This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and

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1 This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier s archiving and manuscript policies are encouraged to visit:

2 Mutation Research 722 (2011) 7 19 Contents lists available at ScienceDirect Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: Community address: Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test Damian E. Bowen a,1, James H. Whitwell b,, Lucinda Lillford b, Debbie Henderson b, Darren Kidd b, Sarah Mc Garry b, Gareth Pearce b, Carol Beevers b, David J. Kirkland c, Work conducted at Covance Laboratories Ltd., Harrogate a JSC International Ltd., Simpson House, Windsor Court, Clarence Drive, Harrogate HG1 2PE, UK b Covance Laboratories Ltd., Otley Road, Harrogate HG3 1PY, UK c Kirkland Consulting, PO Box 79, Tadcaster LS24 0AS, UK article info abstract Article history: Received 28 April 2010 Received in revised form 1 November 2010 Accepted 6 February 2011 Available online 25 February 2011 Keywords: Genotoxicity Carcinogenicity Comet assay Bone-marrow micronucleus Peripheral blood micronucleus Flow cytometry 2-Acetylaminofluorene Benzo[a]pyrene Carbendazim Cyclophosphamide Dimethylnitrosamine Ethyl methanesulfonate Ethyl nitrosurea Mitomycin C With the publication of revised draft ICH guidelines (Draft ICH S2), there is scope and potential to establish a combined multi-end point in vivo assay to alleviate the need for multiple in vivo assays, thereby reducing time, cost and use of animals. Presented here are the results of an evaluation trial in which the bone-marrow and peripheral blood (via MicroFlow flow cytometry) micronucleus tests (looking at potential chromosome breakage and whole chromosome loss) in developing erythrocytes or young reticulocytes were combined with the Comet assay (measuring DNA strand-breakage), in stomach, liver and blood lymphocytes. This allowed a variety of potential target tissues (site of contact, site of metabolism and peripheral distribution) to be assessed for DNA damage. This combination approach was performed with minimal changes to the standard and regulatory recommended sampling times for the stand-alone assays. A series of eight in vivo genotoxins (2-acetylaminofluorene, benzo[a]pyrene, carbendazim, cyclophosphamide, dimethylnitrosamine, ethyl methanesulfonate, ethyl nitrosourea and mitomycin C), which are known to act via different modes of action (direct- and indirect-acting clastogens, alkylating agents, gene mutagens, cross-linking and aneugenic compounds) were tested. Male rats were dosed at 0, 24 and 45 h, and bone marrow and peripheral blood (micronucleus endpoint), liver, whole blood and stomach (Comet endpoint) were sampled at three hours after the last dose. Comet and micronucleus responses were as expected based on available data for conventional (acute) stand-alone assays. All compounds were detected as genotoxic in at least one of the endpoints. The importance of evaluating both endpoints was highlighted by the uniquely positive responses for certain chemicals (benzo[a]pyrene and 2-acetylaminofluorene) with the Comet endpoint and certain other chemicals (carbendazim and mitomycin C) with the micronucleus endpoint. The data generated from these investigations demonstrate the suitability of the multi-endpoint design Elsevier B.V. All rights reserved. 1. Introduction The draft revision of the ICH (International Conference on Harmonisation) S2 genotoxicity guidelines [1] has been accomplished under ICH s ongoing commitment to promote the 3Rs agenda to Replace, Reduce and Refine animal testing in non-clinical safety Corresponding author. Tel.: address: james.whitwell@covance.com (J.H. Whitwell). 1 Current address. studies. Within the proposed revision, animal reduction can be achieved by integration of in vivo genotoxicity testing into repeatdose toxicity assays, but there is also opportunity to reduce animal usage by combination of endpoints into a single acute assay design. This would also reduce cost, and speed-up the generation of relevant data. The combination assay-design evaluated in this paper is modified only slightly from the recommended guidance for conduct of the stand-alone in vivo Comet [2,3] and in vivo bonemarrow micronucleus [4] assays (Figs. 1 and 2). Furthermore, from this combination approach there is scope to incorporate further micronucleus analysis in peripheral blood reticulocytes via flow /$ see front matter 2011 Elsevier B.V. All rights reserved. doi: /j.mrgentox

3 8 D.E. Bowen et al. / Mutation Research 722 (2011) 7 19 Fig. 1. Standard bone marrow micronucleus and Comet assay design. cytometry, allowing analysis of micronuclei in two differing compartments. Although this type of combination approach has been recognised and recently investigated by other workers [5,30], it was considered valuable to conduct a more extensive evaluation with a number of different genotoxins acting via different modes of action. Within the test battery for genotoxicity, a positive response in any in vitro assay necessitates more in vivo work (second tissue/endpoint) in addition to the standard mammalian bonemarrow micronucleus test. A combination endpoint approach should consider the most appropriate secondary in vivo assay for inclusion. Recently published literature [6] has shown the Comet (CMT) assay to be a good complement to the bone-marrow micronucleus test. In this review [6], the published in vivo results of the unscheduled DNA synthesis (UDS) test, the transgenic mutation (TG) assay and the DNA-damage (Comet, CMT) assay for 67 rodent carcinogens that were negative or equivocal in the micronucleus test were investigated. Between 30 and 41 chemicals were evaluated in each of the in vivo tests with some overlap. The UDS and TG assays gave positive results with less than 20% or slightly greater than 50% of these rodent carcinogens, respectively, with the CMT assay detecting approximately 90%. Furthermore, based on a small number of publications with non-carcinogens, the TG and CMT assays gave negative results with non-carcinogens in 69% and 78% of the cases, respectively. This