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Toxicokinetic testing strategies to demonstrate bone marrow exposure in in vivo micronucleus study for genotoxicity assessment of agrochemicals
Journal Pre-proof Toxicokinetic testing strategies to demonstrate bone marrow exposure in in vivo micronucleus study for genotoxicity assessment of agrochemicals Gopinath C. Nallani, Zhiwei Liu, Appavu Chandrasekaran PII:
S0273-2300(19)30316-2
DOI:
https://doi.org/10.1016/j.yrtph.2019.104552
Reference:
YRTPH 104552
To appear in:
Regulatory Toxicology and Pharmacology
Received Date: 11 July 2019 Revised Date:
6 December 2019
Accepted Date: 9 December 2019
Please cite this article as: Nallani, G.C., Liu, Z., Chandrasekaran, A., Toxicokinetic testing strategies to demonstrate bone marrow exposure in in vivo micronucleus study for genotoxicity assessment of agrochemicals, Regulatory Toxicology and Pharmacology (2020), doi: https://doi.org/10.1016/ j.yrtph.2019.104552. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
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Title
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Toxicokinetic testing strategies to demonstrate bone marrow exposure in in vivo micronucleus study for genotoxicity assessment of agrochemicals
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Authors
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Gopinath C Nallani*, Zhiwei Liu and Appavu Chandrasekaran
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Global Regulatory Sciences, FMC Corporation, 1090 Elkton Rd, Newark, DE 19711
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*
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Corresponding Author: FMC Stine Research Center, 1090 Elkton Rd, Newark, DE 19711
Email: [email protected]; Phone: (302) 318-9698
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Abstract
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Following adoption of the new OECD test guideline (TG) 474 for the in vivo mammalian
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erythrocyte micronucleus (MN) test (29 July 2016), demonstration of exposure of target tissue
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(bone marrow) is required, if the test result is negative i.e. no cytogenetic damage. It implies that
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for many active ingredients, relevant metabolites or significant impurities with existing in vivo
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MN tests resulting in negative genotoxicity findings, evidence of target tissue exposure may be
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lacking and is considered a data gap in regulatory reviews. We present here toxicokinetic (TK)
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testing strategies for the design and conduct of studies that would demonstrate evidence of
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delivery of the test substance to the bone marrow. To illustrate this, three examples are presented
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with methods utilized under each scenario. We also propose a decision tree that may help design
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suitable TK studies to establish evidence of bone marrow exposure.
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Key words: genotoxicity; in vivo micronucleus; bone marrow; target tissue exposure; toxicokinetics.
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1. Introduction
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As part of the standard genotoxicity test battery or as a follow-up to a positive test result
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from an in vitro mammalian cell micronucleus (MN) or an in vitro mammalian chromosomal
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aberration study (OECD, 2010a, 2016a), an in vivo mammalian erythrocyte MN test is routinely
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conducted for genotoxicity assessment of industrial chemicals including agrochemicals (Booth et
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al., 2017; Hayashi, 2016). In July 2016, the revised in vivo MN test guideline (OECD 474) was
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adopted into the regulatory framework. One of the key components of the revision included
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incorporation of ‘target tissue exposure’ as part of the test (OECD, 2016b). According to this, a
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positive result from the test does not need evidence of target tissue exposure, since a positive test
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result by itself is indicative of bone marrow (BM) exposure. It is manifested via reduction in the
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proportion of immature erythrocytes among total erythrocytes, i.e. statistically significant
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decrease in the PCE/(NCE+PCE) ratio (PCE: polychromatic erythrocytes, immature erythrocytes
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or reticulocytes; NCE: normochromatic erythrocytes or mature erythrocytes) in comparison with
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the corresponding ratio from control group. On the other hand, a negative test result needs to be
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corroborated with evidence that the test substance has reached the BM tissue. In this paper, the
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terms target tissue and bone marrow are used interchangeably.
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All new agrochemical active ingredients (AIs) under development would be expected to
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be tested following the new OECD 474 guideline to develop acceptable genotoxicity data.
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However, there are numerous AIs that have already been registered with existing in vivo MN
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studies conducted prior to the revision. Furthermore, genotoxicity testing including the follow-up
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in vivo MN assessment is also required for ‘relevant metabolites’ and impurities of toxicological
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concern (EU, 2013). Therefore, for a majority of test compounds with negative in vivo
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genotoxicity test results, evidence of target tissue exposure may be lacking and hence a data gap
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is identified during product reauthorizations (EFSA, 2018, 2017). The objective of this paper is
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to provide strategies on the design and conduct of a suitable toxicokinetic (TK) study to address
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BM exposure. Given the number of registered products currently in the market, it is anticipated
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that registrants will be required to conduct these stand-alone exposure studies for acceptability of
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existing in vivo MN studies. Keeping this in mind, it is the intent of the authors to outline the
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methods utilized in conduct of such studies. To this end, three examples are presented to
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illustrate the concept and approach. The studies referenced in these examples complied with all
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the applicable sections of the Final Rules of the Animal Welfare Act (9 CFR) and IACUC
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protocols. To the best of the authors’ knowledge, this is the first paper to describe and report BM
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exposure studies for agrochemicals or their relevant metabolites. Although the paper specifically
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focuses on target tissue exposure testing in support of in vivo MN test, the strategies and methods
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described are also applicable to in vivo genotoxicity studies in general.
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2. Strategies for design and conduct of bone marrow exposure study
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2.1. Plasma or bone marrow exposure
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The mammalian in vivo MN test is conducted in relevant toxicology species (usually rat
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or mouse) to detect damage to the chromosomes or the mitotic apparatus of erythroblasts. This
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test is a regulatory requirement to identify test substances (AI, relevant metabolite or significant
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impurity) that may cause cytogenetic damage through formation of MN arising from
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chromosomal fragments or chromosomes that are not incorporated into daughter nuclei at the
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time of cell division (Heddle et al., 1983; Schmid, 1975; US EPA, 1996). This is performed via
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analysis of erythrocytes sampled from peripheral hematopoietic cells or in the BM. If the test
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substance does not induce MN in erythrocytes, it can be regarded as non-genotoxic, provided
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target tissue exposure is demonstrated. If there is evidence from this test or other toxicity tests
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that the test substance induces toxic effects in BM, then target tissue exposure is considered
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established. In the absence of systemic toxicity or toxic effects in BM tissue, demonstration of
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detectable levels of test substance in blood, plasma or BM may be used as a line of evidence to
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confirm that the test substance has reached the target tissue (Hardy et al., 2017).
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2.2. Bone marrow exposure assessment as part of ADME study
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In the revised OECD 474 test guideline, it is stated: ………… alternatively, ADME data,
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obtained in an independent study using the same route and same species, can be used to
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demonstrate bone marrow exposure (OECD, 2016b). Therefore, for an AI under development
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including understanding of its absorption, distribution, metabolism and excretion (ADME) in
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rodents, it is useful to incorporate assessment of BM exposure as part of the study. The ADME
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study is conducted in representative toxicology species (typically rat) following the OECD 417
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TG and involves assessment of mass balance, tissue distribution, excretion routes, kinetics and
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metabolic pathways of test substance following administration of single low, single high and
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multiple low doses (OECD, 2010b). The high dose chosen in ADME study typically matches
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that in toxicology studies (including in vivo MN test), so rats from this group may be utilized to
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establish evidence of BM exposure to test substance. As part of the tissue distribution
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assessment, a wide range of tissues including BM may be collected at or around Tmax. If
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detectable levels of radioactivity are present in BM, it implies that the test substance has reached
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the target tissue. Therefore, integration of BM exposure as part of ADME study obviates the
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need for a stand-alone exposure study, hence reduces additional animal testing.
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It is important to understand the relevance of collection of the target tissue at Tmax for
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demonstration of exposure, although genotoxicity in the bone marrow is typically assessed
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between 18 and 24 h following exposure to the test chemical. The formation of micronucleated
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PCE in BM does not need a persistent exposure to a clastogen/aneugen. This is because the time
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course of MN production in PCE can be different and it is unlikely to detect any increase in MN
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earlier than 10 h following exposure (Salamone et al., 1980). Therefore, although the highest
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concentration of the test substance in the BM is expected at Tmax, formation and maturation of
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erythrocytes takes longer and hence a time-course TK measurement is not relevant for exposure
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assessment. Accordingly, it is appropriate to collect BM at Tmax to present evidence of target
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tissue exposure in support of negative test result in in vivo MN test.
