Long-term Rodent Studies Research Articles

TELOMERE LENGTH IN RHESUS MACAQUES WITH VOLUNTARY CHRONIC ETHANOL CONSUMPTION

Introduction. Alcohol abuse is associated with telomere shortening. There is no convincing evidence of a “safe” level of alcohol consumption in this regard. Long-term studies in rodents are not feasible, and clinical trials with the administration of alcohol to healthy individuals is not ethically acceptable. An approach based on a relevant model of voluntary alcohol consumption in monkeys under controlled conditions is a significant alternative. The aim of the study. To estimate the length of telomeres at long-term ethanol consumption by male rhesus macaques in under free choice with water Methods. The study was performed on fourteen mature male Rhesus macaques of groups with low (median 0.62 g/kg/day) and high (median 2.71 g/kg/day) ethanol consumption as 4% (v./v.) solution with condition of all-day access and free choice with drinking water. The duration of consumption was 920 days. The relative length of telomeres was determined by quantitative PCR according to Cawthon (2002) in blood leukocytes. Results. The relative average telomere length in the high-consumption group was 1.53±0.57 before the presentation of ethanol in the adaptation period (-32 day of the study), and at the consumption stage it was on 717 day 2.13±0.19 and on 917 day 4.61±0.7. In the low-consumption group, the average relative telomere length constituted 1.42±0.22, 1.55±0.15 and 3.3±0.47, respectively. The absolute count of leukocytes did not change significantly during the study. However, changes in the differential white cells count were revealed representing development of relative monocytosis by 917 day in both groups. Conclusion. The data obtained do not confirm the association of long-term alcohol consumption in moderate doses with telomere length. The completed study has limitations related to the lack of control without consumption and evaluation in one sex.

ICH S1 prospective evaluation study: weight of evidence approach to predict outcome and value of 2-year rat carcinogenicity studies. A report from the regulatory authorities subgroup.

Introduction: The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) initiated a process in 2012 to revise the S1B Guideline "Testing for Carcinogenicity of Pharmaceuticals". Previous retrospective analysis indicated the importance of histopathological risk factors in chronic toxicity studies, evidence of endocrine perturbation, and positive genetic toxicology results as potentially predictive indicators of carcinogenic risk. In addition, a relationship between pharmacodynamic activity and carcinogenicity outcome in long-term rodent studies has been reported. It was postulated that these factors could be evaluated in a Weight-of-Evidence (WoE) approach to predict the outcome of a 2-year rat study. Methods: The ICH S1B(R1) Expert Working Group (EWG) conducted a Prospective Evaluation Study (PES) to determine the regulatory feasibility of this WoE approach. Drug Regulatory Authorities (DRAs) evaluated 49 Carcinogenicity Assessment Documents (CADs), which describe the WoE for submitted pharmaceutical compounds. Each compound was categorized into a carcinogenic risk category including a statement of the value of the 2-year rat study. The outcome of the completed 2-year rat studies was evaluated in relation to the prospective CAD to determine the accuracy of predictions. Results: Based on the results of the PES, the EWG concluded that the evaluation process for assessing human carcinogenic risk of pharmaceuticals described in ICH S1B could be expanded to include a WoE approach. Approximately 27% of 2-year rat studies could be avoided in cases where DRAs and sponsors unanimously agreed that such a study would not add value. Discussion: Key factors supporting a WoE assessment were identified: data that inform carcinogenic potential based on drug target biology and the primary pharmacologic mechanism of the parent compound and major human metabolites; results from secondary pharmacology screens for this compound and major human metabolites that inform carcinogenic risk; histopathology data from repeated-dose toxicity studies; evidence for hormonal perturbation; genotoxicity data; and evidence of immune modulation. The outcome of the PES indicates that a WoE approach can be used in place of conducting a 2-year rat study for some pharmaceuticals. These data were used by the ICH S1B(R1) EWG to write the R1 Addendum to the S1B Guideline published in August 2022.

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project.

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.

Erythritol: An In-Depth Discussion of Its Potential to Be a Beneficial Dietary Component.