review showed a high sensitivity (ability to detect carcinogens as positive) and specificity (ability to give negative results with non-carcinogens) for the CMT. It is for these Fig. 2. Combination assay design. In addition to the above, satellite animals were dosed according to the same regimen, and sampled at 24 and 44 h after the final dose for peripheral blood micronucleus assessment. Note: Peripheral blood taken for flow micronucleus analysis was looking at the reticulocyte population, whilst blood taken for Comet was targeting the lymphocyte population. reasons, along with ease of use, variety of DNA damage detected and the possibility of sampling multiple tissues, that the current investigations have evaluated combining this endpoint with the bone-marrow (BM) and peripheral blood (PB) micronucleus (MN) endpoints. In the BM-MN assay erythroblasts undergoing their last chromosome replication are the target cell [7 9]. Chromosome fragments or whole chromosomes that are unable to attach to the mitotic spindle are left behind as micronuclei (MN) when the main nucleus is extruded in the production of the polychromatic erythrocyte (PCE), a process known as karyorrhexis. Cellular RNA is gradually lost and maturation results in the formation of a further intermediary, the normochromatic (immature) erythrocyte (NCE), prior to release into the peripheral blood circulation as young reticulocytes. As MN can be generated from intact chromosomes, agents that affect mitotic spindle formation or function (that is, result in aneuploidy) can be detected. As expulsion of the nucleus is known to occur within 5 10 h after the last cell division, with the loss of RNA and maturation to NCE status within a further h period, it is currently recommended that sampling of bone marrow and subsequent analysis of the immature PCE population for MN should occur approximately h following the final administration of test chemical [4]. With the single-cell gel electrophoresis assay, more commonly referred to as the Comet (CMT) assay, DNA damage at the level of the single cell is detected. The alkaline CMT assay can detect a range of genotoxic insults including both single- and doublestrand DNA breaks, alkali-labile sites (expressed as single-strand breaks), single-strand breaks associated with incomplete repair, and DNA DNA or DNA protein cross-links (with modification of the protocol). These types of primary DNA lesion are short-lived and may undergo rapid repair [3]. Therefore, in order to prevent any loss of potential DNA damage, it is recommended that sampling for DNA damage via the CMT occurs within the interval of 3 6 h after the final dose (in the case of multiple dosing) [2]. In this way, rapidly absorbed, unstable, or direct-acting compounds may be detected. However, this sampling time is also 24 h after the penultimate dose or 48 h after the pre-penultimate dose, thereby enabling possible detection of slowly absorbed/distributed and metabolised chemicals. From both the BM-MN and CMT assay designs it would appear difficult to arrive at a combination of both endpoints in one set of animals (Fig. 2). In order to achieve this combination, the standard assay-design was modified slightly from the recommended guidance on the performance of the in vivo CMT [2,3] and the BM-MN [4] assays. The combination protocol-design proposed here (Fig. 2) consists of animals dosed at 0, 24 and 45 h, and sampled at 48 h (i.e., three hours after the final dose). Using this dosing regimen, only the first two doses (at 0 and 24 h) could impact upon the bonemarrow micronucleus endpoint, as the final dose is given too close to the sampling time to have any effect on MN-PCE production. Bone marrow was sampled effectively at 24 h after the second dose, and therefore conforms to OECD 474 recommendations [4]. The CMT tissue samples (liver, blood and stomach) were taken three hours after the final dose or 24 h after the second dose, which also conforms with current recommendations for the alkaline version of the assay [10]. As an alternative to measuring MN in bone marrow, analysis of peripheral blood can provide an effective means of measuring clastogenic damage, i.e. chromosome breakage. In the rat, however, micronucleated cells are removed by the spleen and it is important, therefore, to restrict micronucleus analysis to young reticulocytes, to prevent false negative results [11,12]. As the fraction of young erythrocytes in the peripheral blood is small, laborious microscopical examination of cells can be overcome by use of automated flow cytometric methods (FCM) [13 15] by means of targeting the spe-

4 D.E. Bowen et al. / Mutation Research 722 (2011) Table 1 Summary of chemicals assessed. Chemical (CAS number) Chemical classification Mechanism of action (predominant CYP450, where relevant) Vehicle Doses (mg/kg/day) Route of administration 2AAF ( ) a Aromatic amine Clastogen requiring metabolic Corn oil 45, 90, 180 Intraperitoneal injection activation (CYP1A2) B[a]P ( ) a Polycyclic aromatic Clastogen requiring metabolic Saline 50, 125, 250 Intraperitoneal injection hydrocarbon activation (CYP1A1) CBZ ( ) b Benzimidazole Aneugen. Inhibits the 1% (v/v) methylcellulose 1000, 1500, 2000 Oral by gavage (fungicide) polymerisation of tubulin CPA ( ) b Aziridine Clastogen requiring metabolic Saline 10, 25, 40 Oral by gavage activation (CYP2B6/CYP3A4) DMN ( ) b Nitrosamine Clastogen requiring metabolic Purified water 20, 40, 75 c Oral by gavage activation (CYP2E1) EMS ( ) b Alkyl sulfate Direct acting clastogen Saline 150, 250, 350 Oral by gavage ENU ( ) b Nitrosamide Gene mutagen Saline 10, 25, 40 Oral by gavage MMC ( ) b Quinone DNA crosslinker Saline 1, 2, 4 Intraperitoneal injection a Sprague Dawley rats. b Han Wistar rats. c Analysis not conducted due to excessive clinical toxicity and mortality. cific cell-surface marker, the transferrin receptor (CD71), which is active for only a short time after a reticulocyte (RET) enters the peripheral blood from the bone marrow (approximately 24 h). Automated FCM have great potential for improving the sensitivity, reproducibility and throughput of the traditional in vivo rodent