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Although integration of target tissue exposure in ADME studies is plausible, it is critical
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to closely match the study variables (species, strain, sex, age, body weight, dose, route of dosing,
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vehicle etc.) with those used in in vivo MN study. Any variation from these needs a rationale
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substantiated by data for an acceptable study that addresses target tissue exposure. While the
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choice for selection of species, strain, age, body weight, route of dosing and vehicle is limited in
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terms of matching these variables with an existing in vivo study, it is not necessary to
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demonstrate exposure of test substance for all the dose levels tested. In general, in vivo
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genotoxicity tests include testing of up to three dose levels including an MTD or 2000 mg/kg bw,
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whichever is higher (OECD, 2016b). The TK exposure study could use one of the doses (low,
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intermediate, or high) that is enough to achieve the detection limits in the target tissue. However,
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given the advances in improved analytical detection, it may be beneficial to establish exposure
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data (dose-response) for all the doses.
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The TK study to support a negative in vivo MN test result could be conducted with either
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radio-labeled or non-labeled test substance. The major advantage of conducting the study with a
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C-labeled test substance is that it is not necessary to perform verification/validation of an
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analytical method in BM and establish parameters such as calibration range, selectivity,
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specificity, recovery and precision. For tests with radiolabeled material, a liquid scintillation
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counter (LSC) is used to count ‘disintegrations per minute (dpm)’ in target tissues. Furthermore,
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with radiometric detection, analyte (or metabolite) signal in plasma or BM can be differentiated
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well from the endogenous compounds. This is relevant because it is sufficient to show detectable
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levels of unbound parent/metabolite(s) in blood plasma or BM to establish evidence of systemic
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bioavailability (Hardy et al., 2017).
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2.3. Experimental details
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Unless significant differences in toxicity are noted between male and female rats (or
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mice), adult male rats are often the choice of animals for conducting the exposure study (Krishna
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and Hayashi, 2000). The test animals could be dosed with radioactive test substance to achieve
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100-200 µCi/kg. Around Tmax, animals are sacrificed followed by collection of whole blood and
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BM tissue from both femurs. Total radioactive residue (TRR) in plasma and BM can be
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determined using LSC. Pre-dose or control tissues may be used to calculate background
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radioactivity and subtracted from the sample counts. This also helps establish detection limits of
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radioactivity in the target tissue.
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The plasma samples can be further analyzed to determine free (unbound) and bound
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concentrations of test substance using equilibrium dialysis. A positive control for plasma protein
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binding (e.g. warfarin) can be used to determine acceptability of the method. Radioactivity
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measurements from plasma and buffer sides indicate percent test substance bound and free,
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respectively. Furthermore, aliquots from the buffer side may be analyzed to determine unbound
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concentrations of test substance and/or metabolites.
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A complete illustration of a decision tree for design and conduct of bone marrow
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exposure study is presented in Fig 1.
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3. Examples of toxicokinetic test strategies for target tissue exposure
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3.1. Example # 1: Demonstration of BM exposure of test substance HX
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3.1.1. Overview
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HX is an herbicide under development by FMC Corporation. Since this product is a ‘new
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active ingredient’, a BM exposure study was planned and conducted ahead of regulatory
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submission to provide unequivocal evidence of lack of genotoxicity of HX as established from
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the in vivo MN test (data not shown). This section describes the experimental design and
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methods used in this independent study.
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3.1.2 TK study with 14C-HX: Design, test conduct and results
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For this study, adult (6-8 wk) male SD rats (Charles River CRL: CD SD, n=4, mean body
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weight 199 g) that matched with those tested in the in vivo MN study were used. Rats were dosed
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with 500 mg/kg (the lowest of the three doses 500, 1000 and 2000 mg/kg bw tested in the in vivo
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study) via a single oral gavage (10 mL/kg) consisting of appropriate amounts of [14C]-HX (>95%
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radiopurity) and unlabeled HX in 0.5% carboxy methyl cellulose (CMC w/v, medium viscosity
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400 cps) in 5% Tween 80 in water at a final concentration of 50 mg/mL (20 µCi/mL) (Shen,
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2017). The dose formulation at 200 µCi/kg provided detection of radioactivity at ≥ 0.05% of
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dose. From the ADME study, Tmax of
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rats were sacrificed to collect samples. Whole blood (maximum obtainable) was collected into
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heparin tubes and plasma was obtained by centrifugation. Bone marrow from both femurs were
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collected by centrifugation and combined into one tube per animal and the total net weight of
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bone marrow was recorded.
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C-HX was found to be ~ 4 h (data not shown) at which
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The TRR in biological samples was determined by LSC (Perkin Elmer, Tri-Carb
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3100TR). Counting efficiencies were calculated by the external standard method using a series of
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quenched standards to generate the calibration curves. Where appropriate, background
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disintegration rates were measured in pre-dose samples and subtracted from the sample
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disintegration rate. The limit of detection (LOD) of radioactivity was defined as twice the
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background levels of instrument and was 40 dpm for
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programmed to automatically convert counts per minute (cpm) to disintegrations per minute
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(dpm). The TRR results indicated detection of significant amounts of radioactivity in the plasma
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and BM suggesting that the target tissue was exposed to [14C]-HX (Table 1). Furthermore,
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plasma samples were subjected to equilibrium dialysis to determine bound and unbound
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radioactivity. Briefly, aliquots (150 µL) of individual rat plasma samples (n = 4) were sampled
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into one side of 96-well Teflon plate with 12-14kDa HT dialysis membranes. The other side of
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the dialysis membrane was filled with PBS buffer (pH 7.4, 150 µL). Samples were incubated on
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an orbital shaker at 37°C, 5% CO2/95% air for 5 hours. At the end of the incubation, duplicate
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aliquots (50 µL) were removed from the buffer and plasma sides for total radioactivity analysis
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by LSC. [14C]-Warfarin (~1 µM) in blank rat plasma was used as a positive control (>95%
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bound). The results from this assay indicated that up to 88% of radioactivity was bound (Table
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2). From this, it was confirmed that a portion of applied radioactivity was indeed detected as free
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circulation concentration (unbound) in the plasma. Furthermore, radioactivity profiles on the
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buffer side (free) and plasma side (total) determined using HPLC-UV-βRAM-MS following
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extraction of plasma samples with acetonitrile (3-fold) or by direct analysis (buffer samples),
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revealed similar radioactive profiles including presence of unchanged parent compound (Figures
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2A and 2B). Detection of unchanged parent in the buffer suggested that when administered
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C- radioactivity. The LSC was
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orally, HX circulates as unbound (free-fraction), therefore, is systemically available and reaches
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BM in significant amounts. In addition to detection of unchanged parent, five metabolites (M8,
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M10, M11, M14 and M15) were also observed in the buffer indicating the systemic exposure to
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these metabolites. This approach is quite useful and precludes additional TK testing for relevant
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metabolites. Overall, the study presented here confirmed the delivery test substance to the BM
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and hence validate the in vivo MN test finding that HX is not genotoxic.
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3.2. Example # 2: Evidence of BM exposure of a relevant metabolite with known/expected
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plasma kinetics
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3.2.1. Overview
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Benalaxyl oxopropanoic acid (M1) is a soil and groundwater metabolite of benalaxyl, a
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fungicide. Benalaxyl is currently registered for use in the EU. Following the SANCO/221/2000
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guidance on the relevance assessment of metabolites in groundwater, genotoxicity assessment
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including the in vivo MN test was conducted for this metabolite (SANCO, 2003). Based on the
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results, the metabolite was not considered genotoxic (data not shown), but a data gap on
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submission of evidence of BM exposure had been identified. Since the radioactive soil and
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groundwater metabolites of benalaxyl including M1 were available, the exposure test was
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conducted using the radioactive metabolite.
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3.2.2. Test design, conduct and results
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In this test, 14C-radioactivity concentrations in the plasma and BM tissues of NMRI mice
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were determined following a single intraperitoneal (ip) injection of the radioactive metabolite
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(Shen, 2018). The test species, dose tested, route of administration and the vehicle matched with
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those used in the in vivo MN study. It is understood that the current TGs no longer recommend ip
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as a route of exposure to evaluate in vivo genotoxicity (OECD, 2016b). However, since the
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existing in vivo MN study used ip administration, it was necessary to use the same dose route in
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the exposure study. Furthermore, although not currently recommended, ip dosing also supports
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the general principles of systemic absorption of test substances and avoids concerns about lack of
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intestinal absorption. The study design consisted of eight adult (~8-10 wk, mean BW of 40 g)
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male NMRI mice (CRL: NMRI (Han); Saint-Germain-Nuelles, France) administered a single
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500 mg/kg ip injection (10 mL/kg) of [14C]-M1 (200 µCi/kg, 14C purity > 98%) in corn oil. All
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animals had at least five days of acclimation to the laboratory conditions (21±5 °C, 30-70%
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relative humidity and 12 h light/dark cycle) prior to dosing and received powdered rodent diet
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and water (RO purified) at ad lib access. M1 is a primary metabolite (acid) of benalaxyl (ester)
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and is expected to be more polar than the parent and hence Tmax (~0.5 h) of benalaxyl was chosen
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as the timepoint for collection of target tissues. Approximately 30 min post-dosing, the animals
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were sacrificed by overdose of CO2 and blood was collected via cardiac puncture into test tubes
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containing K2EDTA. Bone marrow from both femurs was collected at necropsy. The plasma and
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BM tissues from two animals each were pooled (n=4) to meet the analytical requirements. The
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total radioactivity concentrations were determined using LSC as described in section 3.1.2. The
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mean total radioactivity concentrations in plasma and BM were 587.28 µg Eq/g and 185.62 µg
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Eq/g, respectively (Table 3). The test results therefore demonstrated exposure of M1 in the BM
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of the toxicology species.
