A global pharmaceutical company initiative: an evidence-based approach to define the upper limit of body weight loss in short term toxicity studies.

Short term toxicity studies are conducted in animals to provide information on major adverse effects typically at the maximum tolerated dose (MTD). Such studies are important from a scientific and ethical perspective as they are used to make decisions on progression of potential candidate drugs, and to set dose levels for subsequent regulatory studies. The MTD is usually determined by parameters such as clinical signs, reductions in body weight and food consumption. However, these assessments are often subjective and there are no published criteria to guide the selection of an appropriate MTD. Even where an objective measurement exists, such as body weight loss (BWL), there is no agreement on what level constitutes an MTD. A global initiative including 15 companies, led by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), has shared data on BWL in toxicity studies to assess the impact on the animal and the study outcome. Information on 151 studies has been used to develop an alert/warning system for BWL in short term toxicity studies. The data analysis supports BWL limits for short term dosing (up to 7days) of 10% for rat and dog and 6% for non-human primates (NHPs).

A European pharmaceutical company initiative challenging the regulatory requirement for acute toxicity studies in pharmaceutical drug development.

Regulatory guidelines indicate acute toxicity studies in animals are considered necessary for pharmaceuticals intended for human use. This is the only study type where lethality is mentioned as an endpoint. The studies are carried out, usually in rodents, to support marketing of new drugs and to identify the minimum lethal dose. A European initiative including 18 companies has undertaken an evidence-based review of acute toxicity studies and assessed the value of the data generated. Preclinical and clinical information was shared on 74 compounds. The analysis indicated acute toxicity data was not used to (i) terminate drugs from development (ii) support dose selection for repeat dose studies in animals or (iii) to set doses in the first clinical trials in humans. The conclusion of the working group is that acute toxicity studies are not needed prior to first clinical trials in humans. Instead, information can be obtained from other studies, which are performed at more relevant doses for humans and are already an integral part of drug development. The conclusions have been discussed and agreed with representatives of regulatory bodies from the US, Japan and Europe.

Genetic toxicity assessment: employing the best science for human safety evaluation part IV: a strategy in genotoxicity testing in drug development: some examples.

The minimal three-test battery of the International Conference on Harmonization guideline has been in use since 1997 for the development of new pharmaceuticals (ICH, 1997). After a 10-year experience of this core battery in regulatory genotoxicity testing, everywhere the time has come for reflection about what was learned from this experience. Different aspects of genotoxicity testing are currently being debated under different organizations (HESI, 2006; IWGT, 2007; Kirkland et al., 2007). The main concerns are to develop relevant strategies and adequate complementary tests to the minimal battery, appropriate for each specific case to assess risk for humans when in vitro positive results or findings in rodent bioassays for carcinogenicity are found. In this article, an example of an in-house decision tree is shown, with some options which can contribute to the current reflections. Additionally, tools built for early genotoxicity are presented.

Genetic toxicity assessment: employing the best science for human safety evaluation. Part II: Performances of the in vitro micronucleus test compared to the mouse lymphoma assay and the in vitro chromosome aberration assay.

The in vitro micronucleus test is commonly used in the early stages of pharmaceutical development as a predictive tool for the regulatory mouse lymphoma assay or in vitro chromosome aberration test. The accumulated data from this assay leads to the suggestion that it could be used as an alternative to the chromosome aberration test or the mouse lymphoma assay in the regulatory genotoxicity battery. In this paper, we present the results of the in vitro micronucleus test on L5178Y mouse lymphoma cells with 25 compounds from Servier research and have compared these results to those obtained in the genotoxicity regulatory battery. All the negative compounds were also negative in the in vitro micronucleus assay. Among the 14 positive compounds, two of them, positive in the mouse lymphoma assay, were found negative in the in vitro micronucleus test. However, this apparent discordance was likely to be due to cytotoxicity- or high concentration-related false positive responses in the mouse lymphoma assay. In addition, we confirmed that the in vitro micronucleus assay is useful for detecting aneugens, especially, when cells in metaphasis and multinucleated cells are also scored and when cells are allowed to recover after the long treatment. On this series of compounds, the in vitro micronucleus assay showed high sensitivity and possibly a better specificity than the mouse lymphoma assay. Thus, the in vitro micronucleus assay was shown to be at least as adequate as the mouse lymphoma assay or the in vitro chromosome aberration test to be used in the standard genotoxicity battery.

Value of in vitro models for the assessment of drug-induced haematotoxicity.

BACKGROUND: A new antipsychotic compound induced unexpected red cell hypoplasia (reticulocytopenia, red marrow hypoplasia) in rats dosed orally for 7 days. MATERIALS AND METHODS: Since an erythropoietin-mediated pathogenesis was excluded, in vitro tests on rat and human bone marrow cells were performed with measurement of formation of late erythroid (CFU-E) and granulocyte-macrophage (CFU-GM) colony-forming units after incubation with the drug. CFU-E together with growth factors were cultured for 2 days (rat) or 7 days (human) and CFU-GM was cultured for 7 days (rat) or 10 days (human). RESULTS: The drug induced inhibition of erythroid progenitors and myeloid progenitors for both species from 3 x 10(-5) mol/L, with the concentration inhibiting the growth of 50% (IC50) consistent with drug plasma levels measured in rats. CONCLUSION: These cloning assays on rat bone-marrow cells were shown to be adequate models for determining the haematotoxicity of the agent and to be predictive of human toxicity. With only a small amount of compound required, they can be used as screening tools to detect haematotoxic potential of candidate drugs.

Easy Procedure for Milk Collection in Lactating Rats.

Procedures have been developed for easy collection of milk samples without oxytocin injection. Dams were separated from their pups for 24 h, then were hand-milked with or without aspiration. During the lactation period, the collected milk volume increased up to day 10 of lactation, reached a plateau between days 10 and 18 (range, 2.49 1.49 to 4.27 1.26 g/dam/15 min), then was lower on day 21 (2.12 1.32 g/dam/15 min). We validated our method by administering estradiol (E2) from days 10 to 17 of lactation. Treatment with E2 was associated with a dose-related reduction in the number of dams giving milk at sampling on day 18 of lactation (controls: 14/14; E2 at 0.05 mg/kg/d of body weight/d 10/15; E2 at 0.10 mg/kg/d 5/15) and a severe dose-related reduction in milk volume collected (controls: 3.75 1.40 g/dam/15 min; E2 at 0.05 mg/kg/day: 0.80 1.03 g/dam/15 min; E2 at 0.10 mg/kg/d 0.38 0.68 g/dam/15 min). Our results suggest that days 10 to 18 of lactation appear to be the most suitable period for milk collection in lactating rats in regulatory reproductive toxicologic studies; within this period, we recommend either day 16 or day 18 for milk collection.