peripheral blood micronucleus (PB-MN) assay, which has been validated by the Collaborative Study Group for the Micronucleus Test (CSGMT) of the Mammalian Mutagenesis Study Group (MMS) of the Environmental Mutagen Society of Japan (JEMS) [11]. Stained blood cells fluoresce as they pass through a focused laser beam, and the resulting data are collated for computer analysis. Targeted staining of the CD71 receptor ensures analysis of the young RET population and avoids artefacts due to sequestration of MN-RET by the spleen. Various published data [12,16] have led to the recommendation that for analysis of MN-RET in peripheral blood, where a single administration regime is used, two sample times should be employed (48 and 72 h); if two or more repeated administrations are used, then a single sample time between 24 and 48 h following the last administration should be employed. In this evaluation, to ensure that a robust assessment of micronucleus appearance in peripheral blood was achieved, samples were taken at 24 and 44 h (from satellite dosed animals) as well as three hours (from dosed animals in the main study) after the last dose (Fig. 2). In this way we investigated whether the proposed alternate dosing/sampling regime allowed incorporation of the peripheral blood end-point into the combined CMT/MN(BM + PB) protocol, alleviating the need for inclusion of additional satellite dosed animals. 2. Materials and methods 2.1. Chemicals Details of the chemicals selected and the rationale for their selection are given in Table 1. Analytical grade 2-acetylaminofluorene (2AAF), benzo[a]pyrene (BaP), carbendazim (CBZ), cyclophosphamide (CPA), dimethylnitrosamine (DMN), ethyl methanesulfonate (EMS); ethyl nitrosourea (ENU) and mitomycin C (MMC) were all obtained fromsigma-aldrich Chemical Co., Poole, UK. Dosing solutionswere freshly prepared in saline (Baxter, Illinois, USA) (B[a]P, CPA, EMS, ENU, and MMC), purified water (DMN), corn oil (SAFC, UK) (2AAF) or 1% methylcellulose (Dow Chemical Company, USA) (CBZ). These solvents were also used as the vehicle controls for the corresponding chemicals tested Selection and justification of dose levels This study was approached as though the chemicals to be tested were unknown agents and initial dose setting range-finder experiments were conducted. The rationale was that in order to provide the best opportunity to compare the respective responses for the different endpoints, it would be necessary to use a range of doses for each chemical, from sub-toxic to toxic. These doses were therefore selected from the preliminary range-finder experiments based on the induction of clinical signs of toxicity. As will be seen later, in such toxic dose ranges, day to-day variability occurs and in some instances in the main experiments, limited mortality and cellular toxicity were observed. The range-finding experiments were performed using limited numbers of male animals, with initial doses based on historical or published data. Clinical signs and bodyweight changes were recorded over a 5-day observation period (Day 1 to Day 5) to suggest suitable doses up to a maximum tolerated dose (MTD). The doses used in the main experiments were the MTD and two lower doses as summarised in Table 1. The dose volume used was 10 ml/kg Animals and dosing Young adult male rats (between 7 and 10 weeks of age on the first day of dosing) of the Han Wistar or Sprague Dawley strains, were obtained from either Charles River (UK) Ltd., Margate, UK or Harlan UK Ltd., Oxon, UK. For reasons discussed later, both Han Wistar and Sprague Dawley strains were used at various points in this study (see Section 4). The animals were acclimatised for at least five days before being placed on study. The animals were housed in groups of up to six in sanitary, stainless steel, polypropylene hanging cages (Type TR/24 solid cages). Climatic conditions were controlled as follows temperature, C; humidity, 40 70%; light cycle, 12 h light/dark; air changes, at least 15/h. SQC Rat and Mouse Maintenance Diet No., Expanded (Special Diets Services Ltd., Witham, UK) and tap water were available ad libitum. In order to enrich both the environment and the welfare of the animals, Aspen chew blocks were provided. After acclimatisation, animals for the combined CMT and MN endpoints (six animals/group) were given the test chemicals as three daily administrations at 0, 24 and 45 h. Animals were sacrificed three hours after the final dose administration. Details of the doses and routes of administration are given in Table 1. Animal body-weights were recorded before each dose and on the day of tissue sampling. Clinical signs were monitored daily. For additional investigation of the PB-MN response, satellite animals (five per group) received the same dosing regime as described, but peripheral blood was sampled at 24 and 44 h (via tail vein and cardiac puncture, respectively) after the final dose administration. Blood (for PB-MN) was also sampled from the CMT and MN animals at the time of necropsy, i.e. three hours after the final dose. This scheme is diagrammatically outlined in Fig Necropsy and preparation of tissues Three hours after the final dose administration (i.e. 48 h after the start of dosing) rats for CMT/MN(BM + PB) endpoints were terminally anaesthetised with isofluorane, in the same sequence used for dosing and killed by exsanguination via cardiac puncture. All treated animals were examined internally for signs of unusual coloration or abnormalities to organs/tissues. The stomach and liver from CMT/MN animals were removed, washed thoroughly in ice-cold mincing buffer [Merchants buffer containing 13.2 mmol/l ethylenediaminetetraacetic acid (EDTA) disodium salt and 10% DMSO in PBS, ph 7.4] prior to placement in fresh buffer, and held over ice until required. Blood for CMT analysis was taken from each animal and collected in lithium/heparin tubes. One femur from each of the sacrificed animals was exposed, removed, cleaned of adherent tissue and the ends removed from the shanks for BM- MN analysis. The blood for PB-MN analysis was collected from all animals into tubes containing K 2EDTA anticoagulant (Tech Lab).