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3.3. Example # 3: Evidence of BM exposure of a relevant metabolite with little or no
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information on plasma kinetics
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3.3.1. Overview
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Carfentrazone-cinnamic acid (CA) is a soil metabolite of carfentrazone-ethyl, an
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herbicide with global registrations for use on a variety of crops. As part of the testing for
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metabolite relevance, genotoxicity assessments including in vivo MN study were performed (data
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not shown). To confirm the negative in vivo MN test result, a separate TK study in rats was
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conducted with [14C]-CA. Although detected in animals, CA is a secondary metabolite and no
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specific information on its systemic availability was known. Therefore, the exposure study with
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the metabolite in rats was conducted in two parts. In the first part of the study, plasma kinetics of
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the metabolite were determined. Information from the kinetics study was then used to select
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optimum sample collection time for plasma and BM tissue.
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3.2.2. Test design, study conduct and results
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In the first part of the study, four adult male SD rats after overnight fasting were
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administered a single 500 mg/kg bw oral gavage dose of [14C]-CA in distilled water (100 µCi/kg;
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10 mL/kg) and aliquots of blood (~ 0.3 mL) were collected at different time intervals (0.25, 0.5,
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1, 2, 4, 6 8 h) after dosing (Theerman, 2016a). At 24 h, whole blood was collected via cardiac
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puncture under CO2 anesthesia. Whole blood samples collected in tubes with sodium heparin
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were processed for plasma (centrifuged at 10,000 rpm for 10 min) within an hour of collection.
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Aliquots of plasma from each time interval were analyzed for radioactivity using LSC. The TK
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parameters in plasma samples were determined using WinNonlin version 6.3 (Pharsight Corp,
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Mountain View, CA). The results are presented in Table 4. The plasma kinetics results indicated
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that the metabolite was readily absorbed in rats with moderate Tmax and T1/2 values.
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In the second part (Theerman, 2016b), four adult male SD rats were administered a
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single 500 mg/kg oral gavage dose of [14C]-CA under the same conditions as described above.
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Following termination of the study around 4 h after dosing, blood was collected via cardiac
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puncture and the BM tissues were collected. Because of the high variability in Tmax values from
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four rats, the maximum time required to achieve Cmax (4 h as opposed to the mean value of 2.8 h)
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was considered for sample collection. The samples were analyzed for total radioactivity
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concentrations using LSC, and the results are presented in Table 5. Both plasma and the BM
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tissues had [14C]-CA concentrations well above the measurable levels, implying that the
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metabolite, following oral administration, is bioavailable and reaches the target tissue in
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significant amounts.
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4. Additional considerations
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Although testing of in vivo MN genotoxicity assays in rodent tissues other than the BM
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have previously been conducted with liver, lung, colon, skin, spleen, testes, GI tract etc.,
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acceptable/standard (e.g. OECD) study guidelines have not been established yet for testing in
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these organs/tissues. Furthermore, scoring of MN in reticulocytes derived from BM is relatively
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easier compared to other tissues (Morita et al., 2011). Therefore, in regulatory setting, the in vivo
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MN test with BM (OECD 474) has been a standard assay for assessing cytogenetic damage.
265
However, it is important to recognize that if toxicokinetic data indicate lack of exposure, the in
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vivo MN test should be conducted in other tissues. This is particularly true for test substances
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metabolized in liver/intestine and forming reactive metabolites that are too short-lived to reach
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the BM, and hence demonstration of bioavailability of parent compound does not necessarily
269
indicate that BM is an appropriate target (EFSA, 2011). However, as indicated earlier, currently
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there are no accepted test guidelines on assessment for other tissues.
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The TK studies for target tissue exposure conducted with C-14 label test substance may
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utilize whole body autoradiography or quantitative determinations via radioactivity
273
measurements. In general, results obtained from either approach are in agreement, although
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quantitative radioactivity measurements in blood/plasma and BM are routinely used (Dereszlay I,
275
1997). Although bone marrow is a highly perfused tissue, only unbound (free) fraction in
276
circulation is available for transport/perfusion into this tissue. Therefore, lower concentrations of
277
radioactivity or the parent test substance are expected in BM compared to plasma, as detailed in
278
the examples presented in this paper. However, currently, there is no established minimum level
279
of bone marrow exposure to be demonstrated in in vivo genotoxicity assays.
280
5. Conclusions
281
1. The in vivo rodent erythrocyte micronucleus test is a core study in genotoxicity test
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battery and this test is valid only if there is evidence that the test substance reaches the
283
target tissue.
284 285
2. The paper describes the design and conduct of stand-alone toxicokinetic studies to determine bone marrow exposure to the test chemical.
286
3. In addition, the approaches on integration of bone marrow exposure as part of ADME
287
study and determination of radio-chromatographic profiles in unbound fraction of
288
plasma radioactivity are discussed which help reduce additional animal testing.
289
Conflict of interest
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Nallani G, Liu, Z and Chandrasekaran, A are employed by FMC Corporation. All test items and
291
metabolites referenced in this paper are products of FMC.
292
Acknowledgment
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The authors would like to acknowledge Dr. Li Shen (Frontage Laboratories, Exton, PA); Dr.
294
Manik Desai and Gina Theerman (WuXi AppTec, Plainsboro, NJ) for their contribution to the
295
experiments.
296
Funding
297
The presented work did not receive any specific grant from funding agencies in the public or not-
298
for-profit sectors. The examples presented in this paper were obtained from studies conducted on
299
behalf of FMC Corporation.
300
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OECD, 2016b. Test No. 474: Mammalian Erythrocyte Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. https://doi.org/10.1787/9789264264762-en
341 342 343
OECD, 2010a. Guidelines for the Testing of Chemicals, Section 4, Test No. 487: In Vitro Mammalian Cell Micronucleus Test. OECD Guidel. Test. Chem. 1–23. https://doi.org/http://dx.doi.org/10.1787/9789264091016-en
344 345
OECD, 2010b. Test No. 417: Toxicokinetics. OECD Environ. Heal. Saf. Publ. 1–20. https://doi.org/10.1787/9789264070882-en
346 347 348
Salamone, Michael, Heddle, John, Stuart Earl, Katz, M., 1980. Towards an improved micronucleus test: studies on 3 model agents, mitomycin C, cyclophosphamide and dimethylbenzathracene. Mutat. Reserach 74, 347–356.
349 350
SANCO, 2003. Guidance document on the assessment of the relevance of metabolites in groundwater of substances regulated under council directive 91/414/EEC., October.
351
Schmid, W., 1975. The micronucleus test. Mutat. Reserach 31, 9–15.
352 353
Shen, L., 2018. Radioactivity concentrations in plasma and bone marrow following intra peritoneal injection of [14C]-M1 to male NMRI mice (unpublished). Exton, PA, USA.
354 355
Shen, L., 2017. Radioactivity concentrations in plasma and bone marrow at T-max after oral administration of [14C]-HX to Sprague-Dawley rats (unpublished). Exton, PA, USA.
356 357
Theerman, G., 2016a. Plasma radio pharmacokinetics of [14C]-MA in male Sprague-Dawley rats following a single oral (gavage) administration (unpublished). Plainsboro, NJ, USA.
358 359 360
Theerman, G., 2016b. Pharmacokinetics and radioanalysis in bone marrow of [14C]-MA in male Sprague-Dawley rats following single oral (gavage) administration (unpublished). Plainsboro, NJ, USA.
361 362
US EPA, 1996. In Vivo Mammalian Cytogenetics Tests : Erythrocyte Micronucleus As say. Heal. Eff. Guid. OPPTS.
