Sheila M. Galloway

A quantitative assessment of the cytotoxicity associated with chromosomal aberration detection in Chinese hamster ovary cells

Regulatory guidelines suggest testing chemicals up to cytotoxic doses in chromosomal-aberration assays. To investigate the utility and limitations of various cytotoxicity indicators we used Chinese hamster ovary (CHO) cells to test 8 chemicals with differing ratios of cytotoxicity to clastogenicity. We measured immediate or delayed cell killing and growth inhibition (ATP levels, cell counts, colony-forming efficiency, CFE) and cell-cycle perturbations (mitotic index, MI; average generation time, AGT). Aberrations (abs) were scored 10 and 24 h from the beginning of the 3-h treatment. All 8 compounds induced abs at concentrations that reduced cell growth at 24 h by 50% or less. Concentrations of each chemical which induced at least 15% cells with abs, gave little loss of CFE (0-20%) for mitomycin C, adriamycin, cadmium sulfate and 2,6-diaminotoluene in contrast to the marked loss of CFE (70-80%) for eugenol (EUG), 2-aminobiphenyl and 8-hydroxyquinoline (8-HQ). 2,4-Diaminotoluene (2,4-DAT) was intermediate. Higher aberration yields were found at 24 h than at 10 h, even when minimal cell-cycle delay was detected by AGT estimates from BrdUrd-labeled cells. Cells with multiple abs were seen at 24 but not at 10 h, and often confirmed clastogenicity when there was only a weak increase in the percentage of cells with aberrations. Total ATP per culture did not always correlate with cell number, especially at later times after treatment. This is likely due to metabolic perturbations or altered cell biomass that are known to affect cell ATP content. MI suppression often did not correlate with AGT, e.g., only small increases in AGT were seen for 8-HQ, 2,4-DAT and EUG despite severe mitotic suppression at 10 h. By 24 h the MI for all chemicals had recovered, sometimes exceeding control levels. Marked mitotic accumulation was seen at 10 h for 2,4-DAT, indicating cell synchrony. Thus, the MI has limited value for dose selection. In conclusion, even weakly active chemicals were detected at a single time without exceeding a 50% growth reduction at 24 h.

Effect of sampling time on chromosome aberration yield for 7 chemicals in Chinese hamster ovary cells

Choice of harvest time is one of the most important variables in the assessment of whether a compound is clastogenic and in establishing a dose relation. We examined the effects of sampling time on aberration yield for 7 diverse chemicals in CHO-WBL cells by harvesting at intervals from 9 to 30 h after treatment for 3 h with or without S9 metabolic activation. We observed both the percentage of aberrant cells and the total number of aberrations. Our data suggest that for most compounds a single harvest time approximately 17-21 h after the beginning of a 3-h treatment is optimal for aberration detection in CHO cells. Maximal aberration yields were observed for 2,4-diaminotoluene, 2,6-diaminotoluene and cytosine beta-D-arabinofuranoside from 17 to 21 h, eugenol from 15 to 21 h, cadmium sulfate from 15 to 24 h and 2-aminobiphenyl, from 17 to 24 h. For adriamycin at 1 microM, the % aberrant cells remained elevated throughout the period from 9 to 29 h, while small increases at 0.1 microM ADR were found only at 13 and at 25 h. For most chemicals the maximal aberration yield occurred at a different time for each concentration tested. However, the use of 3 or more closely spaced concentrations, carefully selected to yield up to 50% toxicity, allowed detection of a positive response at a single harvest time for all 7 chemicals.

Overcoming mutagenicity and ion channel activity: optimization of selective spleen tyrosine kinase inhibitors.

Development of a series of highly kinome-selective spleen tyrosine kinase (Syk) inhibitors with favorable druglike properties is described. Early leads were discovered through X-ray crystallographic analysis, and a systematic survey of cores within a selected chemical space focused on ligand binding efficiency. Attenuation of hERG ion channel activity inherent within the initial chemotype was guided through modulation of physicochemical properties including log D, PSA, and pKa. PSA proved most effective for prospective compound design. Further profiling of an advanced compound revealed bacterial mutagenicity in the Ames test using TA97a Salmonella strain, and subsequent study demonstrated that this mutagenicity was pervasive throughout the series. Identification of intercalation as a likely mechanism for the mutagenicity-enabled modification of the core scaffold. Implementation of a DNA binding assay as a prescreen and models in DNA allowed resolution of the mutagenicity risk, affording molecules with favorable potency, selectivity, pharmacokinetic, and off-target profiles.

