Rodent Toxicity Studies Research Articles

Response to Letter to Editor by Dr. J. C. Wolf on “Analysis of unbiased histopathology data from rodent toxicity studies (or, are these groups different enough to ascribe it to treatment?)”

Response to Letter to Editor by Dr. J. C. Wolf on “Analysis of unbiased histopathology data from rodent toxicity studies (or, are these groups different enough to ascribe it to treatment?)”

Quantified tracking and monitoring of diazepam treated socially housed cynomolgus monkeys

Quantitative assessment of behavioural patterns is frequently used in rodent toxicity studies, however only limited approaches are available for monkeys. Often qualitative behavioural scoring using functional observation batteries (FOBs) is performed, with difficulties like poor reproducibility or lack of sensitivity. In this study, we investigated whether quantitative behavioural monitoring can be applied to group-housed cynomolgus monkeys. Video-tracking EthoVision® XT system and special analysis software were used to evaluate diazepam (i.v. 1mg/kg) related behavioural changes in group-housed animals. Recordings were made predose and at the anticipated time of maximum drug exposure (Tmax). General parameters such as distance travelled and velocity did not reveal the known sedative effects of diazepam. However, inspection of the automatically generated track images indicated that diazepam-treated animals had more a meandering movement pattern suggesting that diazepam induced a loss of balance which was regained by corrective movements. Therefore, parameters revealing specific aspects of the meandering movement pattern such as velocity profiles and turn angles have been analyzed and revealed an increase in the curvature and in the number of directional changes of the movement path.

Integration of Pig‐a, micronucleus, chromosome aberration, and comet assay endpoints in a 28‐day rodent toxicity study with 4‐nitroquinoline‐1‐oxide

As part of the Stage III Pig-a multilaboratory validation trial, we examined the induction of CD59-negative reticulocytes and total red blood cells (RET(CD59-) and RBC(CD59-) , respectively) in male Sprague Dawley(®) rats treated with 4-nitroquinoline-1-oxide (4NQO), for 28 consecutive days by oral gavage, at doses of 1.25, 2.50, 3.75, 5.00, and 7.50 mg kg(-1) day(-1) (the high dose group was sacrificed on Day 15 due to excessive morbidity/mortality). Animals also were evaluated for: micronucleated reticulocytes (mnRET) by flow cytometry; DNA damage in peripheral blood, liver, and stomach using the Comet assay; and chromosome aberrations (CAb) in peripheral blood lymphocytes (PBL). All endpoints were analyzed at two or more timepoints where possible. Mortality, body and organ weights, food consumption, and clinical pathology also were evaluated, and demonstrated that the maximum tolerated dose was achieved at 5.00 mg kg(-1) day(-1) . The largest increases observed for the genetic toxicology endpoints (fold-increase compared to control, where significant; all at 5.00 mg kg(-1) day(-1) on Day 29) were: RET(CD59-) (21X), RBC(CD59-) (9.0X), and mnRET (2.0X). In contrast, no significant increases were observed for the CAb or Comet response, in any tissue analyzed, at any timepoint. Because 4NQO is a well known mutagen, clastogen, and carcinogen, the lack of response for these latter endpoints was unexpected. These results emphasize the extreme care that must betaken in dose and endpoint selection when incorporating genotoxicity endpoints into routine toxicity studies as has been recommended or is under consideration by various regulatory and industrial bodies.

Counterpoint to “Analysis of Unbiased Histopathology Data from Rodent Toxicity Studies (or, Are These Groups Different Enough to Ascribe to Treatment?)”

Counterpoint to “Analysis of Unbiased Histopathology Data from Rodent Toxicity Studies (or, Are These Groups Different Enough to Ascribe to Treatment?)”

Analysis of Unbiased Histopathology Data from Rodent Toxicity Studies (or, Are These Groups Different Enough to Ascribe It to Treatment?)

It is a common problem to distinguish a minor treatment change from background variation, especially when establishing no observed effect levels. Toxicological histopathologists use a wide range of methods at the microscope for comparing groups to help them form their opinions. Although the data produced by these methods can be subjective, all of these methods produce data that can be formally analyzed to give an objective, probabilistic result, provided the observations are unbiased. Other important experimental disciplines make extensive use of completely subjective data to produce objective results, for example, clinical trials using patients' symptoms. It is argued here that pathological experimental data too should be analyzed before an expert opinion (along with the objective evidence for that opinion) is formally reported. The Ordering Method, based on ranking the severity of a putative toxic change, is the most sensitive, robust, and analytically flexible method currently available.