363 364
Acronyms
ADME AI AUC BM CFR Cmax CMC cpm cps dpm EU GI HPLC HT IACUC ip LOD LOQ LSC MN MS MTD NCE OECD PBS PCE PPP RF RO SD rat T1/2 TG TK Tmax TRR
Absorption, Distribution, Metabolism, and Excretion Active ingredient Area under the curve Bone marrow Code of Federal Regulations Maximum concentration in plasma Carboxy methyl cellulose Counts per minute Centipoise Disintegrations per minute European Union Gastrointestinal High performance liquid chromatography High-throughput Institutional Animal Care and Use Committee intra peritoneal Limit of detection Limit of quantitation Liquid scintillation counter Micronucleus Mass spectrometry Maximum tolerated dose Normochromatic erythrocytes Organization for Economic Cooperation and Development Phosphate buffer saline Polychromatic erythrocytes Plant protection product(s) Range-finding Reverse osmosis Sprague-Dawley rat Half-life of test item in plasma Test Guideline Toxicokinetics Time at which the maximum concentration in plasma observed Total radioactive residue
Figure Captions Figure 1. Decision tree and strategy for conducting a toxicokinetic study to demonstrate exposure of bone marrow tissue in support of a negative test result from an in vivo mammalian erythrocyte micronucleus (MN) test (OECD 474). Figure 2A. Radio-chromatographic profile of radioactivity (plasma side) in plasma of adult male Sprague-Dawley rats administered a single 500 mg/kg bw oral gavage dose of [14C]-HX Figure 2B. Radio-chromatographic profile of unbound radioactivity (buffer side) in plasma of adult male Sprague Dawley rats administered a single 500 mg/kg bw oral gavage dose of [14C]HX
Tables Table 1. Mean (±SD, n=4)) total radioactivity concentrations (µg Eq/g) in plasma and bone marrow (~ 4 h post dosing) of adult male Sprague-Dawley rats following a single 500 mg/kg/bw oral gavage dose of [14C]-HX Tissue
Mean (±SD) concentration (µg Eq/g; n=4)
Plasma
153.20 ± 32.46
Bone marrow
49.73 ± 11.94
Table 2. Mean (±SD, n=4) radioactive concentrations (µM) and percent bound and unbound radioactivity in rat plasma (~ 4 h post dosing) in adult male Sprague-Dawley rats following a single 500 mg/kg/bw oral gavage dose of [14C]-HX Mean (±SD, n=4)
14
C-HX
14
C-Warfarin**
PBS* buffer side (µM) 56.14 ± 14.26
Plasma side (µM) 451.17 ± 102.81
% unbound
% bound
12.4 ± 2.2
87.5 ± 2.2
37
819
4.5
95.4
*phosphate buffer saline; ** positive control for protein binding (single replicate only) Table 3. Mean (±SD, n=4*) total radioactivity concentrations (µg Eq/g) in plasma and bone marrow (~0.5 h post dosing) of adult male NMRI mice (n=8) following a single 500 mg/kg/bw intraperitoneal injection of [14C]-benalaxyl oxopropanoic acid (M-1) Tissue
Mean (±SD) concentration (µg Eq/g; n=4)
Plasma
587.28 ± 67.16
Bone marrow
185.62 ± 17.97
* samples from two animals each pooled Table 4. Mean (±SD, n=4) kinetic parameters of [14C]-carfentrazone-cinnamic acid (CA) in plasma of adult male Sprague-Dawley rats following a single 500 mg/kg oral gavage administration. Mean (±SD) kinetic parameter (n=4) Cmax (µg Eq/ml)
AUCinf (h. µg Eq/ml)
Tmax (h)
T1/2 (h)
472 ± 177
3627 ± 1453
2.75 ± 1.50
3.78 ± 1.57
Table 5. Mean (±SD, n=4)) total radioactivity concentrations (µg Eq/g) in plasma and bone marrow (~ 4 h post dosing*) of adult male Sprague-Dawley rats following a single 500 mg/kg/bw oral gavage dose of [14C]-carfentrazone-cinnamic acid (CA) Tissue
Mean (±SD) concentration (µg Eq/g; n=4)
Plasma
2270.25 ± 1336.49
Bone marrow
575.75 ± 340.35
*Due to variability in measurements (SD: 1.75 h, n=4), bone marrow was collected at the highest T-max value (4 h).
Demonstration of target tissue exposure in rodent in vivo micronucleus (MN) test (OECD TG 474, July 2016)
Product registered?
Conduct in vivo MN test on parent, relevant metabolite or impurity following revised OECD TG 474
No
Yes
In vivo MN test on parent, relevant metabolite or impurity conducted following pre-revised TG OECD 474
No
MN study on Parent Does ADME study indicate the parent >10% in urine, bile or urine + bile and/or detected in unbound fraction of plasma?
Yes
MN study on relevant metabolite or impurity
Yes
Does ADME study indicate the metabolite/impurity > 10% in urine, bile or urine + bile and/or detected in unbound fraction of plasma?
No Yes
Evidence of BM exposure established
Conduct TK study (rodents of relevant sex to be administered appropriate dose tested in MN test) with C-14 material (parent, relevant metabolite or impurity). Collect plasma and/or bone marrow around Tmax and determine TRR. Radioactivity levels in BM or unbound plasma above detection limit?
C-14 test substance available?
Yes
Yes
BM exposure not established Yes
No
No
No
Analytical method capable to detect test substance in target tissue?
Figure 1. Demonstration of target tissue exposure in rodent in vivo micronucleus (MN) test (OECD TG 474, July 2016)
Product registered?
Conduct in vivo MN test on parent, relevant metabolite or impurity following revised OECD TG 474
No
Yes
In vivo MN test on parent, relevant metabolite or impurity conducted following pre-revised TG OECD 474
No
MN study on Parent Does ADME study indicate the parent >10% in urine, bile or urine + bile and/or detected in unbound fraction of plasma?
Yes
MN study on relevant metabolite or impurity
Yes
Does ADME study indicate the metabolite/impurity > 10% in urine, bile or urine + bile and/or detected in unbound fraction of plasma?
No Yes
Evidence of BM exposure established
Conduct TK study (rodents of relevant sex to be administered appropriate dose tested in MN test) with C-14 material (parent, relevant metabolite or impurity). Collect plasma and/or bone marrow around Tmax and determine TRR. Radioactivity levels in BM or unbound plasma above detection limit?
C-14 test substance available?
Yes
Yes
BM exposure not established Yes
No
No
No
Analytical method capable to detect test substance in target tissue?
Figure 2
28.6
A) 900 800 700 600
300
35.4
29.9
400
38.4
CPM
28.1
500
200 100 0 0
5
10
15
20
25 Time (min)
30
35
40
45
50
45
50
B) M11 29.4
300
250
M10
Parent 38.4
30.4
28.9
100
M15
35.9
M12
M8 150
24.4
CPM
200
50
0 0
5
10
15
20
25 Time (min)
30
35
40
Designation of radioactive peaks (retention time in min) in Fig 2 A and 2B; 24.4: M8; 28.1-28.9: M10; 28.6-29.4: M11; 29.9-30.4: M12; 35.4-35.9: M15; 38.4: HX (unchanged parent)
Funding The presented work did not receive any specific grant from funding agencies in the public or notfor-profit sectors. The examples presented in this paper were obtained from studies conducted on behalf of FMC Corporation.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0273-2300(19)30316-2
DOI:
https://doi.org/10.1016/j.yrtph.2019.104552
Reference:
YRTPH 104552
To appear in:
Regulatory Toxicology and Pharmacology
Received Date: 11 July 2019 Revised Date:
6 December 2019
Accepted Date: 9 December 2019
Please cite this article as: Nallani, G.C., Liu, Z., Chandrasekaran, A., Toxicokinetic testing strategies to demonstrate bone marrow exposure in in vivo micronucleus study for genotoxicity assessment of agrochemicals, Regulatory Toxicology and Pharmacology (2020), doi: https://doi.org/10.1016/ j.yrtph.2019.104552. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
1
Title
2 3
Toxicokinetic testing strategies to demonstrate bone marrow exposure in in vivo micronucleus study for genotoxicity assessment of agrochemicals
4
Authors
5
Gopinath C Nallani*, Zhiwei Liu and Appavu Chandrasekaran
6
Global Regulatory Sciences, FMC Corporation, 1090 Elkton Rd, Newark, DE 19711
7
*
8 9
Corresponding Author: FMC Stine Research Center, 1090 Elkton Rd, Newark, DE 19711
Email: [email protected]; Phone: (302) 318-9698
10
Abstract
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Following adoption of the new OECD test guideline (TG) 474 for the in vivo mammalian
12
erythrocyte micronucleus (MN) test (29 July 2016), demonstration of exposure of target tissue
13
(bone marrow) is required, if the test result is negative i.e. no cytogenetic damage. It implies that
14
for many active ingredients, relevant metabolites or significant impurities with existing in vivo
15
MN tests resulting in negative genotoxicity findings, evidence of target tissue exposure may be
16
lacking and is considered a data gap in regulatory reviews. We present here toxicokinetic (TK)
17
testing strategies for the design and conduct of studies that would demonstrate evidence of
18
delivery of the test substance to the bone marrow. To illustrate this, three examples are presented
19
with methods utilized under each scenario. We also propose a decision tree that may help design
20
suitable TK studies to establish evidence of bone marrow exposure.
