Assessing the genotoxicity of N-nitrosodiethylamine with three in vivo endpoints in male Big Blue® transgenic and wild-type C57BL/6N mice.

The detection of N-nitrosamines in drug products has raised global regulatory interest in recent years due to the carcinogenic potential of some nitrosamines in animals and a need to identify a testing strategy has emerged. Ideally, methods used would allow for the use of quantitative analysis of dose-response data from in vivo genotoxicity assays to determine a compound-specific acceptable intake for novel nitrosamines without sufficient carcinogenicity data. In a previous study we compared the dose-response relationships of N-nitrosodiethylamine (NDEA) in three in vivo genotoxicity endpoints in rats.

Experiments in the EpiDerm 3D Skin In Vitro Model and Minipigs In Vivo Indicate Comparatively Lower In Vivo Skin Sensitivity of Topically Applied Aneugenic Compounds.

Risk management of in vitro aneugens for topically applied compounds is not clearly defined because there is no validated methodology to accurately measure compound concentration in proliferating stratum basale keratinocytes of the skin. Here, we experimentally tested several known aneugens in the EpiDerm reconstructed human skin in vitro micronucleus assay and compared the results to flow cytometric mechanistic biomarkers (phospho-H3; MPM2, DNA content). We then evaluated similar biomarkers (Ki-67, nuclear area) using immunohistochemistry in skin sections of minipigs following topical exposure the potent aneugens, colchicine, and hesperadin.

2-Hydroxypyridine N-Oxide is not genotoxic in vivo.

2-Hydroxypyridine N-oxide (HOPO) is an important coupling reagent used in pharmaceutical synthesis. Our laboratory previously reported HOPO as equivocal in the Ames assay following extensive testing of multiple lots of material. Given the lack of reproducibility between lots of material and the weak increase in revertants observed, it was concluded that it would be highly unlikely that HOPO would pose a mutagenic risk in vivo.

Compensatory erythropoiesis has no impact on the outcome of the in vivo Pig-a mutation assay in rats following treatment with the haemolytic agent 2-butoxyethanol.

The Pig-a assay has rapidly gained international interest as a useful tool for assessing the mutagenic potential of compounds in vivo. Although a large number of compounds, including both mutagens and non-mutagens, have been tested in the rat Pig-a assay in haematopoietic cells, there is limited understanding of how perturbations in haematopoiesis affect assay performance. Of particular concern is the possibility that regenerative haematopoiesis alone, without exposure to a genotoxic agent, could result in elevated Pig-a mutant cell frequencies.

Evaluation of the in vivo mutagenicity of isopropyl methanesulfonate in acute and 28-day studies.

Understanding the mutagenic dose response could prove beneficial in the management of pharmaceutically relevant impurities. For most alkyl ester impurities, such as isopropyl methanesulfonate (IPMS), little in vivo mutagenicity data exist for dose analysis. The likelihood of a sublinear dose response for IPMS was assessed by comparing the Swain Scott constant, the SN 1/SN 2 reaction mechanism and the O(6) :N(7) guanine adduct ratio to that of more well-known alkyl esters.

Evaluation of the Pig-a, micronucleus, and comet assay endpoints in a 28-day study with ethyl methanesulfonate.

Ethyl methanesulfonate (EMS) was evaluated as part of the validation effort for the rat Pig-a mutation assay and compared with other well-established in vivo genotoxicity endpoints. Male Sprague-Dawley (SD) rats were given a daily dose of 0, 6.25, 12.

Development and validation of an in vitro micronucleus assay platform in TK6 cells.

The Organization for Economic Co-operation and Development (OECD) has recently adopted Test Guideline 487 (TG487) for conducting the in vitro micronucleus (MNvit) assay. The purpose of this study is to evaluate and validate treatment conditions for the use of p53 competent TK6 human lymphoblastoid cells in a TG487 compliant MNvit assay. The ten reference compounds suggested in TG487 (mitomycin C, cytosine arabinoside, cyclophosphamide, benzo-a-pyrene, vinblastine sulphate, colchicine, sodium chloride, nalidixic acid and di(2-ethylhexyl)phthalate and pyrene) and noscapine hydrochloride were chosen for this study.

Report on stage III Pig-a mutation assays using N-ethyl-N-nitrosourea-comparison with other in vivo genotoxicity endpoints.

N-Ethyl-N-nitrosourea (ENU) was evaluated as part of the Stage III trial for the rat Pig-a gene mutation assay. Groups of six- to eight-week-old male Sprague Dawley (SD) or Fischer 344 (F344) rats were given 28 daily doses of the phosphate buffered saline vehicle, or 2.5, 5, or 10 mg/kg ENU, and evaluated for a variety of genotoxicity endpoints in peripheral blood, spleen, liver, and colon.

The rat gut micronucleus assay: a good choice for alternative in vivo genetic toxicology testing strategies.

The in vivo bone marrow (BM) micronucleus assay is one of the three tests in the standard test battery to assess the genotoxic potential of a pharmaceutical candidate. In some cases, depending on results of in vitro studies, the route of administration or the degree of systemic exposure, in vivo assessment of genotoxicity in the BM alone may not be sufficient. Based on the potential for high gut exposures to orally administered compounds with low systemic exposures as well as the potential susceptibility of rapidly dividing cells of the intestinal tissues, we have developed a modified technique for evaluating micronuclei formation in both the duodenum and colon of rats based on earlier publications.

Genotoxicity of 2-(3-chlorobenzyloxy)-6-(piperazinyl)pyrazine, a novel 5-hydroxytryptamine2c receptor agonist for the treatment of obesity: role of metabolic activation.

2-(3-Chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine (3) is a potent and selective 5-HT(2C) agonist that exhibits dose-dependent inhibition of food intake and reduction in body weight in rats, making it an attractive candidate for treatment of obesity. However, examination of the genotoxicity potential of 3 in the Salmonella Ames assay using tester strains TA98, TA100, TA1535, and TA1537 revealed a metabolism (rat S9/NADPH)- and dose-dependent increase of reverse mutations in strains TA100 and TA1537. The increase in reverse mutations was attenuated upon coincubation with methoxylamine and glutathione.

Anilinodialkoxyquinazolines: screening epidermal growth factor receptor tyrosine kinase inhibitors for potential tumor imaging probes.

The epidermal growth factor receptor (EGFR), a long-standing drug development target, is also a desirable target for imaging. Sixteen dialkoxyquinazoline analogues, suitable for labeling with positron-emitting isotopes, have been synthesized and evaluated in a battery of in vitro assays to ascertain their chemical and biological properties. These characteristics provided the basis for the adoption of a selection schema to identify lead molecules for labeling and in vivo evaluation.

Five carboxyl-terminal residues of neuregulin2 are critical for stimulation of signaling by the ErbB4 receptor tyrosine kinase.

The neuregulins (NRGs) are members of the epidermal growth factor (EGF) family of peptide growth factors. These hormones are agonists for the ErbB family of receptor tyrosine kinases, a family that includes the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/HER4. We recently observed that the EGF family hormone NRG2beta is a potent agonist for ErbB4.

Neuregulin isoforms exhibit distinct patterns of ErbB family receptor activation.

During the last decade, several novel members of the Epidermal Growth Factor family of peptide growth factors have been identified. Most prominent among these are the Neuregulins or Heregulins. To date, four different Neuregulin genes have been identified (Neuregulin1-4) and several different splicing isoforms have been identified for at least two of these genes (Neuregulin1 and Neuregulin2).