The sugar alcohol erythritol is a relatively new food ingredient. It is naturally occurring in plants, however, produced commercially by fermentation. It is also produced endogenously via the pentose phosphate pathway (PPP). Consumers perceive erythritol as less healthy than sweeteners extracted from plants, including sucrose. This review evaluates that perspective by summarizing current literature regarding erythritol's safety, production, metabolism, and health effects. Dietary erythritol is 30% less sweet than sucrose, but contains negligible energy. Because it is almost fully absorbed and excreted in urine, it is better tolerated than other sugar alcohols. Evidence shows erythritol has potential as a beneficial replacement for sugar in healthy and diabetic subjects as it exerts no effects on glucose or insulin and induces gut hormone secretions that modulate satiety to promote weight loss. Long-term rodent studies show erythritol consumption lowers body weight or adiposity. However, observational studies indicate positive association between plasma erythritol and obesity and cardiometabolic disease. It is unlikely that dietary erythritol is mediating these associations, rather they reflect dysregulated PPP due to impaired glycemia or glucose-rich diet. However, long-term clinical trials investigating the effects of chronic erythritol consumption on body weight and risk for metabolic diseases are needed. Current evidence suggests these studies will document beneficial effects of dietary erythritol compared to caloric sugars and allay consumer misperceptions.

AAV Joins the Rank of Genotoxic Vectors

AAV Joins the Rank of Genotoxic Vectors

Potential Applications of Clay-Based Therapy for the Reduction of Pesticide Exposures in Humans and Animals.

The risk of pesticide exposure in humans and animals may be magnified following natural and man-made disasters such as hurricanes and floods that can result in mobilization and redistribution of contaminated sediments. To develop broad-acting sorbents for mixtures of diverse toxins, we have processed calcium and sodium montmorillonite clays with high concentrations of sulfuric acid. These acid-processed montmorillonite clays (APMs) have shown limited hydration and swelling in water, higher surface areas, and lower trace metal levels than the parent clays, prior to processing. Isothermal analyses have indicated that newly developed APMs are highly active sorbents, with significantly increased binding capacities for a wide range of pesticides, including pentachlorophenol (PCP), 2,4,6-trichlorophenol (2,4,6-TCP), lindane, diazinon, linuron, trifluralin and paraquat. The safety and protective effects of APMs, against pesticide design mixtures, were confirmed in a living organism (Hydra vulgaris). Further work is planned to confirm the safety of the APMs in long-term rodent studies. This is the first report of a sorbent material (other than carbon) with high binding efficacy for mixtures of these pesticides. Based on our results, APMs (and similar clays), may be able to decrease human and animal pesticide exposures during disasters and emergencies.

Housing conditions modulate spontaneous physical activity, feeding behavior, aerobic running capacity and adiposity in C57BL/6J mice

There is evidence of reduced adiposity in rodents living in a large cages (LC) as compared to animals housed in small cages (SC). Because spontaneous physical activity (SPA) provides an important portion of the total daily energy expenditure, an increase of SPA in rodents kept in LC could explain their reduced body fat accumulation. The relationship between SPA and components of physical fitness (i.e. aerobic and anaerobic fitness and body leanness) has not been previously determined. We examined the effects of eight weeks of LC exposure on SPA, body composition, feeding behavior, as well as aerobic and anaerobic running capacity in adult C57BL/6J mice. Male mice were housed in cages of two different sizes for 8 weeks: a small (SC, n = 10) and large (LC n = 10) cages with 1320 cm2 and 4800 cm2 floor space, respectively. SPA was measured gravimetrically, and food and water intake were recorded daily. Mice had critical velocity (CV) and anaerobic running capacity (ARC) evaluated at the beginning, middle course (4th week) and at the end of study (8th week). Despite non-significant differences in each week LC-mice were more active than SC-mice by considering all SPA values obtained in the entire period of 8 weeks. The difference in SPA over the whole day was mainly due to light phase activity, but also due to activity at dark period (from 6 pm to 9 pm and from 5 am to 6 am). LC-mice also exhibited higher food and water intake over the entire 8-wk period. LC-mice had lower content of fat mass (% of the eviscerated carcass) than SC-mice (SC: 8.4 ± 0.4 vs LC: 6.3 ± 0.3, p < 0.05). LC-mice also exhibited reduced epididymal fat pads (% of body mass) compared to SC-mice (SC: 1.3 ± 0.1 vs LC: 0.9 ± 0.1, p < 0.05) and retroperitoneal fat pads (SC: 0.4 ± 0.05 vs LC: 0.2 ± 0.02, p < 0.05). The LC-group showed significantly higher critical velocity than SC-group at the fourth week (SC: 14.9 ± 0.6 m·min−1 vs LC: 18.0 ± 0.3 m·min−1, p < 0.05) and eighth week (SC: 17.1 ± 0.5 m·min−1 vs LC: 18.8 ± 0.6 m·min−1, p < 0.05). Our findings demonstrate that eight weeks of LC housing increases SPA of C57BL/6J mice, and this may lead to reduced fat accumulation as well as higher aerobic fitness. Importantly, our study implies that SC limits SPA, possibly generating experimental artifacts in long-term rodent studies.