21 22
Key words: genotoxicity; in vivo micronucleus; bone marrow; target tissue exposure; toxicokinetics.
23
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1. Introduction
25
As part of the standard genotoxicity test battery or as a follow-up to a positive test result
26
from an in vitro mammalian cell micronucleus (MN) or an in vitro mammalian chromosomal
27
aberration study (OECD, 2010a, 2016a), an in vivo mammalian erythrocyte MN test is routinely
28
conducted for genotoxicity assessment of industrial chemicals including agrochemicals (Booth et
29
al., 2017; Hayashi, 2016). In July 2016, the revised in vivo MN test guideline (OECD 474) was
30
adopted into the regulatory framework. One of the key components of the revision included
31
incorporation of ‘target tissue exposure’ as part of the test (OECD, 2016b). According to this, a
32
positive result from the test does not need evidence of target tissue exposure, since a positive test
33
result by itself is indicative of bone marrow (BM) exposure. It is manifested via reduction in the
34
proportion of immature erythrocytes among total erythrocytes, i.e. statistically significant
35
decrease in the PCE/(NCE+PCE) ratio (PCE: polychromatic erythrocytes, immature erythrocytes
36
or reticulocytes; NCE: normochromatic erythrocytes or mature erythrocytes) in comparison with
37
the corresponding ratio from control group. On the other hand, a negative test result needs to be
38
corroborated with evidence that the test substance has reached the BM tissue. In this paper, the
39
terms target tissue and bone marrow are used interchangeably.
40
All new agrochemical active ingredients (AIs) under development would be expected to
41
be tested following the new OECD 474 guideline to develop acceptable genotoxicity data.
42
However, there are numerous AIs that have already been registered with existing in vivo MN
43
studies conducted prior to the revision. Furthermore, genotoxicity testing including the follow-up
44
in vivo MN assessment is also required for ‘relevant metabolites’ and impurities of toxicological
45
concern (EU, 2013). Therefore, for a majority of test compounds with negative in vivo
46
genotoxicity test results, evidence of target tissue exposure may be lacking and hence a data gap
47
is identified during product reauthorizations (EFSA, 2018, 2017). The objective of this paper is
48
to provide strategies on the design and conduct of a suitable toxicokinetic (TK) study to address
49
BM exposure. Given the number of registered products currently in the market, it is anticipated
50
that registrants will be required to conduct these stand-alone exposure studies for acceptability of
51
existing in vivo MN studies. Keeping this in mind, it is the intent of the authors to outline the
52
methods utilized in conduct of such studies. To this end, three examples are presented to
53
illustrate the concept and approach. The studies referenced in these examples complied with all
54
the applicable sections of the Final Rules of the Animal Welfare Act (9 CFR) and IACUC
55
protocols. To the best of the authors’ knowledge, this is the first paper to describe and report BM
56
exposure studies for agrochemicals or their relevant metabolites. Although the paper specifically
57
focuses on target tissue exposure testing in support of in vivo MN test, the strategies and methods
58
described are also applicable to in vivo genotoxicity studies in general.
59
2. Strategies for design and conduct of bone marrow exposure study
60
2.1. Plasma or bone marrow exposure
61
The mammalian in vivo MN test is conducted in relevant toxicology species (usually rat
62
or mouse) to detect damage to the chromosomes or the mitotic apparatus of erythroblasts. This
63
test is a regulatory requirement to identify test substances (AI, relevant metabolite or significant
64
impurity) that may cause cytogenetic damage through formation of MN arising from
65
chromosomal fragments or chromosomes that are not incorporated into daughter nuclei at the
66
time of cell division (Heddle et al., 1983; Schmid, 1975; US EPA, 1996). This is performed via
67
analysis of erythrocytes sampled from peripheral hematopoietic cells or in the BM. If the test
68
substance does not induce MN in erythrocytes, it can be regarded as non-genotoxic, provided
69
target tissue exposure is demonstrated. If there is evidence from this test or other toxicity tests
70
that the test substance induces toxic effects in BM, then target tissue exposure is considered
71
established. In the absence of systemic toxicity or toxic effects in BM tissue, demonstration of
72
detectable levels of test substance in blood, plasma or BM may be used as a line of evidence to
73
confirm that the test substance has reached the target tissue (Hardy et al., 2017).
74
2.2. Bone marrow exposure assessment as part of ADME study
75
In the revised OECD 474 test guideline, it is stated: ………… alternatively, ADME data,
76
obtained in an independent study using the same route and same species, can be used to
77
demonstrate bone marrow exposure (OECD, 2016b). Therefore, for an AI under development
78
including understanding of its absorption, distribution, metabolism and excretion (ADME) in
79
rodents, it is useful to incorporate assessment of BM exposure as part of the study. The ADME
80
study is conducted in representative toxicology species (typically rat) following the OECD 417
81
TG and involves assessment of mass balance, tissue distribution, excretion routes, kinetics and
82
metabolic pathways of test substance following administration of single low, single high and
83
multiple low doses (OECD, 2010b). The high dose chosen in ADME study typically matches
84
that in toxicology studies (including in vivo MN test), so rats from this group may be utilized to
85
establish evidence of BM exposure to test substance. As part of the tissue distribution
86
assessment, a wide range of tissues including BM may be collected at or around Tmax. If
87
detectable levels of radioactivity are present in BM, it implies that the test substance has reached
88
the target tissue. Therefore, integration of BM exposure as part of ADME study obviates the
89
need for a stand-alone exposure study, hence reduces additional animal testing.
90
It is important to understand the relevance of collection of the target tissue at Tmax for
91
demonstration of exposure, although genotoxicity in the bone marrow is typically assessed
92
between 18 and 24 h following exposure to the test chemical. The formation of micronucleated
93
PCE in BM does not need a persistent exposure to a clastogen/aneugen. This is because the time
94
course of MN production in PCE can be different and it is unlikely to detect any increase in MN
95
earlier than 10 h following exposure (Salamone et al., 1980). Therefore, although the highest
96
concentration of the test substance in the BM is expected at Tmax, formation and maturation of
97
erythrocytes takes longer and hence a time-course TK measurement is not relevant for exposure
98
assessment. Accordingly, it is appropriate to collect BM at Tmax to present evidence of target
99
tissue exposure in support of negative test result in in vivo MN test.
100
Although integration of target tissue exposure in ADME studies is plausible, it is critical
101
to closely match the study variables (species, strain, sex, age, body weight, dose, route of dosing,
102
vehicle etc.) with those used in in vivo MN study. Any variation from these needs a rationale
103
substantiated by data for an acceptable study that addresses target tissue exposure. While the
104
choice for selection of species, strain, age, body weight, route of dosing and vehicle is limited in
105
terms of matching these variables with an existing in vivo study, it is not necessary to
106
demonstrate exposure of test substance for all the dose levels tested. In general, in vivo
107
genotoxicity tests include testing of up to three dose levels including an MTD or 2000 mg/kg bw,
108
whichever is higher (OECD, 2016b). The TK exposure study could use one of the doses (low,
109
intermediate, or high) that is enough to achieve the detection limits in the target tissue. However,
110
given the advances in improved analytical detection, it may be beneficial to establish exposure
111
data (dose-response) for all the doses.
112
The TK study to support a negative in vivo MN test result could be conducted with either
113
radio-labeled or non-labeled test substance. The major advantage of conducting the study with a
114
14
C-labeled test substance is that it is not necessary to perform verification/validation of an
115
analytical method in BM and establish parameters such as calibration range, selectivity,
116
specificity, recovery and precision. For tests with radiolabeled material, a liquid scintillation
117
counter (LSC) is used to count ‘disintegrations per minute (dpm)’ in target tissues. Furthermore,
118
with radiometric detection, analyte (or metabolite) signal in plasma or BM can be differentiated
119
well from the endogenous compounds. This is relevant because it is sufficient to show detectable
120
levels of unbound parent/metabolite(s) in blood plasma or BM to establish evidence of systemic
121
bioavailability (Hardy et al., 2017).
