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`The International Journal of Toxicology publishes timely, peer-reviewed papers on current topics important to
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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Safety Pharmacology and Genotoxicity
`Evaluation of AVl-4658
`
`Peter Sazani,' Doreen L. Weller, 1 and Stephen B. Shrewsbury 1
`
`International Journal of Toxicology
`29(2) 143-156
`© The Author(s) 2010
`Reprints and permission:
`sagepub.com/journalsPermissions.nav
`DOI: I0. 1177/1091581809359206
`http://ijt.sagepub.com
`®SAGE
`
`Abstract
`Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations. Restoration of dystrophin by exon skipping was
`demonstrated with the phosphorodiamidate morpholino oligomers (PMO) class of splice-switching oligomers, in both mouse and
`dog disease models. The authors report the results of Good Laboratory Practice-compliant safety pharmacology and
`genotoxicity evaluations of AVl-4658, a PMO under clinical evaluation for DMD. In cynomolgus monkeys, no test article-related
`effects were seen on cardiovascular, respiratory, global neurological, renal, or liver parameters at the maximum feasible dose
`(320 mg/kg). Genotoxicity battery showed that AVl-4658 has no genotoxic potential at up to 5000 ~1g/mL in an in vitro mamma(cid:173)
`lian chromosc;,me aberration test and a bacterial reverse mutation assay. In the mouse bone marrow erythrocyte micronucleus
`test, a single intravenous injection up to 2000 mg/kg was generally well tolerated and resulted in no mutagenic potential. These
`results allowed initiation of systemic clinical trials in DMD patients in the United Kingdom.
`
`Keywords
`Exon skipping, phosphorodiamidate morpholino oligomer, genotoxicity, antisense oligonucleotide, safety pharmacology
`
`in every
`Duchenne muscular dystrophy (DMD) affects I
`3500 male and, in rare cases, female newborns worldwide 1
`and results from mutations of the dystrophin gene. Lack of
`dystrophin leads to reduced sarcolemmal stability with actin
`intracellular calcium
`increased
`filament contraction and
`influx followed by muscle fiber degeneration. The clinical
`effect of a disrupted reading frame in the dystrophin gene is
`dramatic and lethal. 1•2 In DMD patients, the first symptoms
`involve the lower limbs'and appear between the third and fifth
`year. These boys develop hypertrophic calves, show difficulty
`in running and climbing stairs, run on their tiptoes, and
`frequently fall. Muscle weakness progresses to the shoulder
`girdle upper arm and trunk muscles and loss of ambulation
`before the age of 12 years. Histological changes are readily
`apparent with light microscopy analysis of cross sections from
`patient muscle biopsies. They involve variation in fiber size
`with atrophic and hypertrophic fibers, degeneration and
`regeneration of the muscle fibers, infiltration of inflammatory
`cells and fibrosis, and characteristic central location of the
`nuclei within muscle cells. The fiber membrane destabiliza(cid:173)
`tion results in leakage of the enzyme creatine kinase (CK),
`resulting in _very high serum CK levels (20 000 to 50 000
`U/L compared with 80 to 250 U/L in unaffected individuals).
`These levels decline as the patients get older, and the overall
`muscle mass decreases progressively. One-third of all
`affected boys are mentally impaired, and learning difficulties
`arc common. Due primarily to the loss of muscle strength and
`integrity, DMD patients usually die in their 20s from cardior(cid:173)
`espiratory failure. 1 •2
`
`Despite extensive effort, no effective disease-modifying
`therapy for DMD is yet available. However, a delay of the
`onset of disease manifestations and an improvement in quality
`of life can be achieved by drugs that decelerate progression of
`the DMD pathology. These include glucocorticoids,3 most
`commonly prednisolone and deflazocort, which can improve
`muscle strength and delay loss of ambulation by up to 2 to
`3 years. 1 The mechanisms of their beneficial effect arc not well
`include anti-inflammatory activity,
`likely
`understood but
`which may prevent the additional damage caused by the infil(cid:173)
`tration of mononuclear cells into the muscle upon necrosis.4
`However, the benefits of glucocorticoid therapy come at a price
`of frequent side effects, which can include obesity, spine defor(cid:173)
`mities, bone loss, and growth retardation. 5·6 To date, the most
`effective treatment for prolonging the life of DMD boys has
`been assisted ventilation with portable ventilators. Ventilation
`has been shown to increase the average life expectancy of
`DMD boys from 19 to 24 years. All of the above treatments are
`palliative and do not address the underlying cause of the dis(cid:173)
`ease: loss of dystrophin expression. No current treatment
`reverses or arrests the progression of DMD.