5 10 D.E. Bowen et al. / Mutation Research 722 (2011) Sampling and slide preparation for the bone-marrow micronucleus test Using a syringe and needle, marrow was flushed from the bone cavity with foetal bovine serum (FBS, Gibco). More FBS was added, and samples were centrifuged at 200 g for 5 min. The serum was aspirated to leave several drops and the cell pellet was gently re-suspended. From this suspension, several drops were placed on the end of each of two appropriately labelled glass microscope slides. A smear was made from the cell suspension by drawing the end of a clean slide along the labelled slide. Following preparation of bone-marrow smears, slides were allowed to air-dry prior to fixation of cells in absolute methanol (10 min). Following rinsing in distilled water, slides were allowed to air-dry prior to staining with acridine orange (12.5 g/ml, prepared in 0.1 M phosphate buffer ph 7.4 (Severn Biotech)) for 5 min. Slides were rinsed in phosphate buffer and stored in the dark at room temperature (15 25 C) Isolation of single-cell suspensions for the Comet assay Liver A portion of the left lateral lobe ( 2cm 3 ) was excised and washed in cold mincing buffer. The liver was then cut into small pieces, and 2 ml Merchants buffer was pipetted over the tissue before being filtered (via forcing) through bolting cloth (150 m). The cell suspension obtained was mixed with a further 2 ml of buffer and held on ice until slide preparation Stomach The stomach was cut open (along the greater curvature) and contents were washed free with buffer. The forestomach was removed, and the remaining glandular stomach was then placed into a clean Petri dish, with the inner surface uppermost, covered in buffer and incubated on ice for 15 min. Following incubation the stomach was removed from the buffer, and the inside surface carefully scraped twice (to remove the mucosal layer) with the back of a scalpel blade. The scraped material was discarded. Two hundred microliter buffer was pipetted onto the inner surface of the stomach and again gently scraped, as previously described. The cells released were pipetted into appropriately labelled cryotubes and held on ice until slide preparation. Prior to any single-cell processing, tissue samples from the left lateral lobe of the liver and glandular region of the stomach were fixed and preserved in buffered formalin in case histopathological examination was required Blood Blood obtained by cardiac puncture was collected as described, mixed thoroughly and held on ice until required. For the whole blood CMT, DNA damage was assessed in leukocytes Slide preparation Slides with frosted edges were pre-coated with 1% normal-melting agarose and allowed to dry at room temperature overnight. For each tissue, cell suspensions were mixed with 0.7% low-melting agarose and 100- L aliquots were added to four slides, then covered with a 22 mm 50 mm glass coverslip. The gels were allowed to set on a cold plate, and then the coverslips were removed. Slides were immersed in lysis solution (2.5 mol/l NaCl (Fisher), 100 mmol/l EDTA disodium salt, 10 mmol/l Tris (VWR), 10% DMSO and 1% Triton X-100 (BDH), ph xx) and stored at 4 C overnight. Following lysis, three slides/animal/tissue were transferred to the electrophoresis buffer (300 mmol/l NaOH, 1 mmol/l EDTA disodium salt, ph > 13), in which DNA was allowed to unwind for 20 (stomach) or 30 (liver and blood) min, before electrophoresis (0.7 V/cm, 300 ma) for a further 20 (stomach) or 40 (liver and blood) min. DNA unwinding and electrophoresis were performed in the dark, in a cold unit set at 4 C. At the end of the electrophroesis period, slides were immersed in three changes of neutralising buffer (0.4 mol/l Tris, HCl (Fluka), ph 7.0) and then allowed to dry at room temperature, protected from light. These slides were used for CMT analysis. After the lysis step (overnight), the fourth slide from each animal was placed in neutralising buffer and then dried and stored (as described previously). This diffusion slide was used to estimate the degree of highly damaged cells in the cell suspension Sample preparation for flow cytometry Blood samples for flow cytometry were processed immediately upon collection (i.e. 3 h after the final dose from animals in the main study, and 24 or 44 h after the final dose from satellite animals) as described in the MicroFlow BASIC kits (for mouse and rat) from Litron Laboratories (Rochester, NY). The kits contained all the supplies and reagents necessary to process blood samples, which were prepared and analysed according to Torous et al., [15] Analysis of data Comet analysis Scoring was carried out under blinded conditions by an individual not connected with the dosing phase of the study. Prior to CMT scoring, the slides were stained with 100 L of ethidium bromide (2 g/ml) and coverslipped. One hundred cells/animal were scored (50 cells/gel) by use of the Perceptive Instruments Comet III data-capture system. Tail intensity (TI % DNA migrated from the head to the tail) was used as the measure of damage. Comet cytotoxicity measures: From 100 cells/slide the number of clouds / hedgehogs were scored [2]. To avoid undue influence on the results, these cells were not used for CMT analysis. For the diffusion slides, 100 cells/slide were categorised into one of two groups: (1) little or no diffusion from the nuclear head or (2) marked diffusion with either a small or no head. Data from animals where neutral diffusion exceeded 24% or for which the percentage of clouds was 30% or higher, indicating high cytotoxicity, were interpreted with caution. Statistical analysis was performed on the median tail-intensity data as follows: Vehicle and dose groups for each chemical were analysed by means of one-way analysis of variance (ANOVA). An overall dose-response test was performed along with Dunnett s test for pairwise comparisons of each treated group with the concurrent vehicle control. Levene s test for equality of variances between groups was performed and where this showed evidence