Scientific Opinion on Flavouring Group Evaluation 86, Revision 1 (FGE.86Rev1): Consideration of aliphatic and aromatic amines and amides evaluated by JECFA (65th meeting)

Scientific Opinion on Flavouring Group Evaluation 86, Revision 1 (FGE.86Rev1): Consideration of aliphatic and aromatic amines and amides evaluated by JECFA (65th meeting)

Differences in Rat Models Used in Routine Toxicity Studies

The discussion on whether the Sprague Dawley (SD), the Fischer F344, or the Hannover Wistar rat is the most appropriate model for toxicity studies in rodents is ongoing. A substantial quantity of data on these strains concerning their source, diet, and housing conditions have been published. Generally, before starting a toxicology program in rodents, it should be taken into account that oncogenicity studies will be required for the majority of compounds successfully completing development. Survival, body weight development, incidence, type, time of onset of age-dependent lesions and neoplasms, as well as some special considerations of the rat model selected may be decisive. Therefore, an understanding of the historical background data is essential. These aspects demonstrate why the use of a specific rat model should be carefully considered at the beginning of the toxicology program.

The role of animal psychology in developmental neurotoxicity assessment for environmental chemicals

This article describes background information for the symposium held at the 70th Annual meeting of the Japanese Society for Animal Psychology. The main theme of this symposium was to provide updated information on neurodevelopmental toxicity caused by environmental chemicals, through lectures by three distinguished researchers, and to discuss possible contributions and expected roles of animal psychology in toxicological studies designed to protect children's health. The Ministry of the Environment has commenced a large-scale, long-term epidemiological study entitled the “Environment and Children's Study (Eco-Chil)”, to elucidate health effects caused by chemical exposures of children including pervasive developmental disorders. Neurobehavioral assessments have been included in globally accepted guidelines for neurotoxicity and reproductive toxicity studies in rodents as effective tools for collecting experimental evidence. Findings from these experiments provide a scientific basis for the epidemiological studies, like Eco-Chil. However, currently available behavioral testing methods are insufficient for detecting neurodevelopmental effects of environmental chemicals, especially for emotionality and social aspects of behavior. Appropriate animal behavioral models are needed to advance toxicological studies further. Animal psychologists could greatly contribute to the development of these animal models and the interpretation of behavioral test results.

Veterinary and toxicological applications for the detection of cardiac injury using cardiac troponin

The use of cardiac troponin (cTn), the 'gold-standard' biomarker of myocardial injury in humans, is growing in veterinary medicine and in animal safety studies, although there are differences in its application in animals. In this study six new assays for the marker were assessed in 619 animals of six different species (dog, cat, horse, cattle, rat and rabbit), in clinical and drug-safety studies. Healthy animals and clinical cases without cardiac disease served as controls. Several of the tested assays had poor analytic or diagnostic sensitivity and only one test was effective in all species and in all models of cardiac injury. This assay had the highest sensitivity and widest dynamic range, and identified cardiac injury due to anaemia, pancreatitis, uncontrolled Addison's and Cushing's disease, old age, renal disease, severe colic, lymphoma and neoplasia. Detection of the cTnI and cTnT forms correlated with loss of cardiac function in toxicity studies in rodents and rabbit. Increased serum cTnI was not found to correlate with disease aetiology or pathogenesis, but was effective in detecting, monitoring and quantifying ongoing cardiac injury. Cardiac injury, as demonstrated by elevated cTnI in blood, appears to be a common sequel to a wide variety of both primarily cardiac disease and of other diseases that do not primarily involve the cardiovascular system.