`
`1 AVI BioPharma, Bothell, WA
`
`Corresponding Author:
`Peter Sazani, 3450 Monte Villa Parkway, Bothell, WA 98021
`Email: psazani@avibio.com
`
`

`

`144
`
`International Journal of Toxicology 2 9(2)
`
`In addition to DMD, a milder, allelic form of muscular dys(cid:173)
`trophy called Becker muscular dystrophy (BMD) exists. 1
`2 In
`•
`BMD, unlike DMD, the reading frame is not disrupted, and
`an internally truncated yet functional dystrophin protein is pro(cid:173)
`duced. Most BMD patients remain ambulant for life and have a
`near-normal life expectancy. Work by Kole and others pro(cid:173)
`posed the use of antisense oligonucleotides to modulate dystro(cid:173)
`phin mRNA splicing and convert out-of-frame DMD mutations
`into the nearest in-frame BMD-like mutation, to produce
`an internally deleted Becker-like functional dystrophin pro(cid:173)
`tein. 7-10 The mechanism of splice-switching oligomer (SSO)
`modulation of dystrophin pre-mRNA splicing involves hybridi(cid:173)
`zation to specific motifs involved in splicing and exon recogni(cid:173)
`tion in the pre-mRNA. This prevents normal spliceosome
`assembly and results in skipping of the target exon in the
`mature RNA transcript. 11
`12 In the case of out-of-frame dystro(cid:173)
`•
`phin gene deletions, selective removal of specific flanking
`exons should result in in-frame mRNA transcripts that may
`be translated into an internally deleted, BMD-like, and
`functionally active dystrophin protein.9· 10
`Unmodified DNA and RNA oligonucleotides have poor in .
`vivo stability and therefore are ineffective as drugs. The most
`common chemical modification used in early-generation oligo(cid:173)
`nucleotides to improve the stability and pharmacokinetics was
`the introduction of the phosphorothioate linkage in place of the
`natural phosphodiester linkage. These phosphorothioate com(cid:173)
`pounds have been used extensively in both preclinical and clin(cid:173)
`ical evaluations, and the dose-limiting toxicities and adverse
`effects are well established. 13 Initial evaluations in primates led
`to mortality following intravenous bolus injections of phos(cid:173)
`phorothioate oligonucleotides at doses as low as IO mg/kg, 14
`while other effects included lethargy, central hypotension, and
`reduced cardiac output. Other notable toxic effects associated
`with phosphorothioate oligonucleotides include complement
`activation and prolonged coagulation times. 15 The latter, spe(cid:173)
`cifically linked to the phosphorothioate component of the oli(cid:173)
`gonucleotides, is therefore considered a class effect, 16 as
`were observed hepatotoxic effects. 17 These dose-limiting toxi(cid:173)
`cities are most likely due to high Cmax achieved with bolus
`injections. Studies subsequent to those in which mortality was
`observed have typically been limited to lower doses and
`increased infusion times to reduce the toxic effects. 13
`To be effective in modulating splicing, SSOs must not
`activate RNA cleavage by RNasc 1-1, which would destroy the
`pre-mRNA before splicing can occur. 18 Phosphorodiamidate
`morpholino oligomers (PMOs) productively compete with the
`splicing factors for target sequences in prc-mRNA during spli(cid:173)
`cing, and in addition, their stability and in vivo uptake and
`bioavailability arc improved compared with natural oligonu(cid:173)
`cleotides. PMOs have standard nucleic acid bases attached to
`the morpholino-phosphoroamidatc backbone (Figure
`I),
`which, unlike other sugar-phosphate backbone oligonuclco(cid:173)
`tides, is uncharged. 19 PMOs are very resistant to enzymatic
`degradation in vivo, providing unparalleled stability and some(cid:173)
`what different biodistribution than other oligonucleotides.