of heterogeneity (p < 0.01), a rank transformation was applied to the data prior to analysis. All tests were performed with a two-sided risk. In accordance with the IWGT [10] and other recent [17] recommendations, statistical significance as well as indications of dose responses, increased migration over control and comparison to the laboratory s historical control range (where relevant) were used to determine positive responses Bone-marrow micronucleus analysis Scoring was carried out under blinded conditions by use of fluorescence microscopy, by an individual not connected with the dosing phase of the study. Initially the relative proportions of PCE, seen as bright-orange enucleate cells, and NCE, seen as smaller dark-green enucleate cells, were determined until a total of at least 1000 cells (PCE plus NCE) had been analysed (under dry-mount coverslip conditions). Then at least 2000 PCE/animal were examined for the presence of MN (under PBS wet-mount coverslip conditions). After completion of the microscopical analysis and decoding of the data, the following were calculated: The percentage PCE for each animal and the mean for each group. The group mean % PCE values were examined to see if there was any decrease in groups of treated animals compared with concurrent control and historical control values that could be taken as evidence of bone-marrow toxicity. Frequency of MN-PCE (i.e. MN/2000 PCE) and percentage of MN-PCE for each animal and the group mean percentage MN-PCE. The numbers of MN-PCE in each treated group were compared with the numbers in each concurrent vehicle control by using a 2 2 contingency table to determine chi-square statistics. Where this showed no evidence of heterogeneity (p > 0.05) at any dose level, pair-wise comparisons with the concurrent control were made using Fisher s exact test. The Cochran Armitage test for a dose related trend was also performed [18]. Where evidence of heterogeneity (p < 0.05) was apparent for at least one dose level, the data were analysed by use of the Wilcoxon rank-sum test for pairwise comparisons with the concurrent control [19]. Where at least three doses were analysed, the Terpstra Jonckheere test for a dose-related trend was also conducted. For the pair-wise comparisons, the Bonferroni correction was used to make adjustments for multiple comparisons. All statistical tests were interpreted with one-sided risk for increasing response with increasing dose Peripheral blood micronucleus analysis Blood samples were analysed by high-speed flow cytometry by Covance Laboratories Ltd. with a Becton Dickinson (BD) FACSCanto II Software version (BD FACSCanto II). The stained cells were moved at high velocity past an argon laser (488 nm excitation). The fluorescence emitted by each cell was collected by photomultipler tubes with the staining procedure previously described by Dertinger et al. [20]. The DNA of the MN, stained with propidium iodide (PI), emitted red fluorescence, the anti-cd71-fitc antibody emitted a strong green-fluorescent signal and platelets were excluded based on their yellow fluorescence. The FITC fluorescence (CD71+) was detected by use of a band-pass filter of 530/30 nm and the PI was detected at wavelengths greater than 650 nm. Debris was excluded from counting, based on forward-angle light and side-scatter profiles. Up to 20,000 RET(CD71+) were analysed for the presence of MN (PI+) in each blood sample. The analysis for some samples was stopped before the required 20,000 RET were collected due to a low RET frequency (indication of toxicity). The following statistical analyses were performed on the data obtained three hours after the final dose:

6 D.E. Bowen et al. / Mutation Research 722 (2011) Table 2 Overall summary of results. Chemical Comet endpoint Micronucleus endpoint Liver Stomach Blood Bone marrow Peripheral blood 2AAF + E a B[a]P + CBZ E b + + CPA DMN + + E c E c EMS ENU MMC + + : negative; +: positive; E: equivocal. a Lack of a statistically significant increase at any dose level (as compared to concurrent vehicle control). b High toxicity (% clouds) associated with the maximum dose level that provided an equivocal response. c Clinical toxicity and mortality associated with the maximum dose level exceeding an MTD. Lower dose response within historical vehicle control values. For each chemical the numbers of MN-RET in each treated group were compared with the numbers in the concurrent vehicle-control group by using a 2 2 contingency table to determine chi-square statistics. Where this showed evidence of heterogeneity (p < 0.05) for at least one dose level, the data were analysed with the Wilcoxon rank-sum test for pair-wise comparisons with the concurrent control [19]. It should be noted that significant heterogeneity was observed for all chemicals tested in this study. For the pair-wise comparisons, the Bonferroni correction was used to make adjustments for multiple comparisons. Where at least three doses were analysed, the Terpstra Jonckheere test for a doserelated trend was also conducted. All statistical tests were interpreted with one-sided risk for increasing response with increasing dose. Increases in the percentage of MN-RET higher than 2-fold over the concurrent control which exceeded the 95% confidence interval of the laboratory s historical control range and showed evidence of a dose-response trend in the proportion of the percentage of MN-RET were also used alongside the statistical analysis as indicators of a positive induction of MN. 3. Results In the main studies no mortality, clinical signs of toxicity or reduction in body-weight gain were seen in any of the vehicle control, or B[a]P-, CBZ-, CPA-, ENU- or MMC-treated rats. For all rats dosed with DMN, piloerection and excessive urination was observed. One of the six rats died following dosing at 40 mg/kg/day, with the remaining five rats showed reduced body-weight gain relative to animals in the control group. At 75 mg/kg/day of DMN excessive signs of clinical toxicity were seen, resulting in either death