Design of a novel pyrrolidine scaffold utilized in the discovery of potent and selective human β3 adrenergic receptor agonists.

A novel class of human β(3)-adrenergic receptor agonists was designed in effort to improve selectivity and metabolic stability versus previous disclosed β(3)-AR agonists. As observed, many of the β(3)-AR agonists seem to need the acyclic ethanolamine core for agonist activity. We have synthesized derivatives that constrained this moiety by introduction of a pyrrolidine. This unique modification maintains human β(3) functional potency with improved selectivity versus ancillary targets and also eliminates the possibility of the same oxidative metabolites formed from cleavage of the N-C bond of the ethanolamine. Compound 39 exhibited excellent functional β(3) agonist potency across species with good pharmacokinetic properties in rat, dog, and rhesus monkeys. Early de-risking of this novel pyrrolidine core (44) via full AMES study supports further research into various new β(3)-AR agonists containing the pyrrolidine moiety.

International regulatory requirements for genotoxicity testing for pharmaceuticals used in human medicine, and their impurities and metabolites

The process of developing international (ICH) guidelines is described, and the main guidelines reviewed are the ICH S2(R1) guideline that includes the genotoxicity test battery for human pharmaceuticals, and the ICH M7 guideline for assessing and limiting potentially mutagenic impurities and degradation products in drugs. Key aspects of the guidelines are reviewed in the context of drug development, for example the incorporation of genotoxicity assessment into non‐clinical toxicity studies, and ways to develop and assess weight of evidence. In both guidelines, the existence of “thresholds” or non‐linear dose responses for genotoxicity plays a part in the strategies. Differences in ICH S2(R1) protocol recommendations from OECD guidelines are highlighted and rationales explained. The use of genotoxicity data during clinical development and in assessment of carcinogenic potential is also described. There are no international guidelines on assessment of potentially genotoxic metabolites, but some approaches to safety assessment are discussed for these. Environ. Mol. Mutagen. 58:296–324, 2017.

Potential impurities in drug substances: Compound-specific toxicology limits for 20 synthetic reagents and by-products, and a class-specific toxicology limit for alkyl bromides

ABSTRACT This paper provides compound‐specific toxicology limits for 20 widely used synthetic reagents and common by‐products that are potential impurities in drug substances. In addition, a 15 &mgr;g/day class‐specific limit was developed for monofunctional alkyl bromides, aligning this with the class‐specific limit previously defined for monofunctional alkyl chlorides. Both the compound‐ and class‐specific toxicology limits assume a lifetime chronic exposure for the general population (including sensitive subpopulations) by all routes of exposure for pharmaceuticals. Inhalation‐specific toxicology limits were also derived for acrolein, formaldehyde, and methyl bromide because of their localized toxicity via that route. Mode of action was an important consideration for a compound‐specific toxicology limit. Acceptable intake (AI) calculations for certain mutagenic carcinogens assumed a linear dose‐response for tumor induction, and permissible daily exposure (PDE) determination assumed a non‐linear dose‐response. Several compounds evaluated have been previously incorrectly assumed to be mutagenic, or to be mutagenic carcinogens, but the evidence reported here for such compounds indicates a lack of mutagenicity, and a non‐mutagenic mode of action for tumor induction. For non‐mutagens with insufficient data to develop a toxicology limit, the ICH Q3A qualification thresholds are recommended. The compound‐ and class‐specific toxicology limits described here may be adjusted for an individual drug substance based on treatment duration, dosing schedule, severity of the disease and therapeutic indication. HighlightsCompound‐specific toxicology limits were developed for common potential impurities in drug substances.A class‐specific limit of 15 &mgr;g/day was developed for monofunctional alkyl bromides.All compound‐specific toxicology limits were based on existing data, current regulatory guidance, and scientific knowledge.

The Evolution, Scientific Reasoning and Use of ICH S2 Guidelines for Genotoxicity Testing of Pharmaceuticals

Two ICH guidances on genotoxicity (ICH S2A and ICH S2B) have been put into practice in the ICH regions in 1995 and 1997. At the end of 2011, these were replaced by the revised single ICH S2(R1) guidance. In the context of safety testing of pharmaceuticals, genotoxicity testing is mainly associated with the goal to remove potentially genotoxic carcinogens early in the process of drug development, and this goal requires a battery of different tests to address the various genotoxic mechanisms involved in carcinogenesis. In the years of use of the first ICH S2 guidelines, it has been recognised that the extreme focus on sensitivity for in vitro genotoxicity tests, as well as general improvements in various test systems, requires a revision of the principles of S2A and S2B. Thus, an ICH expert working group was established, which merged the two ICH S2 guidances into one, the ICH S2(R1) guidance. Essential changes in the way to conduct genotoxicity testing of pharmaceuticals include a reduction in the top concentrations used for testing of pharmaceutical candidate compounds in in vitro genotoxicity tests and an option to omit in vitro genotoxicity tests with mammalian cells in vitro from the test battery with inclusion of a more comprehensive in vivo testing. The revised ICH S2(R1) will enable a better risk-based assessment for genotoxicity of pharmaceuticals.

Sheldon Wolff 1928–2008

On May 24, 2008, Dr. Sheldon Wolff died at his home in Mill Valley, California, his wife and children by his side. He was just 79. With his death, the world lost one of its great pioneers in cytogenetics and radiation biology, and the Environmental Mutagen Society lost one of its former presidents. Many of us also lost a valued teacher, colleague, and friend. Sheldon Wolff was born in Peabody, Massachusetts on September 22, 1928. He obtained his B.S. degree (Magna cum Laude) in Biology from Tufts College in 1950, an M.A. (Biology) from Harvard in 1951, and his Ph.D. (Biology) from Harvard in 1953. He then joined the staff of the Biology Division of the Oak Ridge National Laboratory, where he ran a leading research laboratory in radiation biology and cytogenetics (at that time relying heavily on plant cytogenetics). He was recruited to the faculty of the Univeristy of California at San Francisco (UCSF) in 1966 to join Harvey Patt and Robert Painter at the newly established Laboratory of Radiobiology (later renamed as Laboratory of Radiobiology and Environmental Health), an Organized Research Unit of the Department of Energy (DOE). He later served as the Director of the Laboratory of Radiobiology and Environmental Health (LREH) from 1983–1995. Shelly retained his appointment as Professor of Cytogenetics and Radiology at UCSF with a joint appointment in the Department of Anatomy through the end of his career. In 1996, he was appointed as the Vice Chairman and Chief of Research of the Radiation Effects Research Foundation (RERF) in Hiroshima, Japan, where he remained until his retirement in 2000. Shelly and his laboratory made many key contributions to the field of cytogenetics and radiation biology. He was the first to demonstrate that repair of chromosome damage in cells was a metabolic process requiring the synthesis of chemical bonds. Before this time, chromosome breaks were thought to represent simple physical fusions of breaks. Shelly’s work led directly to our understanding of the complex biochemistry associated with the processing and repair of DNA/chromosome breaks. In the early 1970s, Shelly helped pioneer the sister chromatid exchange (SCE) assay, one of the most sensitive assays for detection of potentially mutagenic and carcinogenic agents. While on sabbatical in the laboratory of his friend and colleague H. John Evans in Edinburgh, Shelly worked with Paul Perry to develop a simpler staining method to identify SCEs. Shelly’s lab went on to make extensive observations concerning their mechanisms of formation and biological significance. Three of his publications in this area were honored by the Institute for Scientific Information as Citation Classics. The third of Shelly’s major scientific contributions was the identification of an adaptive response to ionizing radiation—the observation that low dose radiation exposures can confer cellular resistance to a subsequent challenge with a higher dose. This report sparked a deluge of studies from around the world and an ongoing debate as to the nature of the Sheldon Wolff