`Other modifications that can be used as SSOs include
`
`previous-generation chemistries such as the 2' -O-substituted
`2'-O-methyl phosphorothioate (2'OMe), and locked nucleic
`acids/phosphorothioate. 20
`A VI-4658 is a PMO dmg with the general structure
`described in Figure I, with the base sequence CTC CAA CAT
`CAA GGA AGA TGG CAT TTC TAG. It is designed to skip
`exon 51 of human dystrophin and thus restore dystrophin
`expression in DMD patients having certain mutations.21 A VI-
`4658 targets the pre-mRNA transcripts of the dystrophin gene,
`causing exon 51 to be skipped from the mature, spliced mRNA.
`In cells from DMD patients with deletions in exons 50, 52,
`52-63, 45-50, 48-50, or 49-50, exon skipping restored or is
`expected to restore the reading frame and produce an internally
`truncated, BMD-like form of dystrophin. Here, we report the
`results of a safety pharmacology evaluation of the PMO
`A VI-4658 in cynomolgus monkeys following intravenous and
`subcutaneous administration at doses up to the maximum
`feasible dose of 320 mg/kg. We also report the results of a
`standard genotoxicity battery evaluation using A VI-4658 at
`concentrations up to 5000 ~tg/mL in an in vitro mammalian cell
`chromosome aberrations test, up to 5000 ~1g/plate in a bacterial
`reverse mutation assay, and up to 2000 mk/kg as a single
`intravenous administration in a mouse micronucleus assay.
`
`Materials and Methods
`The safety pharmacology evaluation was performed by MDS
`Pharma Services (Lyon, France). The testing facility is Associ(cid:173)
`ation for Assessment and Accreditation of Laboratory Animal
`Care International (AAALAC) accredited, and the study plan
`was reviewed by the ethical committee, according to the fol(cid:173)
`lowing animal health and welfare guidelines: guide for the care
`and use of laboratory animals, NRC, 1996, Decree no. 200 I-
`464 regarding the experiments with
`laboratory animals
`described in the Journal Officiel de la Rc!pttblique Fral1(;aise
`on May 29, 2001, Decree no. 2001-486 relating to the protec(cid:173)
`tion of animals used in scientific experiments described in the
`Journal Officiel de la Republique Fran9aise on June 6, 2001.
`The study was conducted according to the following: guideline
`on safety pharmacology studies for human phannaceuticals
`(November 8, 2000, issued as CPMP/ICJ-1/539/00-ICJ-I S7 A,
`published in the Federal Register, vol 66, no. 135, July 13,
`200 I, pp 36791-36792) and guideline on nonclinical evaluation
`of the potential for delayed ventricular repolarization (QT
`interval prolongation) by human pharmaceuticals (May 12,
`2005, issued as CPMP/ICI 1/423/02-ICI-I S7l3, published in the
`Federal Register, vol 70, no. 202, October 20, 2005, pp 61133-
`61134). All phases of this study performed at the testing facili ty
`were conducted in compliance with the following Good
`Laboratory Practice (GLP) regulations: OECD Principles cf
`Good laboratory Practice concerning mutual acceptance of
`data in the assessment of chemicals, dated November 26,
`1997, (C[97] 186 Final), "Principles of Good Laboratory Prac(cid:173)
`tice" described in the French Official Journal on March 23,
`2000, Organization for Economic Co-operation and Develop(cid:173)
`ment (OECD) GLP consensus document (the application of the
`
`

`

`Sazani et al
`
`145
`
`[ 5' ]
`
`I ,,-NMe2
`
`Base=
`
`J:~
`C=ll .. A
`N
`I
`'VVVVV
`
`0
`
`[ 3' ]
`
`0
`
`0
`
`N i :
`
`T = ll .. A
`I ✓.}H
`G = {
`N N~NH
`N
`0
`~ 2 ~
`
`Me~NH
`
`Figure I. Structure of phosphorodiamidate morpholino oligomers.
`
`OECD principles of GLP to the organization and management
`ofmultisites studies, ENV/JM/MONO [2002]9, June 25, 2002).