122
2.3. Experimental details
123
Unless significant differences in toxicity are noted between male and female rats (or
124
mice), adult male rats are often the choice of animals for conducting the exposure study (Krishna
125
and Hayashi, 2000). The test animals could be dosed with radioactive test substance to achieve
126
100-200 µCi/kg. Around Tmax, animals are sacrificed followed by collection of whole blood and
127
BM tissue from both femurs. Total radioactive residue (TRR) in plasma and BM can be
128
determined using LSC. Pre-dose or control tissues may be used to calculate background
129
radioactivity and subtracted from the sample counts. This also helps establish detection limits of
130
radioactivity in the target tissue.
131
The plasma samples can be further analyzed to determine free (unbound) and bound
132
concentrations of test substance using equilibrium dialysis. A positive control for plasma protein
133
binding (e.g. warfarin) can be used to determine acceptability of the method. Radioactivity
134
measurements from plasma and buffer sides indicate percent test substance bound and free,
135
respectively. Furthermore, aliquots from the buffer side may be analyzed to determine unbound
136
concentrations of test substance and/or metabolites.
137
A complete illustration of a decision tree for design and conduct of bone marrow
138
exposure study is presented in Fig 1.
139
3. Examples of toxicokinetic test strategies for target tissue exposure
140
3.1. Example # 1: Demonstration of BM exposure of test substance HX
141
3.1.1. Overview
142
HX is an herbicide under development by FMC Corporation. Since this product is a ‘new
143
active ingredient’, a BM exposure study was planned and conducted ahead of regulatory
144
submission to provide unequivocal evidence of lack of genotoxicity of HX as established from
145
the in vivo MN test (data not shown). This section describes the experimental design and
146
methods used in this independent study.
147
3.1.2 TK study with 14C-HX: Design, test conduct and results
148
For this study, adult (6-8 wk) male SD rats (Charles River CRL: CD SD, n=4, mean body
149
weight 199 g) that matched with those tested in the in vivo MN study were used. Rats were dosed
150
with 500 mg/kg (the lowest of the three doses 500, 1000 and 2000 mg/kg bw tested in the in vivo
151
study) via a single oral gavage (10 mL/kg) consisting of appropriate amounts of [14C]-HX (>95%
152
radiopurity) and unlabeled HX in 0.5% carboxy methyl cellulose (CMC w/v, medium viscosity
153
400 cps) in 5% Tween 80 in water at a final concentration of 50 mg/mL (20 µCi/mL) (Shen,
154
2017). The dose formulation at 200 µCi/kg provided detection of radioactivity at ≥ 0.05% of
155
dose. From the ADME study, Tmax of
156
rats were sacrificed to collect samples. Whole blood (maximum obtainable) was collected into
157
heparin tubes and plasma was obtained by centrifugation. Bone marrow from both femurs were
158
collected by centrifugation and combined into one tube per animal and the total net weight of
159
bone marrow was recorded.
14
C-HX was found to be ~ 4 h (data not shown) at which
160
The TRR in biological samples was determined by LSC (Perkin Elmer, Tri-Carb
161
3100TR). Counting efficiencies were calculated by the external standard method using a series of
162
quenched standards to generate the calibration curves. Where appropriate, background
163
disintegration rates were measured in pre-dose samples and subtracted from the sample
164
disintegration rate. The limit of detection (LOD) of radioactivity was defined as twice the
165
background levels of instrument and was 40 dpm for
166
programmed to automatically convert counts per minute (cpm) to disintegrations per minute
167
(dpm). The TRR results indicated detection of significant amounts of radioactivity in the plasma
168
and BM suggesting that the target tissue was exposed to [14C]-HX (Table 1). Furthermore,
169
plasma samples were subjected to equilibrium dialysis to determine bound and unbound
170
radioactivity. Briefly, aliquots (150 µL) of individual rat plasma samples (n = 4) were sampled
171
into one side of 96-well Teflon plate with 12-14kDa HT dialysis membranes. The other side of
172
the dialysis membrane was filled with PBS buffer (pH 7.4, 150 µL). Samples were incubated on
173
an orbital shaker at 37°C, 5% CO2/95% air for 5 hours. At the end of the incubation, duplicate
174
aliquots (50 µL) were removed from the buffer and plasma sides for total radioactivity analysis
175
by LSC. [14C]-Warfarin (~1 µM) in blank rat plasma was used as a positive control (>95%
176
bound). The results from this assay indicated that up to 88% of radioactivity was bound (Table
177
2). From this, it was confirmed that a portion of applied radioactivity was indeed detected as free
178
circulation concentration (unbound) in the plasma. Furthermore, radioactivity profiles on the
179
buffer side (free) and plasma side (total) determined using HPLC-UV-βRAM-MS following
180
extraction of plasma samples with acetonitrile (3-fold) or by direct analysis (buffer samples),
181
revealed similar radioactive profiles including presence of unchanged parent compound (Figures
182
2A and 2B). Detection of unchanged parent in the buffer suggested that when administered
14
C- radioactivity. The LSC was
183
orally, HX circulates as unbound (free-fraction), therefore, is systemically available and reaches
184
BM in significant amounts. In addition to detection of unchanged parent, five metabolites (M8,
185
M10, M11, M14 and M15) were also observed in the buffer indicating the systemic exposure to
186
these metabolites. This approach is quite useful and precludes additional TK testing for relevant
187
metabolites. Overall, the study presented here confirmed the delivery test substance to the BM
188
and hence validate the in vivo MN test finding that HX is not genotoxic.
189
3.2. Example # 2: Evidence of BM exposure of a relevant metabolite with known/expected
190
plasma kinetics
191
3.2.1. Overview
192
Benalaxyl oxopropanoic acid (M1) is a soil and groundwater metabolite of benalaxyl, a
193
fungicide. Benalaxyl is currently registered for use in the EU. Following the SANCO/221/2000
194
guidance on the relevance assessment of metabolites in groundwater, genotoxicity assessment
195
including the in vivo MN test was conducted for this metabolite (SANCO, 2003). Based on the
196
results, the metabolite was not considered genotoxic (data not shown), but a data gap on
197
submission of evidence of BM exposure had been identified. Since the radioactive soil and
198
groundwater metabolites of benalaxyl including M1 were available, the exposure test was
199
conducted using the radioactive metabolite.
200
3.2.2. Test design, conduct and results
201
In this test, 14C-radioactivity concentrations in the plasma and BM tissues of NMRI mice
202
were determined following a single intraperitoneal (ip) injection of the radioactive metabolite
203
(Shen, 2018). The test species, dose tested, route of administration and the vehicle matched with
204
those used in the in vivo MN study. It is understood that the current TGs no longer recommend ip
205
as a route of exposure to evaluate in vivo genotoxicity (OECD, 2016b). However, since the
206
existing in vivo MN study used ip administration, it was necessary to use the same dose route in
207
the exposure study. Furthermore, although not currently recommended, ip dosing also supports
208
the general principles of systemic absorption of test substances and avoids concerns about lack of
209
intestinal absorption. The study design consisted of eight adult (~8-10 wk, mean BW of 40 g)
210
male NMRI mice (CRL: NMRI (Han); Saint-Germain-Nuelles, France) administered a single
211
500 mg/kg ip injection (10 mL/kg) of [14C]-M1 (200 µCi/kg, 14C purity > 98%) in corn oil. All
212
animals had at least five days of acclimation to the laboratory conditions (21±5 °C, 30-70%
213
relative humidity and 12 h light/dark cycle) prior to dosing and received powdered rodent diet
214
and water (RO purified) at ad lib access. M1 is a primary metabolite (acid) of benalaxyl (ester)
215
and is expected to be more polar than the parent and hence Tmax (~0.5 h) of benalaxyl was chosen
216
as the timepoint for collection of target tissues. Approximately 30 min post-dosing, the animals
217
were sacrificed by overdose of CO2 and blood was collected via cardiac puncture into test tubes
218
containing K2EDTA. Bone marrow from both femurs was collected at necropsy. The plasma and
219
BM tissues from two animals each were pooled (n=4) to meet the analytical requirements. The
220
total radioactivity concentrations were determined using LSC as described in section 3.1.2. The
221
mean total radioactivity concentrations in plasma and BM were 587.28 µg Eq/g and 185.62 µg
222
Eq/g, respectively (Table 3). The test results therefore demonstrated exposure of M1 in the BM
223
of the toxicology species.
224
3.3. Example # 3: Evidence of BM exposure of a relevant metabolite with little or no
225
information on plasma kinetics
226
3.3.1. Overview
227
Carfentrazone-cinnamic acid (CA) is a soil metabolite of carfentrazone-ethyl, an
228
herbicide with global registrations for use on a variety of crops. As part of the testing for
229
metabolite relevance, genotoxicity assessments including in vivo MN study were performed (data
230
not shown). To confirm the negative in vivo MN test result, a separate TK study in rats was
231
conducted with [14C]-CA. Although detected in animals, CA is a secondary metabolite and no
232
specific information on its systemic availability was known. Therefore, the exposure study with
233
the metabolite in rats was conducted in two parts. In the first part of the study, plasma kinetics of
234
the metabolite were determined. Information from the kinetics study was then used to select
235
optimum sample collection time for plasma and BM tissue.