or the need to kill the animals in extremis. The surviving 2/6 animals at this dose (75 mg/kg/day) were therefore not analysed for either CMT or micronucleus analysis. For all rats treated with EMS at 250 and 350 mg/kg/day, reduced body-weight gain relative to the controls was observed throughout the observation period of the study (two days after the first dose), with rats exhibiting minor to moderate signs of clinical toxicity: persistent piloerection at 250 mg/kg/day and persistent piloerection, decreased activity and hunched posture at 350 mg/kg/day. Thus, the observations confirmed that higher doses of these chemicals could not have been used without causing severe toxicity or lethality. For all rats treated with 2AAF at 45, 90 and 180 mg/kg/day, decreased body-weight gain relative to the controls was noted with a dose-dependent increase in the severity of clinical signs apparent Comet CMT data are summarised in Table 2, with detailed data presented in Table 3. The group mean percentage TI values for each vehicle-control group for both liver and stomach tissue fell within the laboratory s 95% historical control range for each respective tissue. Whilst no formal historical control range currently exists for rat blood in this laboratory, both group mean and individual median percentage TI values were consistent with data previously obtained for this tissue in this laboratory. The cytotoxicity data ( cloud assessment and diffusion-slide analysis) from the majority of vehicle-control animals demonstrated acceptable levels of cytotoxicity, i.e. the percentage of clouds not exceeding 30% and the percentage of diffused cells not exceeding 24%. DNA migration in samples from the stomach was higher than in other tissues, which was attributed to the mechanical process required for the preparation of stomach single-cell suspensions. With the exception of CBZ (aneugen) and MMC (cross-linker), all compounds tested resulted in marked increases in percentage TI values in one or more tissues over those for each respective concurrent vehicle control. However, it should be noted that for DMN marked increases in percentage TI were accompanied by increased cytotoxicity (percentage clouds ). For CBZ, negative data were observed in all tissues. A small increase in tail intensity was observed at the high dose (2000 mg/kg/day) in the stomach, which was statistically significant (p 0.05), but this was accompanied by an increase in toxicity not observed at lower doses. These data were therefore interpreted with caution, being possibly related to cytotoxicity. A statistically significant decrease (p 0.05) in percentage TI was observed at the high dose in the liver (% TI, 1.17 compared with 1.53 in the vehicle control), although all the values were within historical control ranges. For MMC a statistically significant increase in percentage TI in the stomach was observed in the high dose group. However, this outcome could be considered to be not indicative of DNA damage, but a result of biological variation: the increases observed were less than 2-fold above the concurrent control (mean TI, 3.71%, compared with 2.11% in the control), and all TI values for the individual animals were within the historical vehicle-control range ( %). In the light of the mechanism of action of MMC, these data were not unexpected. No notable decreases in percentage tail intensity were observed with MMC Bone-marrow micronucleus assay BM MN data are summarised in Table 2, with detailed data presented in Table 4. The group mean percentage MN-PCE values for all vehiclecontrol groups were comparable with the laboratory s historical data. Evidence of bone-marrow toxicity became manifest as a decrease in the percentage PCE in treated animals vs concurrent

7 12 D.E. Bowen et al. / Mutation Research 722 (2011) 7 19 Table 3 DNA damage determined by the Comet assay in various tissues from male rats, 3 h after a triple dose of various known mutagens. Chemical Cell type [Comet] Dose [mg/kg/day] % tail intensity Cytotoxicity Group mean ± SD Fold increase % clouds % diffused cells 2AAF Liver ± ± 0.35 (NS) ± 1.66 (***) ± 1.74 (***) Stomach ± ± 1.66 (NS) ± 0.84 (NS) ± 1.32 (NS) Blood ± ± 1.06 (NS) ± 0.63 (NS) ± 0.53 (NS) B[a]P Liver ± ± 0.53 (NS) ± 0.09 (NS) ± 0.07 (*) (Linear trend: **) Stomach ± ± 2.18 (NS) ± 2.52 (NS) ± 3.59 (*) (Linear trend: **) Blood ± ± 0.29 (NS) ± 0.16 (NS) ± 0.40 (NS) CBZ Liver ± ± 0.14 (NS) ± 0.26 (NS) ± 0.29 (*) (Linear trend: *) Stomach ± ± 1.36 (NS) ± 2.76 (NS) ± 3.68 (*) (Linear trend: **) Blood ± ± 0.29 (NS) ± 0.19 (NS) ± 0.20 (NS) CPA Liver ± ± 0.24 (NS) ± 0.21 (NS) ± 0.81 (**) (Linear trend: **) Stomach ± ± 4.20 (NS) ± 4.26 (**) ± 1.52 (***) Blood ± ± 0.69 (NS) ± 0.52 (***) ± 0.74 (***) DMN Liver ± ± 1.98 (***) ± 1.61 (***) Stomach ± ± 1.42 (NS) ± 0.49 (NS) Blood ± ± 1.74 (***) ± 1.35 (***)

8 D.E. Bowen et al. / Mutation Research 722 (2011) Table 3 (Continued) Chemical Cell type [Comet] Dose [mg/kg/day] % tail intensity Cytotoxicity Group mean ± SD Fold increase % clouds % diffused cells EMS Liver ± ± 0.90 (**) ± 1.43 (***) ± 3.29 (***) Stomach ± ± 1.48 (***) ± 1.43 (***) ± 1.34 (**) (Linear trend: **) Blood ± ± 1.28 (***) ± 0.97 (***) ± 4.40 (***) ENU Liver ± ± 0.80 (***) ± 1.68 (***) ± 1.21 (***) Stomach ± ± 4.16 (**) ± 4.16 (***) ± 4.22 (***) Blood ± ± 0.58 (***) ± 0.57 (***) ± 0.55 (***) MMC Liver ± ± 0.23 (NS) ± 1.19 (NS) ± 0.41 (NS) Stomach ± ± 0.33 (NS) ± 0.47 (NS) ± 0.72 (***) Blood ± ± 0.41 (NS) ± 0.37 (NS) ± 0.16 (NS) Values in parenthesis refer to statistical analysis undertaken. NS not significant, *P < 0.05, **P < 0.01, ***P < The Laboratory s Comet 95% historical vehicle-control ranges (% tail intensity) are calculated from median animal values rather than group mean values: Stomach range = %; liver range = %. There is currently no calculated range available for the Comet assay in blood; the laboratory s highest observed vehicle control value is 2.6% tail intensity. Animals where neutral diffusion or the percentage of clouds reached 30% or greater were interpreted