`The genotoxicity battery was performed by BioReliance
`(Rockville, MD). This study was conducted in compliance with
`the most recent version of the US Food and Drug Administra(cid:173)
`tion GLP regulations, 21 CFR part 58, and the OECD Princi(cid:173)
`ples of Good Laboratory Practice, C(97) 186/Final, and in
`compliance with the testing guidelines ICH S2A (1996), ICH
`S2B (1997), and OECD 474 (1998). The number of mice and
`the procedures and experimental design used for this study
`have been reviewed and were approved by the BioReliance
`Institutional Animal Care and Use Committee 8 and 10. All
`procedures involving mice performed at BioReliance follow
`the specifications recommended in The Guide for the Care
`and Use of Laboratory Animals (National Academy Press,
`Washington, DC, 1996). The mice were housed in an
`AAALAC-accredited facility.
`
`Safety Pharmacology Evaluation of AV/-4658
`Animals and animal husbandry. Six male cynomolgus mon(cid:173)
`keys (Macaca fascicularis) were used in this study, with a
`weight range of 2.7 to 2.9 kg and an age range of 2 to 3 years.
`Animals were housed in I room for the study in an air(cid:173)
`conditioned building with a target temperature of 22°C ±
`2°C, relative humidity >40%, a minimum IO air changes per
`hour, and 12 hours light (artificial)/12 hours dark. Animals
`were housed singly in stainless steel mesh cages. Animals were
`fed expanded complete commercial primate diet at approxi(cid:173)
`mately I 00 g diet/animal per day. In addition, animals received
`fruit or vegetable daily (apple, banana, or carrot). Certificates
`of analysis for the diet and drinking water are maintained in the
`archives of the testing facility, which conducted the tests
`according to current animal welfare guidelines. The normal
`dark cycle was interrupted on occasions (for up to 45 minutes)
`
`

`

`146
`
`Table I. Study Design
`
`Treatment Group
`
`Dose Level, Number of
`mg/kg
`Animals
`
`Dose Volume,
`mUkg
`
`Phase I, subcutaneous
`dosing
`Vehicle (PBS)
`0
`Low dose
`40
`Intermediate dose 160
`High dose
`320
`Phase 2, intravenous
`dosing
`Vehicle (PBS)
`High dose
`
`0
`320
`
`6
`6
`6
`6
`
`6
`6
`
`Abbreviation: PBS, phosphate-buffered saline.
`
`2.67
`2.67
`2.67
`2.67
`
`2.67
`2.67
`
`to allow completion of technical procedures. These differences
`were not considered to have affected the outcome of the study.
`
`Procedures for telemetry and venous catheter implantation.
`Animals were fasted for at least 15 hours before surgical
`procedures. For implantation of the telemetry device, each
`animal was premedicated with a subcutaneous injection of g\y(cid:173)
`copyrrolate (Robinul V, Vetoquinol SA; 0.01 mg/kg) and then
`anesthetized with an intramuscular injection of ketamine
`(Imalgene 500, Merial SAS; 15 mg/kg) and xylazine hydro(cid:173)
`chloride (Rom pun 2%, Bayer AG; 0. 7 mg/kg). In addition,
`local oropharyngeal anesthesia was provided with a spray of
`lidocaine chlorhydrate (Xylocaine 5 % Nebuliseur, AstraZe(cid:173)
`neca). The hair on the abdomen, the inguinal area, and the
`thorax was clipped. During surgery, the level of anesthesia was
`maintained with gaseous anesthetic (I% to 5% isoflurane in
`oxygen, AErrane, Laboratoire Baxter). An antibiotic prophy(cid:173)
`laxis by intramuscular injection with long-acting amoxycillinc
`(Clamoxyl LA, Pfizer Italia SRL) at 30 mg/kg was given 48
`hours before surgery. The transmitter body was implanted into
`the abdominal cavity under aseptic conditions. The pressure
`catheter (polyurethane tubing that extends out of the device
`body) was inserted into the lower abdominal aorta via the
`femoral artery and the bipotential leads then placed. Animals
`received antibiotic prophylaxis by intramuscular injection with
`long-acting amoxycilline (Clamoxyl Li\, Pfizer; 30 mg/kg)
`and an analgesic prophylaxis intramuscular injection of tol fc(cid:173)
`namic acid (4% Tolfcdine, Vctoquinol; 4 mg/kg), once right
`after implantation and then repeated 4 times at 48-hour inter(cid:173)
`vals. The surgical wounds were disinfected with iodine (Vctc(cid:173)
`dine, Vetoquinol) for 7 days. After surgery, animals were
`allowed to recover for approximately 3 weeks before the first
`treatment. Prior to intravenous dosing, the animals were
`implanted with a venous catheter, under anesthesia similar to
`that above. The catheter was attached to the delivery system via
`a tether and a swivel joint.
