal aging, that were characterized by a vacuolization of tubular epithelial cells, detected in 100% of the kidneys.”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Figure 2H, however, is described as “[r]epresentative of silver impregnation that illustrates absence of atrophic lesions or tubular necrosis.”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar However, no vacuolation of tubular cells is visible. This is also true for any other picture presented under Figure 2. In contrast, a focal vacuolar change in the renal tubules is presented in Supplementary Figure S4C and D. Such lesions are, however, often noted changes in mouse kidneys and are considered an incidental finding,S9,S10 especially when not associated with necrosis or atrophy. Although the C57BL/6 mouse is relatively resistant toward developing glomerulosclerosis, proteinuria, and hypertension, it is known to represent a strain that is used to establish nontransgenic models for induced renal injury.S11,S12 In addition, very detailed evaluations have been undertaken to investigate glomerular changes in C57BL/6 mice during live time.S13,S14 Yumura et al.S14 have shown age-related glomerular changes by demonstrating increased glomerular injury associated with a marked deposition of IgG and IgM in the enlarged mesangium from CB57BL/6 mice during ages of 6 to 24 months. In addition, Yabuki et al.S13 have undertaken a very detailed image analysis. They could show that the number of glomeruli per unit area does not change at ages 3 to 15 months, but thereafter, gradually increase in mice aged 24 to 27 months of age. They considered this numerical change as a possible consequence of tubular atrophy. Furthermore, they evaluated the age-related glomerular changes. Briefly, the percentage of glomeruli with a cuboidal parietal layer decreased, and the cuboidal layer of the renal capsule in males displayed atrophic changes during the later stages of life. In addition, glomerular lesions that were heavily or mildly stained with PAS (expanded mesangium) were rarely encountered in 3-month-old animals but became severe and diffuse with age. Other changes consisted of interstitial inflammatory cell infiltration by lymphocytes and plasma cells, amyloid deposition, scar lesions in the cortex, and ultrastructural changes that were observed in mice at 27 months old. Ultrastructural changes revealed in aged mice severe expansion of the mesangial matrix, fusion of podocyte foot processes, and accumulation of lysosomes and large lysosomes with accumulated lipid content. The authors described amyloidosis in the renal glomeruli for 1 of 5 C57BL/6S mice and 1 of 3 C57BL/6 mice. In Figure 2c–e, hyaline deposits in glomeruli are shown. The Congo red stained negative (Figure 2b), and hence, hyaline glomerulopathy can be excluded.S15 There is therefore no doubt, that the cases (see also “liver”) present true amyloidosis. However, amyloidosis is a very common age-related background lesion in mice. In contrast to, for example, A/J mice, the C57BL/6 strains are some of the most susceptible mouse strains.S16,S17 Amyloidosis was reported also by other authors that investigated mouse kidneys in detail.S13,S18 Some others considered amyloidosis the most common age-related non-neoplastic finding with >80% of C57BL/6 males affected.S3 The reported incidence is not deemed to be very high, considering, for example, published incidences by Zurcher et al.S3 of 73% in females and 83% in males. In general, C57BL6 mice are considered to be relatively susceptible to secondary (serum amyloid A [SAA]) amyloidosis as well as to senile (AApoAII) amyloidosis.S19,S20 It is also considered that stressors (e.g., group housing and infections) have an impact on the incidence of amyloidosis.S21,S22 A urine test was included into the study design for transgenic mice (TgHuA53T) only, that is, proteinurea was monitored weekly from the third month of exposure onward using dipstick urinalysis (Albustix, Siemens, Munich, Germany). It was stated “[p]roteinuria suggests glomerular dysfunction without significant glomerular amyloidosis alterations at this stage.”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar This is very likely; however, control animals were not monitored. Therefore, these results are useless. Boudard et al.1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar stated that “[h]istopathological abnormalities in livers from NM-200–exposed mice are provided in detail in Supplementary Table S3 and Supplementary Figure S2”. However, the conclusion of this report is that NM-200 induced renal and liver lesions. It is, therefore, considered necessary to show the results well presented in a main document. Regarding the liver, it was summarized that NM-200 induced liver inflammation and amyloidosis in C57BL/6 mice.1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Amyloidosis