Portable wireless transcranial ultrasound brain stimulation for freely behaving small animals

Conventional transcranial ultrasound stimulation (tUS) for animal studies utilize external bulky transducer systems, requiring the use of wires that limit the study to anesthetized or immobilized subjects. To facilitate behavioral studies with ultrasound, we designed a portable wireless tUS system that can provide acoustic stimulation on a freely behaving rat. Stimulation is given by a transducer attached on the rat skull that is driven by a circuit placed on the back of the rat. The whole system is controlled wirelessly via bluetooth. The designed system can facilitate ultrasound stimulation research for clinical use through behavioral and long-term studies in rodents.

A Mechanically-Adaptive Polymer Nanocomposite-Based Intracortical Probe and Package for Chronic Neural Recording.

Mechanical, materials, and biological causes of intracortical probe failure have hampered their utility in basic science and clinical applications. By anticipating causes of failure, we can design a system that will prevent the known causes of failure. The neural probe design was centered around a bio-inspired, mechanically-softening polymer nanocomposite. The polymer nanocomposite was functionalized with recording microelectrodes using a microfabrication process designed for chemical and thermal process compatibility. A custom package based upon a ribbon cable, printed circuit board, and a 3D-printed housing was designed to enable connection to external electronics. Probes were implanted into the primary motor cortex of Sprague-Dawley rats for 16 weeks, during which regular recording and electrochemical impedance spectroscopy measurement sessions took place. The implanted mechanically-softening probes had stable electrochemical impedance spectra across the 16 weeks and single units were recorded out to 16 weeks. The demonstration of chronic neural recording with the mechanically-softening probe suggests that probe architecture, custom package, and general design strategy are appropriate for long-term studies in rodents.

Cr3O(O2CCH2CH3)6(H2O)3]NO3·H2O (Cr3) Toxicity Potential in Bacterial and Mammalian Cells.

Chromium(III) has generally been considered to be essential for proper carbohydrate and lipid metabolism, and, despite recent evidence to the contrary, chromium(III)-containing compounds remain one of the most popular commercial dietary supplements. Cr3, or [Cr3O(O2CCH2CH3)6(H2O)3]NO3·H2O, is a trivalent chromium compound that is a promising chromium nutritional supplement. Studies with Cr3 have indicated that it is non-toxic in developmental and short- and long-term exposure studies in rodents, but the safety of this compound to chromosomes and cells has not been explored. The current study evaluates the mutagenicity, cytotoxicity, and clastogenicity of Cr3 in bacterial and mammalian cells and compares these results with similar studies using the bestselling Cr(III) nutritional supplement, chromium picolinate (CrPic). The mutagenicity of CrPic and Cr3 was tested in Escherichia coli FX-11 and Salmonella typhimurium (TA 98 and TA 100). Cytotoxicity was measured as a decrease in plating efficiency relative to controls after treatment with Cr3 and CrPic for 24h in CHO K1 cells. Clastogenicity was measured by counting the number of metaphases damaged and of the total number chromosomal aberrations in CHO K1 cells. Mutagenesis assays in E. coli and S. typhimurium were negative. All treatments of Cr3 produced ≥84% plating efficiency except 80μg/cm2, which reduced the plating efficiency to 62%. Cr3 at any treatment level did not produce a significant increase in the number of cells with abnormal metaphases, while treatments using ≥40μg/cm2 of CrPic elevated the number significantly. These data suggest that Cr3 is significantly less mutagenic in bacteria cells and less clastogenic in CHO K1 cells, while CrPic is clastogenic in CHO K1 cells.