Biodistribution and safety assessment of AAV2‐GAD following intrasubthalamic injection in the rat

The steps necessary to translate promising new biological therapies to the clinic are poorly documented. For gene therapy, there are unique aspects that need to be addressed in biodistribution studies. Notably, the spread of the vector beyond the intended target cells or tissue may result in persistent unwanted biological activity or unpredictable biological events; thus, it is critical to evaluate the risks associated with viral vector-mediated gene transfer prior to embarking on human clinical trials. In the present study, we conducted a comprehensive assessment of vector biodistribution throughout the brain, blood and major organs of rats that had been injected via the subthalamic nucleus with recombinant adeno-associated virus (AAV) expressing glutamic acid decarboxylase (GAD). In addition, behavioral and histological analyses were also performed. AAV genomes were not detected in blood or cerebrospinal fluid, and did not disseminate to organs outside of the brain in the majority of animals. In the brain, an average of 97.3% of AAV2-GAD genomes were restricted to the area of the ipsilateral subthalamic nucleus (STN). There were no discernable effects of AAV2-GAD on general health, and a behavioral assessment of the animals did not reveal any alteration in general behavior, exploration, locomotion or motor symmetry. The present study met Food and Drug Administration requirements, in addition to efficacy and toxicity studies in rodents and nonhuman primates, to support and supplement a Phase II clinical trial invloving the gene transfer of AAV2-GAD to the human STN for the potential therapy of Parkinson's disease.

A novel water delivery system for administering volatile chemicals while minimizing chemical waste in rodent toxicity studies

Rodent toxicity studies typically use water bottles to administer test chemicals via drinking water. However, water bottles provide inconsistent exposure of volatile chemicals due to varying headspace, and lead to excessive waste of test material. To refine drinking water toxicity studies in rodents by enhancing sample quality and consistency, and minimizing waste, we designed and implemented a novel water delivery system that keeps the water chilled, headspace free and protected from light. Materials used were resistant to chemical interaction. In this gravity-fed system, a 6-L Teflon water bag, stored in a polystyrene cooler on the cage rack, was connected to a stainless steel manifold delivering water to five cages via specialized drinking valves. Due to the absence of headspace in the water bag, this system allows consistent exposure of volatile chemicals. In addition, small diameter tubing throughout the system reduces the amount of test material residing in the system and minimizes chemical waste.

Subchronic feeding study of grain from herbicide-tolerant maize DP-Ø9814Ø-6 in Sprague-Dawley rats

This 13-week feeding study conducted in Sprague-Dawley rats evaluated the potential health effects from long-term consumption of a rodent diet formulated with grain from genetically modified (GM), herbicide-tolerant maize DP-Ø9814Ø-6 (98140; trade name Optimum® GAT® (Optimum® GAT® is a registered trademark of Pioneer Hi-Bred)). Metabolic inactivation of the herbicidal active ingredient glyphosate was conferred by genomic integration and expression of a gene-shuffled acetylase coding sequence, gat4621, from Bacillus licheniformis; tolerance to acetolactate synthase (ALS) inhibiting herbicides was conferred by overexpression of a modified allele (zm-hra) of the endogenous maize ALS enzyme that is resilient to inactivation. Milled maize grain from untreated (98140) and herbicide-treated (98140+Gly/SU) plants, the conventional non-transgenic, near-isogenic control (091), and three commercial non-transgenic reference hybrids (33J56, 33P66, and 33R77) was substituted at concentrations of 35–38% w/w into a common rodent chow formula (PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002) and fed to rats (12/sex/group) for at least 91 consecutive days. Compared with rats fed diets containing grain from the conventional near-isogenic control maize, no adverse effects were observed in rats fed diets containing grain from 98140 or 98140+Gly/SU maize with respect to standard nutritional performance metrics and OECD 408-compliant toxicological response variables [OECD, 1998. Section 4 (Part 408), Health Effects: Repeated Dose 90-Day Oral Toxicity Study in Rodents, Guideline for the Testing of Chemicals. Organisation of Economic Co-operation and Development, Paris, France]. These results support the comparative safety and nutritional value of maize grain from genetically modified Optimum® GAT® and conventional, non-transgenic hybrid field corn.