is, however, a systemic disease. When amyloidosis occurs in kidneys, it likely occurs in livers as well. Nevertheless, it is a normal background lesion in this mouse strain. It occurs spontaneously at high incidences in mice, as discussed previously. The infiltrates and the vacuolation (very likely fat) are very normal background lesions in mice and rats of any strain. The only change that was slightly outstanding is presented in Figure S2f. This case might be considered a secondary infiltration after necrosis, however, is occasionally also observed in control mice as an idiopathic lesion. It was described that occasionally apoptotic bodies occurred and were associated with lymphocytic infiltrate. This, however, can be observed also in control animals from studies under SPF, and are deemed to be part of the normal hepatocellular turnover in mice (own experience: the author of this letter to the editor has been working daily on the microscope for approximately 30 years). Inflammatory cell foci may be, for example, in rats recorded even in young adult control animals at incidences up to 100%.S23 The term “necroto-inflammatory” lesion for such a condition is not deemed to be adequate. It remains also unclear why an induced liver inflammation was established under uptake of NM-200. In Supplementary Table S3, no real differences are visible between control and treated mice. All illustrations provided for the liver in Supplementary Figure S2 are deemed to be background lesions. In the kidneys from transgenic mice, a vacuolar change is presented in Supplementary Figure S4D.1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar There are vacuoles resembling lipid inclusions in mesangial cells. It is unclear if this is a single glomerulus, and the age of the respective animal is not stated. This change might be consistent with the lipid storage in lysosomes described by Yabuki et al.,S13 and, hence, would fit glomerular changes under control conditions. Boudard et al.1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar stated that “hepatocytes from mice deleted for α-synuclein displayed steatotic-like cytoplasmic vacuolization (micro- or macro[-]vacuoles similar to those observed in human steatosis), independent of their exposure to SAS NM-200.”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Again, fatty change is a common finding in mouse livers.S7 In summary, a low number of transgenic animals were evaluated. It is also questionable if a historical data bank on spontaneous lesions for this mouse strain exists. There were also a few deaths during the study, whereby it is not clear how many exposed and control animals died. The cause of death was not established. Total silicon was determined in tissues using an Agilent 8800 ICP-QQQ mass spectrometer. The following results were obtained: “In kidney, silica levels were higher in C57BL/6 mice compared with both C57BL/6S mice and controls (statistically significant, P < 0.01).”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar This is not understood, because the relationship to control is not clear. Furthermore, it was stated “Silica deposition in liver followed the order C57BL/6 mice > C57BL/6S mice > control mice, although with a high inter-individual variability. The silica concentration was higher in livers compared with kidneys of C57BL/6 mice.”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar However, again, it remains unclear what “control” does mean. In the Methods section it is written that “[s]tatistical analyses for tissue silicon content were carried out comparing exposed to control groups by means of the Mann-Whitney U test.”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Therefore, one would expect to see control bars, 1 for each control group. With “n = 4–6 for control mice”1Boudard D. Aureli F. Laurent B. et al.Chronic oral exposure to synthetic amorphous silica (NM-200) results in renal and liver lesions in mice.Kidney Int Rep. 2019; 4: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar it remains completely unclear how this fit to n = 7 for C57BL6 and n = 8 for C57BL6/S as given in the Supplementary Material. How were animals selected for Si determination or what happened with missing values if all animals were to be processed? Without comprehensive information on the data set used for statistical analysis, the statistical significance remains scientifically useless. The same is true for the nonsignificant trend shown in Figure 1A. Furthermore, in Figure 1B, the difference for the amount of Si in kidneys between “control” and C57BL/6S values seems to be on a biologically similar level. Any statistical relevance should also be checked for a biological relevance. This has not been addressed. Considering these limitations of the data there are several further questions to be asked:(i)How much Si is normally present in the organs of control animals? With having a permanent natural background of silicon exposure (Si is one of the most common elements on earth and is bound to silica minerals that are omnipresent, e.g., feldspars, plagioclases) and various possibilities of silicon contamination during animal treatment and sample preparation, it is of high relevance to have a sound basis for the natural background of silicon in these organs if conclusions are drawn from a limited database.