`
`Safety examinations. Arterial blood pressure, heart rate, elec(cid:173)
`trocardiogram, respiratory parameters, global neurological
`activity, and renal and liver functions were examined following
`
`International Journal of Toxicology 29(2)
`
`3 separate subcutaneous administrations and 1 single intrave(cid:173)
`nous administration of A VI-4658 in the conscious male cyno(cid:173)
`molgus monkey. All observations,
`including respiratory
`parameters, were collected from the freely moving animal.
`A VI-4658 was administered by the subcutaneous route (phase
`1) at O (vehicle), 40, 160, and 320 mg/kg on days 0, 7, 14, and
`21 and by the intravenous route (phase 2) at O (vehicle) and
`320 mg/kg on days 38 and 45 using a dosing volume of
`2.67 mL/kg with an infusion rate of I mL/min (Table I). Sub(cid:173)
`cutaneous and intravenous dosing were examined to support
`both routes for potential clinical administration. The high dose
`(320 mg/kg) was used to support up to 100 mg/kg clinically,
`based on allometric scaling. For intravenous dosing, 320 mg/
`kg was the maximum feasible dose based on dose volume and
`solubility of the compound. The low dose for phase l was
`selected based on the expected therapeutic dose (human equiv(cid:173)
`alent dose of approximately l O mg/kg). For phase l, animals
`were randomized in a Latin square design. For both phases,
`there were at least 6 days between each testing session. Each
`animal served as its own control.
`Telemetry signals (body temperature, cardiovascular and
`respiratory parameters) were recorded. Time points were
`selected to correspond to the times of maximum exposure to the
`drug:
`
`• phase I: on days 0, 7, 14, and 21, starting at least 1.5 hours
`preadministration and for 21 hours postadrninistration
`• phase 2: on days 38 and 45, starting at least 1.5 hours pre-
`administration and for 24 hours postadministration
`
`The telemetric system used consisted of an implantable
`TL! 1M3-O70-CCTP device (OSI, St Paul, MN), an RMC-1
`receiver located on the top of each cage, a DEM data exchange
`matrix that centralizes the signals from all animals, an APR- I
`ambient pressure reference that allows a barometric correction,
`and a microcomputer with acquisition card. For collecting and
`analyzing hemodynamic, cardiac, and respiratory parameters,
`Notocord-hem software (Notocord Systems SA, Croissy-sur(cid:173)
`Seine, France) was used.
`Por cardiovascular analyses, the value for each parameter
`(systolic blood pressure, mean arterial blood pressure, diastolic
`blood pressure, and heart rate) was the mean of the values
`recorded for 5 minutes around the time point (selected times
`± 2.5 minutes). The value for each interval or complex (PR,
`RR, QT, or QRS) was the mean of the IO best quality electro(cid:173)
`cardiogram (ECG) traces at the time point (selected time ± 2.5
`minutes). The value for respiration rate (f), inspiratory time
`(Tl), expiratory time (TE), nnd AUCEMO was the mean of I 0
`values obtained around the time point (selected time ± 2.5 min(cid:173)
`utes). Note that the signals recorded nt the predefined time
`points were occasionally disturbed. In this case, data recorded
`a few minutes before or a few minutes after the theoretical time
`point were used instead. On a few occasions, electromyogram
`(EMG) parameter values were calculated from fewer than I 0
`values. In another few cases, sustained disturbances of the sig(cid:173)
`nal did not allow an accurate evaluation of any data at the
`
`

`

`Sazani et al
`
`147
`
`predefined time point ( detailed in the raw data). The results
`were expressed as mean ± standard error of the mean (SEM).