Request from the European Commission related to the safeguard clause invoked by Austria on maize lines MON863 according to Article 23 of Directive 2001/18/EC

Request from the European Commission related to the safeguard clause invoked by Austria on maize lines MON863 according to Article 23 of Directive 2001/18/EC

Metabonomic Study of Ochratoxin A Toxicity in Rats after Repeated Administration: Phenotypic Anchoring Enhances the Ability for Biomarker Discovery

For early detection of toxicity and improved mechanistic understanding, GC/MS-, 1H NMR-, and LC/MS-based metabonomics were applied to urine samples from a rodent toxicity study on the mycotoxin and renal carcinogen ochratoxin A (OTA). OTA was administered at doses of 0, 21, 70, and 210 microg/kg body wt for up to 90 days. Urine samples were collected at 24 h intervals 14, 28, and 90 days after the start of treatment and analyzed with GC/MS, 1H NMR, and LC/MS. Principal component analysis and orthogonal projection to latent structures discriminate analysis (OPLS-DA) based on GC/MS and 1H NMR data discriminated controls from animals dosed with 210 microg/kg body wt OTA as early as 14 days and animals dosed with 70 microg/kg body wt 28 days after the start of treatment, correlating with mild histopathological changes in the kidney. Integration of histopathology scores as discriminators in OPLS-DA models resulted in better multivariate model predictivity and facilitated marker identification. Decreased 2-oxoglutarate and citrate excretion and increased glucose, creatinine, pseudouridine, 5-oxoproline, and myo-inositol excretion were detected with GC/MS. Decreased 2-oxoglutarate and citrate excretion and increased amino acid excretion were found with 1H NMR. Increased urinary glucose is a well-established indicator of kidney damage, and altered excretion of TCA cycle intermediates (citrate and 2-oxoglutarate) is found as a general response to toxic insult in many metabonomics studies. Other markers are associated with cell proliferation (pseudouridine), changes in renal osmolyte handling (myo-inositol), and oxidative stress (5-oxoproline), established mechanisms of OTA toxicity. LC/MS was also able to discriminate controls and treated animals but contained more noise, and marker annotation was only speculative due to lack of reference databases. Use of multiple analytical platforms for metabonomics analysis may result in a more comprehensive metabolite coverage and may be applied to obtain mechanistic information from conventional rodent toxicity studies.

Acute pulmonary eosinophilia by i.t. carbon nanotubes: a DAMP trigger

Carbon‐based nanoparticles have promising applications in nanomedicine, electronics and drug delivery. However, lung toxicity studies in rodents continue to reflect tissue effects. Our in vivo studies with single wall carbon nanotubes (SWCNT) administered intratracheally in pulmonary surfactant (50 mg in 50 microliters i.t.) produced a rapid pulmonary eosinophilia and airway mucin upregulation within minutes. Nanotube clusters produced pulmonary tissue granulomas within 1 week, at which time mucin‐laden macrophages, some with nanoparticle fibers, traveled to the mesothelial space and the draining lymph nodes. The responses of the SWCNT‐treated rat airway and mesothelium was of interest given the known response of human mesothelium to fibers such as asbestos. Toxicity of SWCNT on Met‐5A human mesothelial cells was investigated in vitro. Viability was evaluated in the presence and absence of surfactant with increasing doses of SWCNT. Cell toxicity, evaluated by WST‐1 assay showed TC50 of 63 μg/mL without and 70 μg/mL with 20% surfactant in the culture medium, a nonsignificant difference. Transmission electron microscopy on mesothelial cells treated with SWCNT showed intracellular nanotube clusters indenting the nuclear membrane with projecting fibers. These data suggested that the SWCNT‐induced rapid eosinophilia might be triggered by such damage, through cellular release of endogenous signals. Our early efforts with nuclear protein HMGB1 in tissue suggests that the SWCNT are responsible for a "DAMP‐type" trigger of eosinophilia in vivo in the rat model. TEM efforts with pleural fluid and nodes continue to trace the path of the SWCNT in the pulmonary‐ mesothelial interface.

Collaborative work on evaluation of ovarian toxicity 3) Effects of 2- or 4- week repeated-dose toxicity and fertility studies with tamoxifen in female rats