(ii)What could be the source of silicon in organs? With ICP-as being a non–substance-specific analytical method, it is not possible to clarify the source of silicon found in organs. Therefore, it becomes extremely important to have exact information on possible sources of silicon exposure from feed and drinking water. Impurification by Si is absolutely normal due to the presence in Si in the environment. This shows up in high silicon contents in food and feed basis material. The European Food Safety Authority provided an estimate of the silicon content in food: “High levels of silicon are found in foods derived from plants, particularly cereals such as oats (3910–4310 mg/kg dry weight), barley (2610–2720 mg/kg dry weight), white wheat flour (81–103 mg/kg dry weight), or polished rice (55–57 mg/kg dry weight).”S24 In drinking water, the silicon level varies from region to region and might be higher in mineral springs. Normal drinking water contains silica at a range from 4.2 to 22.4 mg/L.S25 Bottled silicon-rich water contains up to 85 to 90 mg/l silica. However, also beer contains 0.9 to 3.94 mg silicon/100 g.S26•It should also be mentioned that rodent diet in many cases contains synthetic amorphous silica as food additive (E 551) for the equal distribution of substance as, for example, vitamins. The applied dosage of 4.8 mg SiO2/kg body weight per day to mice by drinking water is very low considering background Si level by normal drinking water and diet and cannot be even considered to induce lesions. Based on the presented study design and the findings established, including the silicon analysis in different organs, no effect of Si on the liver and kidney can be stated. All lesions described are within the range of normal expected background alterations in wild-type mice of this strain and age. No control data are available from the type of transgenic mice used.

BIA 10-2474 (3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide) is a novel fatty acid amide hydrolase (FAAH) inhibitor developed by BIAL for the treatment of medical conditions which would benefit from enhanced levels of endogenous anandamide (AEA) such as pain disorders. During a Phase I clinical trial one subject died after receiving BIA 10-2474 and others displayed neurological signs. As part of series of papers presenting all the toxicology data available prior to the clinical trial we report here the nonclinical toxicology studies performed in cynomolgus monkeys. Maximum Tolerated Dose (MTD) studies and a preliminary 14-day study by oral (capsule) administration of BIA 10-2474 established a dose between 90 and 120 mg/kg/day as a suitable high dose for a subsequent regulatory toxicity studies. An up-titration scheme was used to achieve these doses. The dose-limiting effect was the early sacrifice for ethical reasons of monkeys at doses from 125 mg/kg/day upwards. Thereafter, regulatory 4- and 13-week oral gavage toxicity studies followed by a 2- or a 4-week recovery period, respectively, were performed. In both cases a 3-4-week up-titration period was used prior to repeat dosing with the target doses. One female was euthanized during the up-titration period after receiving 9 administrations of 75 mg/kg as a result of bleeding erosion on the feet and hands and ulceration on the tongue. These signs were not seen in any other monkeys during these studies. Doses of 10, 50 or 100 mg/kg/day were administered during the 4-week study and clinical signs related to the pharmacological action of BIA 10-2474 (e.g., tremors and weakness, incoordination and loss of balance, reduction in food intake and reduced body weight) were observed in several monkeys from the intermediate and high dose. Histological alterations consisted of axonal dystrophy in the fasciculus cuneatus (dorsal medulla oblongata) characterized by swollen axons and myelin sheath edema, edema in the pars nervosa of the pituitary gland and vacuolation of Meissner's plexus ganglia in all gastrointestinal segments. All lesions recovered and a dose of 100 mg/kg/day was considered to be the NOAEL. In the 13-week oral study the monkeys received BIA 10-2474 daily by gavage at a dose of 6.25, 37.5 or 75 mg/kg/day. Similar clinical signs and histological alterations as noted in monkeys of the 28-day study were observed in monkeys at 37.5 or 75 mg/kg/day. All findings recovered, and the dose of 75 mg/kg/day was considered the NOAEL.