An Integrated Experimental Design for the Assessment of Multiple Toxicological End Points in Rat Bioassays.

Background:For nearly five decades long-term studies in rodents have been the accepted benchmark for assessing chronic long-term toxic effects, particularly carcinogenicity, of chemicals. The European Food Safety Authority (EFSA) and the World Health Organization (WHO) have pointed out that the current set of internationally utilized test methods capture only some of the potential adverse effects associated with exposures to these agents over the lifetime.Objectives:In this paper, we propose the adaption of the carcinogenicity bioassay to integrate additional protocols for comprehensive long-term toxicity assessment that includes developmental exposures and long-term outcomes, capable of generating information on a broad spectrum of different end points.Discussion:An integrated study design based on a stepwise process is described that includes the priority end points of the Economic Co-operation and Development and the National Toxicology Program guidelines on carcinogenicity and chronic toxicity and developmental and reproductive toxicity. Integrating a comprehensive set of relevant toxicological end points in a single protocol represents an opportunity to optimize animal use in accordance with the 3Rs (replacement, reduction and refinement). This strategy has the potential to provide sufficient data on multiple windows of susceptibility of specific interest for risk assessments and public health decision-making by including prenatal, lactational, neonatal exposures and evaluating outcomes over the lifespan.Conclusion:This integrated study design is efficient in that the same generational cohort of rats used for evaluating long-term outcomes can be monitored in satellite parallel experiments to measure biomarkers and other parameters related to system-specific responses including metabolic alterations and endocrine disturbances.Citation:Manservisi F, Babot Marquillas C, Buscaroli A, Huff J, Lauriola M, Mandrioli D, Manservigi M, Panzacchi S, Silbergeld EK, Belpoggi F. 2017. An integrated experimental design for the assessment of multiple toxicological end points in rat bioassays. Environ Health Perspect 125:289–295; http://dx.doi.org/10.1289/EHP419

Long-term Local and Systemic Safety of Poly(L-lactide-co-epsilon-caprolactone) after Subcutaneous and Intra-articular Implantation in Rats.

The use of biodegradable materials is gaining popularity in medicine, especially in orthopedic applications. However, preclinical evaluation of biodegradable materials can be challenging, since they are located in close contact with host tissues and might be implanted for a long period of time. Evaluation of these compounds requires biodegradability and biocompatibility studies and meticulous pathology examination. We describe 2 preclinical studies performed on Sprague-Dawley rats for 52 weeks, to evaluate clinical pathology, biocompatibility, biodegradability, and systemic toxicity after implantation of 2-layered films or saline-inflated balloon-shaped implants of downsized InSpace™ devices (termed "test device"). The test devices are made from a copolymer of poly-L-lactide-co-∊-caprolactone in a 70:30 ratio, identical to the device used in humans, intended for the treatment of rotator cuff tears. Intra-articular film implantation and subcutaneous implantation of the downsized device showed favorable local and systemic tolerability. Although the implanted materials have no inherent toxic or tumorigenic properties, one animal developed a fibrosarcoma at the implantation site, an event that is associated with a rodent-predilection response where solid materials cause mesenchymal neoplasms. This effect is discussed in the context of biodegradable materials along with a detailed description of expected pathology for biodegradable materials in long-term rodent studies.

Data mining in the U.S. National Toxicology Program (NTP) database reveals a potential bias regarding liver tumors in rodents irrespective of the test agent.