with caution, indicating high cytotoxicity. controls, and was noted in animals treated with both CBZ (1500 and 2000 mg/kg/day) and CPA (25 and 40 mg/kg/day). For all other treated animals there was no evidence of either bone-marrow toxicity or increased erythropoiesis (i.e. increased percentage PCE) when compared with each respective vehicle-control group. With the exception of 2AAF and B[a]P, all chemicals induced significantly (p 0.05) elevated frequencies of MN-PCE at one or more dose levels, over those in each respective concurrent vehicle control. For DMN a statistically significant (p 0.01) increase in MN-PCE was observed at the high dose (40 mg/kg/day), a dose at which limited mortality and clinical signs of toxicity and body-weight loss were observed. Due to the observed mortality, and the fact that this dose would be considered to exceed a suitable MTD in regulatory compliant studies, data from this dose level were interpreted with caution. No statistical significance was observed at the 20-mg/kg/day dose, with mean MN-PCE values for both 20 and 40 mg/kg/day falling within historical vehicle-control data ranges. A positive linear trend was noted. These data were therefore considered as equivocal for DMN at this endpoint Peripheral blood micronucleus test PB-MN data are summarised in Table 2, with detailed data presented in Table 5. The percentage MN-RET values for all vehicle-control groups (all time points) fell within the laboratory s 95% historical control range. The one exception to this was the group mean vehiclecontrol value for B[a]P, 44-h time point, where the percentage MN-RET exceeded that observed for any of the treated groups. Whilst it cannot be confirmed, cross contamination of samples during storage was suspected. As outlined in the Introduction, current guidance for sampling peripheral blood for RET micronucleus analysis [14,16] recommends a single sample time between 24 and 48 h following the last of two or more repeated administrations. In this evaluation

9 14 D.E. Bowen et al. / Mutation Research 722 (2011) 7 19 Table 4 Bone-marrow micronucleus formation determined by fluorescence microscopy, three hours after a triple dose of various known mutagens. Chemical Dose [mg/kg/day] %PCE MN PCE/2000PCE %MN PCE ± SD 2AAF ± ± 0.04 (NS) ± 0.03 (NS) ± 0.08 (NS) (Linear trend: *) B[a]P ± ± 0.11 (NS) ± 0.09 (NS) ± 0.04 (NS) CBZ ± ± 0.29 (**) ± 0.36 (**) ± 0.28 (**) CPA ± ± 1.09 (**) ± 0.60 (**) ± 0.64 (**) (Linear trend: *) DMN ± ± 0.05 (NS) ± 0.12 (**) EMS ± ± 0.30 (**) ± 0.89 (**) ± 1.12 (**) ENU ± ± 0.10 (*) ± 0.47 (**) ± 0.72 (**) MMC ± ± 0.84 (**) ± 2.08 (NS) ± 2.16 (NS) (Linear trend: *) Values in parentheses refer to statistical analysis undertaken. NS not significant, *P < 0.05, **P < 0.01, ***P < %PCE: 95% confidence interval 40 57%. 95% confidence interval MN PCE/2000 PCE. study, data from a three-hour sampling time, which allows incorporating of peripheral blood sampling of animals in the main study, was compared with data from 24- and 44-h sampling times with satellite-dosed animals. Despite some differences in magnitude of the response, there were no qualitative differences between sampling three hours after the last dose, or sampling 24 or 44 h after the final dose. All chemicals inducing a positive increase in the percentage MN-RET at 24 or 44 h were also clearly positive at the three-hour sampling time. For the purposes of this evaluation study, statistical analyses were consequently restricted to the three-hour data alone. Evidence of cytotoxicity to peripheral blood, expressed as a decrease in the percentage of RET, was evident in animals treated with CBZ (1500 and 2000 mg/kg/day), CPA (10 mg/kg/day), EMS (250 and 350 mg/kg/day) and ENU (40 mg/kg/day), when sampled three hours after the final administration. For these chemicals, with the exception of EMS, data on the later sampling times (24 and 44 h) also showed marked toxicity to the extent that the number of RET was insufficient to allow analysis of MN-RET. For the chemicals CBZ, CPA, EMS, ENU, and MMC at least a twofold increase in the percentage MN-RET which also exceeded the 95% confidence interval of the laboratory s historical control range was observed at one or more dose levels, accompanied by a dose-response. Furthermore, statistically significant increases in MN-RET were seen at one or more dose levels, indicating a clearly positive induction of the percentage of MN-RET. For 2AAF, although increases in the percentage of MN-RET reached two-fold at the intermediate dose alone (90 mg/kg/day), this was in the absence of any cytotoxicity or dose-response. Also, none of the dose levels tested resulted in a statistically significant increase in the percentage of MN-RET. As such, the data for 2AAF were considered equivocal for this endpoint. For B[a]P, the percentage of MN-RET was comparable with the concurrent vehicle control at all time points sampled. Whilst a dose-related increase in the percentage of MN-RET was observed in animals dosed with DMN, this was accompanied by excessive signs of clinical toxicity, resulting in limited mortality at 40 mg/kg/day, suggesting that the MTD had been exceeded. Statistical analysis confirmed a weak but statistically significant (p 0.05) increase in the percentage of MN-RET (less than 2-fold over the concurrent vehicle control) at the lower dose of 20 mg/kg/day, with a stronger response at 40 mg/kg/day. However, the increase observed at the lower dose was small and did not exceed the laboratory s historical vehicle-control range ( % MN-RET). In hindsight, this chemical may not have been detected as positive in this endpoint had a true MTD been used. The result for DMN is therefore considered as equivocal for this endpoint. 4. Discussion Half of the chemicals tested produced positive or equivocal responses for both MN and CMT endpoints. Therefore, either endpoint could have been chosen for these chemicals. However, the