BIA 10-2474 is a novel fatty acid amide hydrolase (FAAH) inhibitor developed for the treatment of medical conditions which would benefit from enhanced levels of endogenous anandamide (AEA) such as pain disorders. During a Phase I clinical trial one subject died after receiving BIA 10-2474 and four other subjects displayed neurological signs. As part of series of papers presenting all the toxicology data available prior to the clinical trial, we report here the preclinical toxicology studies examining once-a-day oral administration of BIA 10-2474 to male and female Wistar rats. These included a 14-day dose range finding (150, 200 and 250 mg/kg/day), a 4-week study (30, 90 and 150 mg/kg/day) and 13- and 26-week studies (both at 10, 30 and 90 mg/kg/day). The 13- and 26-week studies also included a 4-week recovery arm and a toxicokinetic arm for the parent compound, BIA 10-2474, and the two major metabolites (BIA 10-2445 and BIA 10-2583) were also measured in the 26-week study. At 150 mg/kg and below, all animals survived the scheduled treatment periods although neurological side-effects (abnormal or stiff gait, dragging of fore- or hind-limbs) were seen at 150 mg/kg in both the dose-range finding and 4-week studies. At 90 mg/kg/day, even up to 26-weeks treatment, no clinical signs were seen apart from some decreases in body weight gain. A number of consistent hematological and biochemical changes were noted which were considered related to treatment with BIA 10-2474. Morphologically, in the 4-week study, except for a slight gliosis in the hippocampus of one female at 150 mg/kg, no CNS histopathology was observed; hippocampus gliosis was not observed in subsequent studies. In the 13-week study axonal swelling was present in the medulla oblongata in about half the animals at 90 mg/kg/day and this increased to nearly all the rats at 90 mg/kg/day in the 26-week study. Additional signs seen only in the 26-week study at 90 mg/kg/day included axonal swelling of the fasiculus gracilis and vacuolar changes in the medulla oblongata and ventral commissure of the 3rd ventricle. Other findings included vacuolar degeneration in the ganglia of the GI tract, salivary glands, prostate gland, uterus, and parathyroid glands. The pituitary gland showed edema and mitotic figures in the pars nervosa. These observations outside the CNS were seen in most rats at 90 and 150 mg/kg/day independent of study duration. At 30 mg/kg/day, most of these observations were only seen in isolated cases except for the vacuolar degeneration in GI tract ganglia, which was absent at this dose after 4 weeks treatment but was present in almost all rats at 13 and 26 weeks. Hepatocellular hypertrophy and nephropathy were seen across all studies and the extent of these changes was similar in the 13- and 26-week studies. Most findings resolved after the 4-week recovery periods except for the axonal swelling seen in the medulla oblongata and spinal cord. BIA 10-2474 exposure was markedly higher than the exposure to either metabolite, BIA 10-2445 (19- to 192-fold) and BIA 10-2583 (63- to 526-fold). Exposure to metabolites differed between sexes with higher concentrations of BIA 10-2445 in females compared to males, but the inverse for BIA 10-2583. Although a No Observed Adverse Effect Level (NOAEL) of 30 mg/kg/day was concluded following the 4-week study, the histopathological findings at that dose in the 13- and 26-week studies resulted in the NOAEL being determined to be 10 mg/kg/day.