Long-term studies in rodents are the benchmark method to assess carcinogenicity of single substances, mixtures, and multi-compounds. In such a study, mice and rats are exposed to a test agent at different dose levels for a period of two years and the incidence of neoplastic lesions is observed. However, this two-year study is also expensive, time-consuming, and burdensome to the experimental animals. Consequently, various alternatives have been proposed in the literature to assess carcinogenicity on basis of short-term studies. In this paper, we investigated if effects on the rodents’ liver weight in short-term studies can be exploited to predict the incidence of liver tumors in long-term studies. A set of 138 paired short- and long-term studies was compiled from the database of the U.S. National Toxicology Program (NTP), more precisely, from (long-term) two-year carcinogenicity studies and their preceding (short-term) dose finding studies. In this set, data mining methods revealed patterns that can predict the incidence of liver tumors with accuracies of over 80%. However, the results simultaneously indicated a potential bias regarding liver tumors in two-year NTP studies. The incidence of liver tumors does not only depend on the test agent but also on other confounding factors in the study design, e.g., species, sex, type of substance. We recommend considering this bias if the hazard or risk of a test agent is assessed on basis of a NTP carcinogenicity study.

GMOs in animal agriculture: time to consider both costs and benefits in regulatory evaluations

In 2012, genetically engineered (GE) crops were grown by 17.3 million farmers on over 170 million hectares. Over 70% of harvested GE biomass is fed to food producing animals, making them the major consumers of GE crops for the past 15 plus years. Prior to commercialization, GE crops go through an extensive regulatory evaluation. Over one hundred regulatory submissions have shown compositional equivalence, and comparable levels of safety, between GE crops and their conventional counterparts. One component of regulatory compliance is whole GE food/feed animal feeding studies. Both regulatory studies and independent peer-reviewed studies have shown that GE crops can be safely used in animal feed, and rDNA fragments have never been detected in products (e.g. milk, meat, eggs) derived from animals that consumed GE feed. Despite the fact that the scientific weight of evidence from these hundreds of studies have not revealed unique risks associated with GE feed, some groups are calling for more animal feeding studies, including long-term rodent studies and studies in target livestock species for the approval of GE crops. It is an opportune time to review the results of such studies as have been done to date to evaluate the value of the additional information obtained. Requiring long-term and target animal feeding studies would sharply increase regulatory compliance costs and prolong the regulatory process associated with the commercialization of GE crops. Such costs may impede the development of feed crops with enhanced nutritional characteristics and durability, particularly in the local varieties in small and poor developing countries. More generally it is time for regulatory evaluations to more explicitly consider both the reasonable and unique risks and benefits associated with the use of both GE plants and animals in agricultural systems, and weigh them against those associated with existing systems, and those of regulatory inaction. This would represent a shift away from a GE evaluation process that currently focuses only on risk assessment and identifying ever diminishing marginal hazards, to a regulatory approach that more objectively evaluates and communicates the likely impact of approving a new GE plant or animal on agricultural production systems.

AAV Provides an Alternative for Gene Therapy of the Peripheral Sensory Nervous System

Recombinant adeno-associated virus (rAAV) is a promising vector for applications in the central nervous system (CNS). Clinical phase I/II studies have demonstrated safety of rAAV-expressing therapeutic transgenes for a variety of benign (i.e., noncancer) CNS disorders, namely, Canavan disease,1 Parkinson’s disease,2,3,4 and Batten disease.5 Human studies were also recently reported targeting the retina.6,8,9,9 From the early stages of preclinical development, rAAV was appealing because it was found to be neurotrophic and capable of long-term activity without immune interference or toxicity when injected into the CNS.10

Concentrations of the Propylene Metabolite Propylene Oxide in Blood of Propylene-Exposed Rats and Humans—a Basis for Risk Assessment

Propylene (PE) was not carcinogenic in long-term studies in rodents. However, its biotransformation to propylene oxide (PO) raises questions about a carcinogenic risk. PO alkylates macromolecules, is a direct mutagen, and caused tumors in rodents at high concentrations. In order to acquire knowledge on the species-specific PO concentrations in blood resulting from PE exposure, we exposed male Fischer 344/N rats in closed exposure chambers to constant PE concentrations, between 20.1 and 3000 ppm (7 h at least), and four male volunteers to mean constant PE concentrations of 9.82 and 23.4 ppm (180 min) in inhaled air. In the animal experiments, PE and PO were measured in the chamber atmosphere, PE by gas chromatography with flame ionization detection (GC/FID), PO by GC/FID or GC with mass-selective detection (GC/MSD). In the human studies, PE was measured in inhaled and exhaled air by GC/FID. PO was quantified by GC/MSD from exhaled breath collected in gasbags. Blood concentrations of PO were calculated based on the measured PO concentrations in air using the blood-to-air partition coefficients of 60 (rat) and 66 (human). In rats, PO blood concentrations ranged from 53 nmol/l at 20.1 ppm PE to 1750 nmol/l at 3000 ppm PE. In humans, mean blood concentrations of PO were 0.44 and 0.92 nmol/l at mean PE concentrations of 9.82 and 23.4 ppm, respectively. These findings should be taken into consideration when estimating the carcinogenic risk of PE to humans based on carcinogenicity studies in PE- or PO-exposed rats.