To assess whether ovarian histopathological examination in repeated-dose rodent toxicity study could reliably anticipate toxic effects on female reproductive function and to assess whether ovarian change could be detected in a 2-week repeated-dose toxicity study, tamoxifen was administrated orally to female rats at 0.005, 0.03, or 0.2 mg/kg/day for 2 and 4 weeks in the repeated-dose toxicity studies, and for 2 weeks prior to cohabitation, during cohabitation, and through Gestation Day 7 in a female fertility study. The relationship between ovarian histopathological findings and fertility results was investigated. Findings at 0.03 and 0.2 mg/kg/day included decreases in body weight gains associated with decreases in food consumption, in 2- and 4-week repeated-dose toxicity studies and fertility study. The ovarian histopathological findings included increases in large atretic follicles, increases in interstitium cells and absence of newly-formed corpus lutea at 0.2 mg/kg/day in the 2-week study and at 0.03 and 0.2 mg/kg/day in the 4-week study. The treatment induced estrogenic and antiestrogenic reactions in the uterus, while mucinous degeneration was detected in the vagina. Effects on female fertility consisted primarily of disturbance of estrus cycle and decreases in numbers of pregnant rats which were considered to be related to ovarian histopathological changes. Based on these findings, ovarian histopathological evaluation in the repeated-dose toxicity study could anticipate the effects of tamoxifen on female fertility via ovarian dysfunction at slightly toxic doses, and 2-week treatment of tamoxifen at appropriate dose could be sufficient to detect ovarian toxicity by microscopic examination.

Comparing single and repeated dosimetry data for perfluorooctane sulfonate in rats

Perfluorooctane sulfonate (PFOS) is a member of a class of perfluorinated chemicals used in a variety of consumer and industrial applications because of their oleophobic and hydrophobic properties. It has been shown to cause toxicity in adult and developing laboratory animals. Because PFOS has also been shown to be widely distributed throughout the environment, there have been concerns about its potential health risk to humans. Limited pharmacokinetic data for PFOS are available in rodents and humans, while epidemiological studies of workers and extensive toxicity studies in rodents have been performed. The existing pharmacokinetic and toxicity database in rodents can be useful in the cross-species extrapolations needed to evaluate and interpret internal dosimetry in humans. A mathematical model that describes the disposition of PFOS in adult rats following intravenous, oral, and chronic dietary exposures was developed to gain a better understanding of the pharmacokinetics of PFOS and to determine whether single-dose kinetics are predictive of repeated-dose kinetics. In order to characterize existing time-course data, time-dependent and concentration-dependent changes in the pharmacokinetic parameters for urinary and biliary clearance and liver distribution were needed. Whether these time-dependent changes represent inconsistencies across experiments, effects of aging in the rats, or chemically induced changes in pharmacokinetics remains to be determined.

Safety evaluation to support First-In-Man investigations I: Kinetic and safety pharmacology studies

Kinetic ( in vitro, pharmacokinetic, mass balance and toxicokinetic studies) and safety pharmacology data available in 34 Investigator’s Brochures used to support First-In-Man clinical trials over a 10 year period have been evaluated to give an insight into the types of study designs used and how these have changed over the period analysed (1997–2006). For kinetic evaluation, study packages often had single dose pharmacokinetic studies in the rodent and non-rodent by oral and intravenous dose routes as well as protein binding measurement accompanied more recently by rodent mass balance studies and an examination of in vitro metabolism across species and/or enzyme activity with hepatocytes, liver microsomes or recombinantly expressed enzymes. Toxicokinetic data were examined from key repeat dose toxicity studies in rodents and non-rodents with the recent inclusion of sampling in control animals to check for drug contamination. For safety pharmacology evaluation, study packages included “core battery” rodent central nervous system as well as anaesthetised non-rodent cardiovascular and respiratory function studies. More recently, cardiovascular assessment occurred in conscious non-rodents, necessitating stand-alone rodent respiratory function studies although conscious models assessing both cardiovascular and respiratory function were also seen in some of the most recent packages. Recent packages also had an in vitro electrophysiology study, usually the hERG assay. Considerations for kinetic and safety pharmacology study designs are discussed.

ESTP comments on the draft updated OECD test guideline 407

The revision of the OECD TG 407 test guideline (repeated dose 28-day oral toxicity study in rodents) focuses on endpoints to detect endocrine activities of chemicals. The new endpoints are likely to influence other previously established core endpoints of this study type. An expert group of pathologists and toxicologists within the European Society of Toxicologic Pathologists (ESTP) has contributed to the scientific discussion of the draft guideline. The advantages and disadvantages of methodical changes as necropsy of all females in dioestrus, blood collection for clinical chemistry and haematology at the same cycle stage, weighing of the thyroid gland and separate weighing of ventral and dorsolateral lobes of the prostate are considered. Possible alternatives are pointed out covering scientific as well as practical aspects.

Risk assessment of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) on the Smoke Flavouring Primary Product ‐ FF‐B

Risk assessment of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) on the Smoke Flavouring Primary Product ‐ FF‐B