We independently and retrospectively reviewed three studies that evaluated the toxicity of BIA 10-2474 (3-(1-(cyclohexyl(methyl)carbamoyl)-lH-imidazol-4-yl)pyridine 1-oxide), a novel fatty acid amide hydrolase (FAAH) inhibitor in male and female CD-1 mice based upon raw data obtained from Bial Portela & Companhia S.A. (São Mamede do Coronado, Portugal). These studies were carried out prior to the clinical trial with BIA 10-2474 and formed part of the regulatory submission. An initial oral dose range-finding study with BIA 10-2474 showed that doses from 600 mg/kg/day were poorly tolerated with a high mortality rate and signs of weakness, prostration, labored breathing, clear lacrimation, tachypnea/bradypnea and decreased activity. At lower doses (100 and 300 mg/kg/day) there were few signs but post-mortem analysis showed increased liver weight. In a 28-day study a third of the animals receiving 500 mg/kg/day died or required euthanasia, with similar signs to those seen in the dose-range finding study. At lower doses (i.e. 100 and 300 mg/kg/day) there were few clinical signs although there were dose-related decreases in erythrocyte count and hemoglobin. Histopathology was seen in the 300 and 500 mg/kg/day groups and included hepatocellular hypertrophy (with increased liver weight), nephropathy and enterocyte vacuolation. Finally, in the 13-week oral gavage study, BIA 10-2474 was administered to CD-1 mice of both sexes at dose levels of 25, 75 and 150 mg/kg/day. Under these conditions, there were almost no clinical signs apart from a tendency to increase body-weight. Cholesterol was increased at 75 and 150 mg/kg and remained high after recovery. Liver and spleen weights increased at 75 and 150 mg/kg/day. Histopathologically, there was a dose-dependent increase in sciatic nerve and myofiber degeneration, hepatocellular hypertrophy, nephropathy and inflammatory loci in the bladder. The nerve damage and nephropathy seen at 150 mg/kg/day persisted after a 4-week recovery period. Toxicokinetic analysis in the 4- and 13-week studies showed that exposure was broadly dose-proportional with no evidence of accumulation. On the basis of the changes seen during the 13-week study, the NOAEL was established at 75 mg/kg/day.

A series of regulatory studies were carried out to investigate the effects of the FAAH inhibitor BIA 10-2474 on fertility, embryo-fetal toxicity and pre- and post-natal development in rats and rabbits. Despite some reductions in sperm count in rats from 50 mg/kg, there were no major changes in male fertility up to 100 mg/kg. In female rats administered up to GD6, there were increases in pre-implantation loss at 50 and 100 mg/kg but neither post-implantation loss nor early embryonic development was affected. In contrast, when administered to female rats during pregnancy (GD6-GD17), BIA 10-2474 at 75 mg/kg/day reduced food consumption resulting in weight loss, increased post-implantation loss and reduced mean fetal body weight. In rabbits, the same maternal toxicity was seen but there were no effects in this species on post-implantation loss or fetal body weights. There were no teratological effects clearly due to BIA 10-2474 and developmental milestones and behavior of offspring were not affected. When administered during pregnancy and lactation (GD6-PND20), some post-implantation loss was seen from 20 mg/kg/day, but developmental milestones and behavior of the offspring were not affected, although males tended to have lower body weight. Based on these data the NOAEL for parental fertility was established as 50 mg/kg/day, the maternal NOAEL during pregnancy was 25 mg/kg/day in rats and developmental NOAEL was 25 and 75 mg/kg/day in rats and rabbits, respectively. When administered during post-natal development to rats the maternal NOAEL was 6 mg/kg/day. The parental reproductive NOAEL, the NOAEL for viability and growth of the F1 offspring, the F1 parental NOAEL and the F1 reproductive NOAEL were all considered to be 20 mg/kg/day.

Using the acute dextran sulfate sodium (DSS)-induced colitis model, studies have demonstrated that intestinal inflammation is accompanied by major changes in the composition of the intestinal microbiota. Only little is known about the microbial changes and more importantly their functional impact in the chronic DSS colitis model. We used a refined model of chronic DSS-induced colitis that reflects typical symptoms of the human disease without detrimental weight loss usually observed in DSS models. We sampled cecum and colon content as well as colon mucus from healthy and diseased mouse cohorts ( n = 12) and applied 16S rRNA gene sequencing and metaproteomics. An increase of Prevotella sp. in both colon content and mucus was observed. Functional differences were observed between sample types demonstrating the importance of separately sampling lumen content and mucus. The abundance of Desulfovibrio, a sulfate-reducing bacterium, was positively associated with the carbon metabolism. Lachnoclostridium was positively correlated to both vitamin B6 and tryptophan metabolism. In summary, functional changes in the distal colon caused by DSS treatment were more pronounced in the mucus-associated microbiota than in the microbiota present in the distal colon content.