Human carcinogenic risk evaluation, part IV: assessment of human risk of cancer from chemical exposure using a global weight-of-evidence approach.

In the early 1960s, the National Cancer Institute developed procedures to formalize the process for the evaluation of the human risk of cancer from chemical exposure (Boorman, 1994; Weisburger, 1983). This approach was based on decades of research that demonstrated that chemicals that were known to cause cancer in humans could be shown to induce cancer in laboratory animals as well. Despite the fact that this approach by the NCI was envisioned as an initial assessment of carcinogenic activity to identify those chemicals for which further study was needed, the use of two rodent species exposed for 2 years as the primary mechanism to identify potential human hazards was in widespread use by the early 1970s (Boorman, 1994). In 1975, this process was developed into a recommendation that has formed the basis of regulatory guidance (Sontag, 1976). This guidance represented the best thinking of the time about how to accomplish the critical but difficult task of prospectively identifying chemicals that might pose a carcinogenic risk to humans. Despite significant progress in our understanding of the biology of the carcinogenic response in animals and humans in the nearly 30 years since this guideline was published, these recommendations continue to form the foundation of how we do cancer risk assessment today. We have a large and extensive database today from which to develop a more rational, science-based approach to this process. A careful consideration of the available data suggests an alternative approach to this important task. Much of the early work done in this field that led to the use of the 2-year bioassay was done with reactive chemicals, most of which interacted with DNA (Miller, 1978). Ample evidence accumulated that compounds in classes like the polycyclic aromatic hydrocarbons that were known or suspected human carcinogens produced tumors in rodents. It was logical to assume that rodent tumor data could predict the hazard associated with human exposure. The concept of genetic versus epigenetic or alternative mechanisms of carcinogenesis originally proposed in the late 1980s is now well accepted. While much is understood about the mechanism of tumor induction by DNA-reactive carcinogens, assessment of human risk from exposure to nongenotoxic agents is more problematic. Careful and comprehensive evaluation of the modes of action of chemicals that produce tumors in rodents has led to the understanding that positive findings in long-term rodent studies do not necessarily predict human hazard. We now understand, for example, why urinary bladder tumors in rats exposed to high doses of saccharin or melamine do not imply a hazard for humans (Cohen et al., 1991; Rodent Bladder Carcinogenesis Working Group, 1995). Similarly, a broad and very comprehensive array of data on male rat kidney tumors induced by compounds like d-limonene that produce a characteristic male rat hydrocarbon nephropathy do not predict a carcinogenic hazard for humans following exposure to these chemicals (Swenberg and Lehman-McKeeman, 1999). In the pharmaceutical arena, there are numerous similar examples. Rat mammary tumors produced in response to b-adrenergic blocking agents in the 1970s caused significant disruption to drug development programs as concern was raised over the possibility of a similar tumorigenic response in humans. It is now clear that the biology surrounding the tumor promoting activity in rodents (hyperprolactinemia) is dissimilar between the two species and does not predict human hazard (Schyve et al., 1978; Welsch and Nagasawa, 1977). A similar major concern was raised over the appearance of the unusual mesovarial leiomyoma in rats exposed to b-agonists like salbutamol in this same time frame. It is now well accepted that this rodent tumorigenic response is secondary to a unique pharmacodynamic effect in this tissue in the rat and is not an indicator of human risk (Jack et al., 1983). To whom correspondence should be addressed: Schering-Plough Research Institute, 2015 Galloping Hill Rd., Kenilworth, NJ 07033. Fax: (973) 940-4159. E-mail: james.madonald@spcorp.com.