`For respiration parameters, inspiratory time (Tl, millise(cid:173)
`conds) was defined as the duration of the diaphragmatic
`EMG burst. Expiratory time (TE, milliseconds) was defined
`as the time elapsed between 2 diaphragmatic EMG bursts,
`and respiration rate (f, breath/min) was calculated from the
`averaged respiratory cycle duration (Tl + TE). The AUC of
`the rectified diaphragmatic EMG burst (AUCEMo) is that for
`which variation in amplitude has been shown to be corre(cid:173)
`lated in humans and animals with variation of the tidal vol(cid:173)
`ume.22·24 For this evaluation, the raw EMG signal is filtered
`and rectified. Moreover, AUCEMG is normalized such that
`AUCEMG values are expressed as a percentage change from
`the pretest value.
`For neurological evaluations, all animals were examined at
`pretest and approximately 4 hours and 8 hours after treatment.
`Evaluated parameters included level of consciousness, motor
`function, and eye movements. Normal versus abnormal results
`were recorded and graded for each major parameter at each
`time point. All results for all animals were normal and noted
`as follows: level of consciousness: -1 = alert (nonnal); motor
`function: -1 = normal; eye movements: -1 = nonnal fixation
`and following of stimulus. Hepatic function evaluation, hema(cid:173)
`tology, and other clinical chemistry analyses were perfonned
`on day-4 and day 24. Renal function evaluation and urine anal(cid:173)
`ysis were performed on day -3 and day 25.
`
`Genotoxicity Evaluation of AVl-4658
`Bacterial reverse mutation assay. The tester strains used were
`typhimurium histidine auxotrophs T A98,
`the Salmonella
`TA 100, TA 1535, and TA 153 7 as described by Ames et al25 and
`Escherichia coli WP2 uvrA as described by Green and
`Muriel.26 Salmonella
`received from
`tester strains were
`Dr Bruce Ames' designated distributor, Discovery Partners
`International (San Diego, CA). The E coli tester strain was
`received from the National Collection of Industrial and Marine
`Bacteria (Aberdeen, Scotland). Overnight cultures were pre(cid:173)
`pared by inoculating from the appropriate master plate or from
`the appropriate frozen pennanent stock into a vessel containing
`~ 50 mL of culture medium. To ensure that cultures were har(cid:173)
`vested in the late log phase, the length of incubation was con(cid:173)
`trolled and monitored. Aroclor 1254-induced rat liver S9 was
`used as the metabolic activation system. The S9 was prepared
`from male Sprague-Dawley rats induced with a single intraper(cid:173)
`itoneal injection of Aroclor 1254, 500 mg/kg, 5 days prior to
`sacrifice. The S9 was prepared by and purchased from MolTox
`(Boone, NC). Upon arrival at BioReliance, the S9 was stored at
`-60°C or colder until used. Each bulk preparation of S9 was
`assayed for its ability to metabolize at least 2 promutagens to
`forms mutagenic to S typhimuriwn TA I 00. The S9 mix was
`prepared immediately before its use and contained 10% S9,
`5 mM glucose-6-phosphate, 4 mM P-nicotinamide-adenine
`dinucleotide phosphate, 8 mM MgC12, and 33 mM KC! in a
`100 mM phosphate buffer at pH 7.4. The sham S9 mixture
`
`(sham mix), containing 100 mM phosphate buffer at pH 7.4,
`was prepared immediately before its use. To confirm the steri(cid:173)
`lity of the S9 and sham mixes, a 0.5-mL aliquot of each was
`plated on selective agar.
`In the initial toxicity-mutation assay, the maximum dose of
`A Vl-4658 tested was 5000 ~•g per plate; this dose was achieved
`using a concentration of 50 mg/mL and a 100-~,L plating ali(cid:173)
`quot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500,
`1500, and 5000 µg per plate. The test article formed soluble and
`clear solutions in sterile water for injection from 0.015 to
`50 mg/mL. Neither precipitate nor background lawn toxicity
`was observed. No positive mutagenic responses were observed
`with any of the tester strains in either the presence or absence of
`S9 activation.