BIA 10-2474 is a novel fatty acid amide hydrolase inhibitor developed for the treatment of medical conditions which would benefit from enhanced levels of endogenous anandamide (AEA) such as pain disorders. During a Phase I clinical trial one subject died after receiving BIA 10-2474 and others displayed neurological signs. We describe here the toxicology studies in beagle dogs that supported phase I testing of BIA 10-2474 in humans. A Maximum Tolerated Dose (MTD) study using once-a-day oral (capsule) application of BIA 10-2474 was first conducted to establish suitable dose levels for subsequent studies. Based on these results, 100 mg/kg/day was considered to be the MTD. The 4-week oral (capsule) toxicity study with a 3-week recovery period for BIA 10-2474 was therefore carried out at 20, 50 or 100 mg/kg/day. There were no changes recorded at 50 mg/kg/day and this was considered the oral No Observed Effect Level (NOEL) for four-week once-a-day capsule administration to Beagle dogs. At 100 mg/kg/day, the dose-limiting findings consisted of clinical symptoms including tremor, loss of balance, abnormal gait, decreased motor activity, weakness, vomits, salivation increase and miosis, increased severity of thymic atrophy/involution, and moderate acute, focal/multifocal bronchopneumonia in lungs of three animals. In a 13-week oral (capsule) toxicity study in the Beagle dog with a 6-week recovery period, using the same dose levels, clinical signs were recorded during treatment with BIA 10-274 at 50 and 100 mg/kg/day. The most frequent signs included difficulty breathing, respiratory sounds (with or without auscultation) and cough. Incoordination of the hind limbs with absence of correction reflex were also observed on some occasions. As a result, the 50 and 100 mg/kg/day doses were reduced to 35 and 50 mg/kg/day respectively on day 37. Because of the continued signs, the doses in both groups were further reduced to 20 mg/kg/day from day 77. Under the conditions of this study and given the severe signs recorded in groups treated at 100-50-20 and 50-35-20 mg/kg/day and only very occasional presence of signs in the group treated for the 13-week period at 20 mg/kg/day (abnormal respiratory sounds once in two animals), the dose of 20 mg/kg/day was considered the No Observed Adverse Effect Level (NOAEL).

Weber, K. (2017). Differences in Types and Incidence of Neoplasms in Wistar Han and Sprague-Dawley Rats. J Toxicol Pathol45, 64-75. (Original DOI: 10.1177/0192623316672075) In the January 2017 issue of Toxicologic Pathology, a number of in text Table citations were incorrectly labeled. The following are the sentences with corrected Table number (this has also been corrected in the online version): Many fewer neoplasms are recorded for the endocrine pancreas compared with islet cell tumor incidences (Table 3). The data presented in Table 4 are consistent with the high incidences mentioned for keratoacanthomas by various authors for SD (M. Chandra, Riley, and Johnson 1992; Nakazawa et al. 2001) and Wistar (Poteracki and Walsh 1998) rats. Mammary gland neoplasms are considered a major cause of morbidity/death (Weber et al. 2011; Table 6). The most common tumor in male F344 rats is the interstitial (Leydig) cell tumor at a mean incidence of 81% (Mitsumori and Elwell 1998; NTP 2010) but is also reported to reach almost 100% (Nolte et al. 2010; Table 6). Incidences at >0.5% are only reported for granulosa cell tumors or theca granulosa cell tumors, and the highest incidences are noted in Wistar strains (Table 7). Stromal sarcoma and leiomyoma were noted also at incidences >0.5% (Table 9). In Table 9, adenomas of the pars intermedia and anterior are combined due to the fact that several pathologists did not differentiate between these tumor types.