Toxicology of progestogens of implantable contraceptives for women

There are currently four progestogens used in implantable contraceptives marketed or tested in clinical trials: levonorgestrel in Norplant and Jadelle, etonogestrel (3-keto-desogestrel) in Implanon, nestorone in Elcometrine, and nomegestrol acetate in Uniplant and Surplant. Each progestogen was evaluated for hormonal activity and for safety in a wide variety of tests in vitro and in animals prior to their use in women. All four progestogens underwent pre-clinical testing that generally followed the format for animal testing of steroidal contraceptives published by the World Health Organization and the US Food and Drug Administration (FDA) [1]. Most of the progestogens have been tested for genotoxicity in bacterial and mammalian cultured cells and in rodents. All were tested for toxicity in short- and long-term toxicology studies in rodents and dogs or monkeys, and all were tested for their effects on reproduction and fetal development. In most cases, the progestogens were tested for carcinogenicity in two rodent species, rats and mice. Early clinical trials in small numbers of women provided additional safety data prior to the exposure of large numbers of women in Phase 3 clinical trials. The published data and data submitted to the FDA demonstrate that the implantable progestogens have no significant or unusual toxicities and have a similar safety profile to the progestogens found in the approved oral contraceptives.

Society of toxicologic pathology position paper: diet as a variable in rodent toxicology and carcinogenicity studies.

Diet is an important variable in rodent toxicology and carcinogenicity studies. Several diet models are now available for selection in rodent toxicology studies. Abundant data have clearly established a signiŽ cant correlation between average food (calorie) consumption, nutrient composition of the diet, adult body weight, the onset and incidences of speciŽ ed pathological endpoints, and survival (2, 3). Ad libitum feeding of common diets to rodents on long-term studies results in overeating, excessive caloric intake, higher body weights, decreased activity patterns, and earlier onset and higher incidences of some spontaneous degenerative diseases (nephropathy and cardiomyopathy), neoplasia (eg, pituitary and mammary tumors), and reduced survival. Reduced survival of the control animals in 2-year studies, often signiŽ cantly less than 50%, has heightened the industry’s awareness of these diet-related issues (2). The overeating problems in long-term rodent studies have been addressed by two different approaches: dietary modiŽ cation (change the nutrient contents of the diet) and dietary restriction (control the amount of food the animals can eat). Some of the early studies using dietary modiŽ cation were inconclusive, but more recently scientists at the National Toxicology Program and others have shown that control of the formulation of an ad libitum, nonpuriŽ ed diet source for laboratory animals (diet modiŽ cation) has a signiŽ cant effect on survival and the incidence and severity of several dietary and possibly age-associated pathology. More speciŽ cally, a diet (NTP-2000) with lower protein and higher fat and Ž ber ( 15% protein, 7–8.5% fat, and 9–14% crude Ž ber), when fed ad libitum to F344 rats, enhanced survival and reduced the incidence and/or severity of several common age-related

Rodent carcinogenicity tests need be no longer than 18 months: an analysis based on 210 chemicals in the IARC Monographs

The IARC Monographs (Vols 1–70) were studied to determine the time of onset of treatment-related tumorigenicity in long-term rodent studies for chemicals classified by IARC as having sufficient evidence of carcinogenicity in animals. The analysis excluded studies on metals and their salts, studies on particulates, studies by parenteral routes of administration that resulted in tumours only at the site of exposure, and studies that did not approximate to the current standard long-term rodent carcinogenicity bioassay, for instance transplacental or multigeneration studies, initiator-promoter studies, lung tumour assays in Strain A mice and studies in newborn animals. Data from a total of 210 chemicals revealed that, overall, evidence of treatment-related tumorigenicity was first apparent within 12 months for 66% of the chemicals and for only 7% were studies of longer than 18 months necessary. All IARC Group 1 chemicals were detected in animals within 18 months, and most within 12 months. Most of the tumour types that required more than 18 months for detection were of dubious relevance to human risk assessment. Termination of rodent carcinogenicity studies at 18 months or earlier would greatly reduce the complications that arise in interpreting the findings in aged animals which often have defective hepatic or renal function and would also markedly reduce the time required for histopathological examination of dozens of tissues taken from the approximately 500 animals routinely employed in these studies.