`Based on the findings of this initial toxicity-mutation assay,
`the maximum dose plated in the confirmatory mutagenicity
`assay was 5000 ~1g per plate. For the confinnatory mutagenicity
`assay, the dose levels tested were 50,150,500, 1500, and 5000
`~lg per plate.
`On the day of its use, minimal top agar, containing 0.8 %
`agar (W/V) and 0.5 % NaCl (W/V), was melted and supple(cid:173)
`mented with L-histidine, D-biotin, and L-tryptophan solution
`to a final concentration of 50 ~1M each. Top agar not used with
`S9 or sham mix was supplemented with 25 mL of water for
`each 100 mL of minimal top agar. For the preparation of media
`and reagents, all references to water imply sterile, deionized
`water produced by the Milli-Q Reagent Water System. Bottom
`agar was Vogel-Bonner minimal medium E (Vogel and
`Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom
`agar was Vogel-Bonner minimal medium E containing 1.5 %
`(W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutri(cid:173)
`ent Broth No. 2 (dry powder). Nutrient Broth was Vogel(cid:173)
`Bonner salt solution supplemented with 2.5% (W/V) Oxoid
`Nutrient Broth No. 2 (dry powder). Each plate was labeled with
`a code system that identified the test article, test phase, dose
`level, tester strain, and activation, as described in detail in
`BioReliance's Standard Operating Procedures. One-half
`(0.5) milliliter of S9 or sham mix, 100 ~1L of tester strain, and
`100 ~LL of vehicle or test article dilution were added to 2.0 mL
`of molten selective top agar at 45°C ± 2°c. After vortexing,
`the mixture was overlaid onto the surface of 25 mL of minimal
`bottom agar. When plating the positive controls, the test article
`aliquot was replaced by a 50-~tL aliquot of appropriate positive
`control. After the overlay had solidified, the plates were
`inverted and incubated for approximately 48 to 72 hours at
`37°C ± 2°c. Plates that were not counted immediately follow(cid:173)
`ing the incubation period were stored at 2°C to 8°C until colony
`counting could be conducted.
`The condition of the bacterial background lawn was evalu(cid:173)
`ated for evidence of test article toxicity by using a dissecting
`microscope. Precipitate was evaluated after the incubation
`period by visual examination without magnification.
`Revertant colonies for a given tester strain and activation
`condition, except for positive controls, were counted either
`entirely by automated colony counter or entirely by hand unless
`the plate-exhibited toxicity.
`
`

`

`148
`
`International Journal o( Toxicology 29(2)
`
`Table 2. Mouse Bone Marrow Micronucleus Test Design
`
`Number of Mice/Sex
`
`Number of
`Mice/Sex
`Dosed
`
`24 Hours 48 Hours
`Postdose Postdose
`
`Treatment
`
`The chromosome aberration assay was performed using
`standard procedures.29 The CHO cells were seeded and treated
`as above. In the absence of both test article precipitation in the
`treatment medium and at least 50% toxicity, the highest dose
`level evaluated was 5000 ~tg/mL. After the exposure period, the
`treatment medium was removed, the cells washed with CMF(cid:173)
`PBS, and refed with complete medium. Two hours prior to the
`scheduled cell harvest, Colcemid was added to duplicate flasks
`for each treatment condition at a final concentration of 0.1 ~1g/
`mL, and the flasks were returned to the incubator until cell col(cid:173)
`lection. A concurrent toxicity test was conducted in both the
`nonactivated and the S9-activated test systems. Two hours after
`the addition of Colcemid, metaphase cells were harvested for
`both the nonactivated and S9-activated studies by trypsiniza(cid:173)
`tion. Cells were collected and chromosome sample slides pre(cid:173)
`pared and analyzed as described previously.30 Slides were
`coded using random numbers by an individual not involved
`with the scoring process. Mitomycin C was used as the positive
`control in the nonactivated study at final concentrations of 0.1
`and 0.2 µg/mL. Cyclophosphamide was used as the positive
`control in the S9-activated study at final concentrations of I 0
`and 20 ~tg/mL.
`
`Mouse bone marrow micronucleus test. ICR mice were
`obtained from Harlan (Freder