236
3.2.2. Test design, study conduct and results
237
In the first part of the study, four adult male SD rats after overnight fasting were
238
administered a single 500 mg/kg bw oral gavage dose of [14C]-CA in distilled water (100 µCi/kg;
239
10 mL/kg) and aliquots of blood (~ 0.3 mL) were collected at different time intervals (0.25, 0.5,
240
1, 2, 4, 6 8 h) after dosing (Theerman, 2016a). At 24 h, whole blood was collected via cardiac
241
puncture under CO2 anesthesia. Whole blood samples collected in tubes with sodium heparin
242
were processed for plasma (centrifuged at 10,000 rpm for 10 min) within an hour of collection.
243
Aliquots of plasma from each time interval were analyzed for radioactivity using LSC. The TK
244
parameters in plasma samples were determined using WinNonlin version 6.3 (Pharsight Corp,
245
Mountain View, CA). The results are presented in Table 4. The plasma kinetics results indicated
246
that the metabolite was readily absorbed in rats with moderate Tmax and T1/2 values.
247
In the second part (Theerman, 2016b), four adult male SD rats were administered a
248
single 500 mg/kg oral gavage dose of [14C]-CA under the same conditions as described above.
249
Following termination of the study around 4 h after dosing, blood was collected via cardiac
250
puncture and the BM tissues were collected. Because of the high variability in Tmax values from
251
four rats, the maximum time required to achieve Cmax (4 h as opposed to the mean value of 2.8 h)
252
was considered for sample collection. The samples were analyzed for total radioactivity
253
concentrations using LSC, and the results are presented in Table 5. Both plasma and the BM
254
tissues had [14C]-CA concentrations well above the measurable levels, implying that the
255
metabolite, following oral administration, is bioavailable and reaches the target tissue in
256
significant amounts.
257 258
4. Additional considerations
259
Although testing of in vivo MN genotoxicity assays in rodent tissues other than the BM
260
have previously been conducted with liver, lung, colon, skin, spleen, testes, GI tract etc.,
261
acceptable/standard (e.g. OECD) study guidelines have not been established yet for testing in
262
these organs/tissues. Furthermore, scoring of MN in reticulocytes derived from BM is relatively
263
easier compared to other tissues (Morita et al., 2011). Therefore, in regulatory setting, the in vivo
264
MN test with BM (OECD 474) has been a standard assay for assessing cytogenetic damage.
265
However, it is important to recognize that if toxicokinetic data indicate lack of exposure, the in
266
vivo MN test should be conducted in other tissues. This is particularly true for test substances
267
metabolized in liver/intestine and forming reactive metabolites that are too short-lived to reach
268
the BM, and hence demonstration of bioavailability of parent compound does not necessarily
269
indicate that BM is an appropriate target (EFSA, 2011). However, as indicated earlier, currently
270
there are no accepted test guidelines on assessment for other tissues.
271
The TK studies for target tissue exposure conducted with C-14 label test substance may
272
utilize whole body autoradiography or quantitative determinations via radioactivity
273
measurements. In general, results obtained from either approach are in agreement, although
274
quantitative radioactivity measurements in blood/plasma and BM are routinely used (Dereszlay I,
275
1997). Although bone marrow is a highly perfused tissue, only unbound (free) fraction in
276
circulation is available for transport/perfusion into this tissue. Therefore, lower concentrations of
277
radioactivity or the parent test substance are expected in BM compared to plasma, as detailed in
278
the examples presented in this paper. However, currently, there is no established minimum level
279
of bone marrow exposure to be demonstrated in in vivo genotoxicity assays.
280
5. Conclusions
281
1. The in vivo rodent erythrocyte micronucleus test is a core study in genotoxicity test
282
battery and this test is valid only if there is evidence that the test substance reaches the
283
target tissue.
284 285
2. The paper describes the design and conduct of stand-alone toxicokinetic studies to determine bone marrow exposure to the test chemical.
286
3. In addition, the approaches on integration of bone marrow exposure as part of ADME
287
study and determination of radio-chromatographic profiles in unbound fraction of
288
plasma radioactivity are discussed which help reduce additional animal testing.
289
Conflict of interest
290
Nallani G, Liu, Z and Chandrasekaran, A are employed by FMC Corporation. All test items and
291
metabolites referenced in this paper are products of FMC.
292
Acknowledgment
293
The authors would like to acknowledge Dr. Li Shen (Frontage Laboratories, Exton, PA); Dr.
294
Manik Desai and Gina Theerman (WuXi AppTec, Plainsboro, NJ) for their contribution to the
295
experiments.
296
Funding
297
The presented work did not receive any specific grant from funding agencies in the public or not-
298
for-profit sectors. The examples presented in this paper were obtained from studies conducted on
299
behalf of FMC Corporation.
300
References
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Booth, E.D., Rawlinson, P.J., Maria Fagundes, P., Leiner, K.A., 2017. Regulatory requirements for genotoxicity assessment of plant protection product active ingredients, impurities, and metabolites. Environ. Mol. Mutagen. https://doi.org/10.1002/em.22084
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Dereszlay I, Patfalusi M, and Klebovich, I., 1997. Study of the bone marrow penetration of radioactivity after oral administration of radiolabelled girisopam (EGIS-5810) in mice. Acta Physiol Hung 85, 139–148.
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EFSA, 2018. Conclusion on the peer review of the pesticide risk assessment of the active substance rimsulfuron. EFSA J. 16(5), 23 pp. https://doi.org/https://doi.org/10.2903/j.efsa.2018.5258
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EFSA, 2017. Conclusion on the peer review of the pesticide risk assessment of the active substance rimsulfuron. EFSA J. 16(5), 28pp. https://doi.org/https://doi.org/10.2903/j.efsa.2018.5258
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EFSA, 2011. Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment. EFSA J. https://doi.org/10.2903/j.efsa.2011.2379.
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EU, 2013. Commission Regulation (EU) No 283/2013 of March 2013 setting out data requirements for active substances, in accordance with Regulation (EC) No 1107/2009 of the European Parliament and of the Council concerning the placing of plant protection products on . Official Journal of the European Union L 93, 3 April 2013, pp. 1-84.
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Hardy, A., Benford, D., Halldorsson, T., Jeger, M., Knutsen, H.K., More, S., Naegeli, H., Noteborn, H., Ockleford, C., Ricci, A., Rychen, G., Silano, V., Solecki, R., Turck, D., Younes, M., Aquilina, G., Crebelli, R., Gürtler, R., Hirsch‐Ernst, K.I., Mosesso, P., Nielsen, E., van Benthem, J., Carfì, M., Georgiadis, N., Maurici, D., Parra Morte, J., Schlatter, J., 2017. Clarification of some aspects related to genotoxicity assessment. EFSA J. 15. https://doi.org/10.2903/j.efsa.2017.5113
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Hayashi, M., 2016. The micronucleus test-most widely used in vivo genotoxicity test. Genes Environ. 38, 4–9. https://doi.org/10.1186/s41021-016-0044-x
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Heddle, J.A., Hite, M., Kirkhart, B., Mavournin, K., MacGregor, J.T., Newell, G.W., Salamone, M.F., 1983. The induction of micronuclei as a measure of genotoxicity. A report of the U.S. environmental protection agency Gene-Tox program. Mutat. Res. Genet. Toxicol. 123, 61–
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118. https://doi.org/10.1016/0165-1110(83)90047-7
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Krishna, G., Hayashi, M., 2000. In vivo rodent micronucleus assay: Protocol, conduct and data interpretation. Mutat. Res. - Fundam. Mol. Mech. Mutagen. 455, 155–166. https://doi.org/10.1016/S0027-5107(00)00117-2
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Morita, T., MacGregor, J.T., Hayashi, M., 2011. Micronucleus assays in rodent tissues other than bone marrow. Mutagenesis. https://doi.org/10.1093/mutage/geq066
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OECD, 2016a. Test No. 473: In Vitro Mammalian Chromosomal Aberration Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
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OECD, 2016b. Test No. 474: Mammalian Erythrocyte Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. https://doi.org/10.1787/9789264264762-en
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OECD, 2010a. Guidelines for the Testing of Chemicals, Section 4, Test No. 487: In Vitro Mammalian Cell Micronucleus Test. OECD Guidel. Test. Chem. 1–23. https://doi.org/http://dx.doi.org/10.1787/9789264091016-en
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OECD, 2010b. Test No. 417: Toxicokinetics. OECD Environ. Heal. Saf. Publ. 1–20. https://doi.org/10.1787/9789264070882-en
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Salamone, Michael, Heddle, John, Stuart Earl, Katz, M., 1980. Towards an improved micronucleus test: studies on 3 model agents, mitomycin C, cyclophosphamide and dimethylbenzathracene. Mutat. Reserach 74, 347–356.