10 D.E. Bowen et al. / Mutation Research 722 (2011) Table 5 Peripheral blood micronucleus formation determined by flow cytometry, 3, 24 and 44 h after a triple dose of various known mutagens. Chemical Dose [mg/kg/day] Sampling time: 3 h post final dose Sampling time: 24 h post final dose Sampling time: 44 h post final dose No. RETs scored [10 3 ] %RET %MN RET ± SD No. RETs scored [10 3 ] %RET %MN RET ± SD No. RETs scored [10 3 ] %RET %MN RET ± SD 2AAF ± ± ± ± 0.04 (NS) ± ± ± 0.40 (NS) ± ± ± 0.04 (NS) ± ± 0.06 (Linear trend: **) B[a]P ± ± ± 0.93 a ± 0.07 (NS) ± ± ± 0.03 (NS) ± ± ± 0.03 (NS) ± ± 0.06 CBZ ± ± ± ± 0.04 (**) ± ± ± 0.08 (**) 25 b b 31 b b ± 0.16 (**) 21 b b 21 b b CPA ± ± ± ± 0.34 (**) ± ± b b 0 b b 0 b b 40 2 b b 0 b b 0 b b (Linear trend: n/a) DMN ± ± ± ± 0.03 (*) ± ± ± 0.07 (**) b b b b EMS ± ± ± ± 0.18 (**) ± ± ± 0.22 (**) 12 b b 3 b b ± 0.38 (**) 2 b b 1 b b ENU ± ± ± ± 0.03 (**) ± ± ± 0.35 (**) ± ± ± 0.45 (**) ± ± 0.18 MMC ± ± ± ± 0.39 (**) ± ± ± 1.00 (NS) ± ± ± 1.28 (NS) ± ± 5.68 (Linear trend: *) a Possible cross contamination during storage, data included for information purposes. b Insufficient RET available for analysis due to toxicity. No RET scored [10 3 ] values rounded to nearest 1 3 Percentage RET 95% confidence interval %. Percentage MN RET 95% confidence interval %. Values in parenthesis refer to statistical analysis undertaken for the 3 h sampling time. NS not significant, *P < 0.05, **P < 0.01, ***P <

11 16 D.E. Bowen et al. / Mutation Research 722 (2011) 7 19 remaining four chemicals were clearly positive only for one of the endpoints: 2AAF and B[a]P gave uniquely positive, unequivocal CMT responses, and CBZ and MMC gave uniquely positive, unequivocal MN responses. These chemicals really define the benefits of the combination approach. The eight chemicals selected for this study are all genotoxic in vitro, but have different profiles of activity for genotoxicity and carcinogenicity in vivo. The pattern of results obtained in this combination study may therefore be explained by a clearer understanding of the issues surrounding their metabolism and tissue specificity. Results with some of the chemicals studied are therefore discussed in more detail below AAF Our initial work on the hepatotoxin 2AAF (dosed orally by gavage to Han Wistar rats at mg/kg/day) indicated a negative result for both CMT and MN endpoints (data not reported). This was initially surprising, as published data [21] have shown that a single oral dose of 2AAF induced weak increases in the frequency of micronuclei. We therefore tested this chemical again, changing the route of administration from oral to intraperitoneal (as a means of increasing exposure) and strain of rat (from Han Wistar to Sprague Dawley). This resulted in increased DNA damage in the liver, which corresponds with one of the known primary tumour sites in the rat, i.e. liver, mammary gland and skin [22]. There was also an equivocal induction of PB-MN, i.e. three-fold increase observed in the peripheral blood, which was restricted to the intermediate dose and which marginally exceeded the laboratory s historical control range; this increase, however, was not statistically significant. Whilst it is known that strain differences do occur [23], it is unknown whether the results observed here were due to the change in route of administration and/or the change in strain. Work by Ashby and Beije [21] confirmed that 2AAF gave a weak positive response in the bone-marrow micronucleus assay in rats following a single oral dose, whereas intraperitoneal injection failed to induce a significantly increased number of MN (Tzros et al., (1978) cited in Maier and Schawakder [24]). To understand the results obtained with 2AAF in this evaluation study with those previously reported, it is important to consider the metabolism of 2AAF in vivo. DNA-reactive metabolites are formed following N- hydroxylation, and the resultant N-hydroxy-AAF may give rise to reactive nitrenium ions. The production of this metabolite (via CYP450) is increased nine-fold following repeated administration of 2AAF compared with a single administration [25]. The DNA adduct formed has at some point lost the acetyl group, which would further suggest that deacetylation is a prerequisite for mutagenesis (Fig. 3). In contrast to the conclusion drawn by Maier and Schawalder [24], repeated intraperitoneal administration would suggest an enhanced formation of a liver-mediated stable 2AAF intermediate. Whilst it is known that the liver metabolite formed is locally stable and highly reactive, the failure to induce MN in the bone marrow or PB (away from the site of contact) is not perhaps surprising.. However, although in this study the CMT cloud data did not indicate any evidence of toxicity, tissue samples were not examined for histopathological changes. As such, it cannot be confirmed whether the positive CMT response observed was linked to toxicity in the liver. Further investigations on this point are to be conducted and reported separately. Fig. 3. Metabolism of 2AAF to the reactive nitrenium ion (not shown is the formation of the carbonium ion from either N-hydroxyaminofluorene (N-hydroxy-AF) or N- hydroxy-aaf. DA, deacteylation). route from oral to intraperitoneal dosing resulted in positive CMT data in samples from the stomach. The primary tumour site in the rat is reportedly the forestomach [22]. B[a]P undergoes a wide variety of metabolic transformations catalysed by CYP450. Formation of the ultimate carcinogen (B[a]P- 7,8-dihydrodiol-9,10-epoxide) is thought to be the critical step in the carcinogenesis of B[a]P (Fig. 4) although it is known that this pathway can be saturated. Other metabolites of B[a]P (3,6-quinone and semiquinone), whilst being both cytotoxic and mutagenic, can inhibit CYP450-mediated oxidation of B[a]P preventing formation of the diol-epoxide ultimate carcinogen [25] BaP Our initial experimental work conducted with B[a]P (dosed via oral gavage to Han Wistar rats at mg/kg/day) yielded negative results for all endpoints (data not reported). Alteration of the Fig. 4. Metabolism of BaP to the ultimate carcinogen. Adapted from [25].