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SANCO, 2003. Guidance document on the assessment of the relevance of metabolites in groundwater of substances regulated under council directive 91/414/EEC., October.
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Schmid, W., 1975. The micronucleus test. Mutat. Reserach 31, 9–15.
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Shen, L., 2018. Radioactivity concentrations in plasma and bone marrow following intra peritoneal injection of [14C]-M1 to male NMRI mice (unpublished). Exton, PA, USA.
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Shen, L., 2017. Radioactivity concentrations in plasma and bone marrow at T-max after oral administration of [14C]-HX to Sprague-Dawley rats (unpublished). Exton, PA, USA.
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Theerman, G., 2016a. Plasma radio pharmacokinetics of [14C]-MA in male Sprague-Dawley rats following a single oral (gavage) administration (unpublished). Plainsboro, NJ, USA.
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Theerman, G., 2016b. Pharmacokinetics and radioanalysis in bone marrow of [14C]-MA in male Sprague-Dawley rats following single oral (gavage) administration (unpublished). Plainsboro, NJ, USA.
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Acronyms
ADME AI AUC BM CFR Cmax CMC cpm cps dpm EU GI HPLC HT IACUC ip LOD LOQ LSC MN MS MTD NCE OECD PBS PCE PPP RF RO SD rat T1/2 TG TK Tmax TRR
Absorption, Distribution, Metabolism, and Excretion Active ingredient Area under the curve Bone marrow Code of Federal Regulations Maximum concentration in plasma Carboxy methyl cellulose Counts per minute Centipoise Disintegrations per minute European Union Gastrointestinal High performance liquid chromatography High-throughput Institutional Animal Care and Use Committee intra peritoneal Limit of detection Limit of quantitation Liquid scintillation counter Micronucleus Mass spectrometry Maximum tolerated dose Normochromatic erythrocytes Organization for Economic Cooperation and Development Phosphate buffer saline Polychromatic erythrocytes Plant protection product(s) Range-finding Reverse osmosis Sprague-Dawley rat Half-life of test item in plasma Test Guideline Toxicokinetics Time at which the maximum concentration in plasma observed Total radioactive residue
Figure Captions Figure 1. Decision tree and strategy for conducting a toxicokinetic study to demonstrate exposure of bone marrow tissue in support of a negative test result from an in vivo mammalian erythrocyte micronucleus (MN) test (OECD 474). Figure 2A. Radio-chromatographic profile of radioactivity (plasma side) in plasma of adult male Sprague-Dawley rats administered a single 500 mg/kg bw oral gavage dose of [14C]-HX Figure 2B. Radio-chromatographic profile of unbound radioactivity (buffer side) in plasma of adult male Sprague Dawley rats administered a single 500 mg/kg bw oral gavage dose of [14C]HX
Tables Table 1. Mean (±SD, n=4)) total radioactivity concentrations (µg Eq/g) in plasma and bone marrow (~ 4 h post dosing) of adult male Sprague-Dawley rats following a single 500 mg/kg/bw oral gavage dose of [14C]-HX Tissue
Mean (±SD) concentration (µg Eq/g; n=4)
Plasma
153.20 ± 32.46
Bone marrow
49.73 ± 11.94
Table 2. Mean (±SD, n=4) radioactive concentrations (µM) and percent bound and unbound radioactivity in rat plasma (~ 4 h post dosing) in adult male Sprague-Dawley rats following a single 500 mg/kg/bw oral gavage dose of [14C]-HX Mean (±SD, n=4)
14
C-HX
14
C-Warfarin**
PBS* buffer side (µM) 56.14 ± 14.26
Plasma side (µM) 451.17 ± 102.81
% unbound
% bound
12.4 ± 2.2
87.5 ± 2.2
37
819
4.5
95.4
*phosphate buffer saline; ** positive control for protein binding (single replicate only) Table 3. Mean (±SD, n=4*) total radioactivity concentrations (µg Eq/g) in plasma and bone marrow (~0.5 h post dosing) of adult male NMRI mice (n=8) following a single 500 mg/kg/bw intraperitoneal injection of [14C]-benalaxyl oxopropanoic acid (M-1) Tissue
Mean (±SD) concentration (µg Eq/g; n=4)
Plasma
587.28 ± 67.16
Bone marrow
185.62 ± 17.97
* samples from two animals each pooled Table 4. Mean (±SD, n=4) kinetic parameters of [14C]-carfentrazone-cinnamic acid (CA) in plasma of adult male Sprague-Dawley rats following a single 500 mg/kg oral gavage administration. Mean (±SD) kinetic parameter (n=4) Cmax (µg Eq/ml)
AUCinf (h. µg Eq/ml)
Tmax (h)
T1/2 (h)
472 ± 177
3627 ± 1453
2.75 ± 1.50
3.78 ± 1.57
Table 5. Mean (±SD, n=4)) total radioactivity concentrations (µg Eq/g) in plasma and bone marrow (~ 4 h post dosing*) of adult male Sprague-Dawley rats following a single 500 mg/kg/bw oral gavage dose of [14C]-carfentrazone-cinnamic acid (CA) Tissue
Mean (±SD) concentration (µg Eq/g; n=4)
Plasma
2270.25 ± 1336.49
Bone marrow
575.75 ± 340.35
*Due to variability in measurements (SD: 1.75 h, n=4), bone marrow was collected at the highest T-max value (4 h).
Demonstration of target tissue exposure in rodent in vivo micronucleus (MN) test (OECD TG 474, July 2016)
Product registered?
Conduct in vivo MN test on parent, relevant metabolite or impurity following revised OECD TG 474
No
Yes
In vivo MN test on parent, relevant metabolite or impurity conducted following pre-revised TG OECD 474
No
MN study on Parent Does ADME study indicate the parent >10% in urine, bile or urine + bile and/or detected in unbound fraction of plasma?
Yes
MN study on relevant metabolite or impurity
Yes
Does ADME study indicate the metabolite/impurity > 10% in urine, bile or urine + bile and/or detected in unbound fraction of plasma?
No Yes
Evidence of BM exposure established
Conduct TK study (rodents of relevant sex to be administered appropriate dose tested in MN test) with C-14 material (parent, relevant metabolite or impurity). Collect plasma and/or bone marrow around Tmax and determine TRR. Radioactivity levels in BM or unbound plasma above detection limit?
C-14 test substance available?
Yes
Yes
BM exposure not established Yes
No
No
No
Analytical method capable to detect test substance in target tissue?
Figure 1. Demonstration of target tissue exposure in rodent in vivo micronucleus (MN) test (OECD TG 474, July 2016)
Product registered?
Conduct in vivo MN test on parent, relevant metabolite or impurity following revised OECD TG 474
No
Yes
In vivo MN test on parent, relevant metabolite or impurity conducted following pre-revised TG OECD 474
No
MN study on Parent Does ADME study indicate the parent >10% in urine, bile or urine + bile and/or detected in unbound fraction of plasma?
Yes
MN study on relevant metabolite or impurity
Yes
Does ADME study indicate the metabolite/impurity > 10% in urine, bile or urine + bile and/or detected in unbound fraction of plasma?
No Yes
Evidence of BM exposure established
Conduct TK study (rodents of relevant sex to be administered appropriate dose tested in MN test) with C-14 material (parent, relevant metabolite or impurity). Collect plasma and/or bone marrow around Tmax and determine TRR. Radioactivity levels in BM or unbound plasma above detection limit?
C-14 test substance available?
Yes
Yes
BM exposure not established Yes
No
No
No
Analytical method capable to detect test substance in target tissue?
Figure 2
28.6
A) 900 800 700 600
300
35.4
29.9
400
38.4
CPM
28.1
500
200 100 0 0
5
10
15
20
25 Time (min)
30
35
40
45
50
45
50
B) M11 29.4
300
250
M10
Parent 38.4
30.4
28.9
100
M15
35.9
M12
M8 150
24.4
CPM
200
50
0 0
5
10
15
20
25 Time (min)
30
35
40
Designation of radioactive peaks (retention time in min) in Fig 2 A and 2B; 24.4: M8; 28.1-28.9: M10; 28.6-29.4: M11; 29.9-30.4: M12; 35.4-35.9: M15; 38.4: HX (unchanged parent)
Funding The presented work did not receive any specific grant from funding agencies in the public or notfor-profit sectors. The examples presented in this paper were obtained from studies conducted on behalf of FMC Corporation.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: