Pig-a

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335
Mutagenesis, 2015, 335–342
doi:10.1093/mutage/gev013
Original Article
Advance Access publication 1 May 2015
Original Manuscript
Integration of Pig-a, micronucleus, chromosome
aberration and comet assay endpoints in a 28-
day rodent toxicity study with urethane
Leon F. Stankowski, Jr1,
*, Marilyn J. Aardema1,2
,Timothy E. Lawlor1
,
Kamala Pant1
, Shambhu Roy1
,Yong Xu1
and Reem Elbekai1
1
BioReliance Corporation, Rockville, MD 20850, USA, 2
Marilyn Aardema Consulting LLC, Fairfield, OH 45014, USA
*To whom correspondence should be addressed. Tel: +1 240 447 2674; Fax: +1 301 738 2362; Email: leon.stankowski@bioreliance.com
Received October 1 2014; Revised January 21 2015; Accepted January 21 2015.
Abstract
As part of the international Pig-a validation trials, we examined the induction of Pig-a mutant
reticulocytes and red blood cells (RETCD59−
and RBCCD59−
, respectively) in peripheral blood of male
Sprague Dawley®
rats treated with urethane (25, 100 and 250 mg/kg/day) or saline by oral gavage
for 29 days. Additional endpoints integrated into this study were: micronucleated reticulocytes
(MN-RET) in peripheral blood; chromosome aberrations (CAb) and DNA damage (%tail intensity
via the comet assay) in peripheral blood lymphocytes (PBL); micronucleated polychromatic
erythrocytes (MN-PCE) in bone marrow; and DNA damage (comet) in various organs at termination
(the 29th dose was added for the comet endpoint at sacrifice). Ethyl methanesulfonate (EMS;
200 mg/kg/day on Days 3, 4, 13, 14, 15, 27, 28 and 29) was evaluated as the concurrent positive
control (PC). All animals survived to termination and none exhibited overt toxicity, but there were
significant differences in body weight and body weight gain in the 250-mg/kg/day urethane group,
as compared with the saline control animals. Statistically significant, dose-dependent increases
were observed for urethane for: RETCD59−
and RBCCD59−
(on Days 15 and 29); MN-RET (on Days 4,
15 and 29); and MN-PCE (on Day 29).The comet assay yielded positive results in PBL (Day 15) and
liver (Day 29), but negative results for PBL (Days 4 and 29) and brain, kidney and lung (Day 29). No
significant increases in PBL CAb were observed at any sample time. Except for PBL CAb (likely due
to excessive cytotoxicity), EMS-induced significant increases in all endpoints/tissues.These results
compare favorably with earlier in vivo observations and demonstrate the utility and sensitivity
of the Pig-a in vivo gene mutation assay, and its ability to be easily integrated, along with other
standard genotoxicity endpoints, into 28-day rodent toxicity studies.
Introduction
The induction and quantification of Pig-a mutant erythrocytes in
rodents were first described in 2008 (1–4). Since then, this assay has
been the subject of intensive study and global validation coordi-
nated by Litron Laboratories and the Genetic Toxicology Technical
Committee of the ILSI Health and Environmental Sciences Institute
(5–16). These studies indicated that the Pig-a endpoint was highly
transferrable and reproducible between laboratories, it could be eas-
ily integrated into standard toxicology studies, and that it held great
promise as an in vivo mutation assay (17,18).
Integration of genotoxicity endpoints into 28-day toxicity studies
is included in new regulatory guidelines and protocols, such as those
from the International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human Use
[ICH; (19)] and the Organization for Economic Co-operation and
Development [OECD; (20–22)], as a way to maximize genotoxicity
assessments while minimizing the use of animals. As part of these
continuing Pig-a validation trials, we examined the induction of
RETCD59−
and RBCCD59−
in peripheral blood of male Sprague Dawley®
rats treated with urethane for 29 consecutive days. For comparison,
MN-RET in peripheral blood, chromosome aberrations (CAb) and
byguestonJanuary18,2017http://mutage.oxfordjournals.org/Downloadedfrom

DNA damage (via the comet assay) in peripheral blood lymphocytes
(PBL), micronucleated polychromatic erythrocytes (MN-PCE) in
bone marrow and DNA damage in various organs were also evalu-
ated. In this study, we demonstrate how every standard in vivo geno-
toxicity endpoint, including Pig-a, can be integrated into standard
28-day toxicity studies.
Urethane is carcinogenic in multiple rodent species by various
routes (23–25), and we use it at BioReliance as a very effective PC in
ras H2 transgenic carcinogenicity studies (26). It is also mutagenic
and clastogenic in vivo, but generally is considered non-genotoxic
in vitro due to its unique metabolism requirements (24,25,27,29).
Metabolism of urethane requires CYP2E1 and carboxylesterase
isozyme hydrolase A and proceeds through vinyl carbamate and
conversion to an epoxide that can interact with DNA (29,30).
Support for this metabolic pathway comes from the observation that
urethane-induced MN-PCE frequencies are reduced in CYP2E1-null
mice (31). Thus, urethane represents a key test chemical to evaluate
the sensitivity and applicability of the Pig-a assay, particularly for
the detection of promutagens (18).
Materials and Methods
Reagents
Urethane (CAS no. 51-79-6) and ethyl methanesulfonate (EMS; CAS no.
62-50-0) were purchased from Sigma-Aldrich (St. Louis, MO). Saline
for injection (USP; saline) was purchased from B. Braun Medical (Irvine,
CA). Prototype MutaFlow®
Rat Pig-a Mutation Assay and MicroFlow®
Rat Blood Micronucleus Kits were supplied by Litron Laboratories
(Rochester, NY). Other reagents and supplies included Lympholyte®
-
Mammal cell separation media from Cedarlane (Burlington, NC);
Anti-PE MicroBeads, LS Columns and a QuadroMACS™ Separator
from Miltenyi Biotec (Bergisch Gladbach, Germany); CountBright™
Absolute Count Beads from Invitrogen (Carlsbad, CA).
Animals, treatment and harvests
The number of animals, procedures and design used for this study
were reviewed and approved by the BioReliance Institutional Animal
Care and Use Committee and followed the specifications recom-
mended in the most current version of The Guide for the Care and
Use of Laboratory Animals (32). In addition, the study was conducted
in compliance with Good Laboratory Practice regulations (33).
Five- to six-weeks old Sprague Dawley®
male rats (Harlan; Frederick,
MD) were singly housed in polycarbonate cages and acclimated for
11 days prior to the start of dosing. Certified Rodent Diet (Global Diet
#2018C; Harlan TEKLAD, Madison, WI) and water were provided ad
libitum. Sani-Chip Hardwood bedding (P.J. Murphy Forest Products,
Montville, NJ) was used to absorb liquids. Animals were housed in an
environmentally controlled room with temperatures between 21 and
24°C,a relative humidity of 30–70%,at least 10 changes of fresh HEPA-
filtered air every hour, and a 12-h light/12-h dark cycle. Animals were
7–8-weeks old at the start of dosing and weighed 176–225 g. General
daily clinical observations, as well as weekly detailed observations, were
performed for each animal. Body weight and food consumption were
monitored throughout the treatment period.
Animals were treated for 29 consecutive days with urethane at
doses of 25, 100 and 250 mg/kg/day, or the saline vehicle control (all
n = 10; Table 1). The EMS PC was administered at a dose of 200 mg/
kg/day on Days 3, 4, 13, 14, 15, 27, 28 and 29 (n = 6). The final
dose administered on Day 29 was necessary for the comet endpoint.
Urethane dose formulations were prepared twice (on Days 0 and
14), aliquoted for daily use, stored at 2–8ºC and maintained at room
temperature for at least 30 min prior to use. EMS dose formulations
were freshly prepared and maintained at room temperature until use.
All dose formulations were prepared in saline, corrected for potency
and administered by oral gavage using a 10-ml/kg dose volume, with
the first day of dosing designated as Day 1.
Blood and tissue collection
Blood for the various endpoints was collected from the retro-orbital
sinus under 70% CO2
/30% O2
anesthesia at the volumes and time
points indicated (Table 2). Each collection was ~2.5–3 h after the appro-
priate daily dose as is typical for the comet endpoint (22). Blood for the
comet and the CAb assays was collected directly into sodium heparin
tubes, whereas blood for the Pig-a and MN-RET assays was initially
drawn into an empty tube and then quickly aliquoted into the appropri-
ate volume of heparin (Litron kit supplied). Samples also were collected
for clinical pathology (Clin Path) analysis (hematology, clinical chemis-
try), as is standard for a 28-day rodent toxicity assay (not shown).
All animals were sacrificed by CO2
overdose on Day 29, immedi-
ately after the final blood collections, and a complete necropsy was
performed. The left kidney, and consistent portions of the left lateral
liver lobe, brain, spleen and lungs were collected for the comet assay.
Similarly, the left femur was reserved for bone marrow micronucleus
assessment. All remaining portions of these tissues, as well as numer-
ous other tissues and the entire carcass including animal identifica-
tion, were collected and fixed in 10% neutral buffered formalin for
histopathologic evaluation (as in a standard 28-day rodent toxicity
assay; data not shown).
Pig-a assay
Blood was processed, labeled and analysed using Prototype In
Vivo MutaFlow kits and instructions (version 120209, with minor
Table 1. Study design
Group Treatment Animals
per groupa
Dose level Dose formulation
(mg/kg/day) (mg/ml)
1 Saline 10 0.00 0.00
2 Urethane 10 25.0 2.50
3 Urethane 10 100 10.0
4 Urethane 10 250 25.0
5 EMS 6 200 20.0
a
Males only. Ten animals per group were treated with urethane or the ve-
hicle control (as per a standard 28-day rodent toxicity study). Eight animals/
group (or all from the EMS positive control group) initially were analysed
prior to dosing to determine the pre-dose Pig-a mutant frequencies. One ani-
mal from the 100 mg/kg/day urethane group was excluded from all subsequent
genetic toxicology endpoint analyses (due to a high spontaneous Pig-a mutant
frequency), and the first six animals remaining in each group were analysed for
all genetic toxicology endpoints.
Table 2. Sampling times and blood volumes (µl)a
Day N Pig-a MN-RET CAb Comet Clin path Total
−5 or −3 8 100 – – – – 100
4 6 – 150 250 100 – 500
15 6 100 150 250 100 – 600
29 6 100 150 250 100 800 1400
a
Blood was collected ~2.5–3 h after dosing, except for the pre-dose samples
on Days −3 or −5.
336 L. F. Stankowski et al., 2015, Vol. 30, No. 3

modifications) and with immunomagnetic enrichment (34). A stand-
ard template was used and data was acquired using a BD FACSCanto
II flow cytometer with FACSDiva Software (BD Biosciences; San
Jose, CA). Whenever possible, ≥100 million total RBCs and ≥3 mil-
lion RETs were analysed. For each animal, the total number of RET
and RBC equivalents analysed, as well as the number and frequency
of RETCD59−
and RBCCD59−
, were determined/calculated.
MN-RET assay
Blood was processed, labeled and analysed according to the instruc-
tion manual (version 120702) received with the MicroFlow®
Rat
Blood Micronucleus Kit. Approximately 20 000 RET were scored
for micronuclei, per animal and time point, where possible. The
same flow cytometer and software were used for data acquisition
and analysis.
MN-PCE assay
Bone marrow was aspirated by syringe, transferred to a centrifuge
tube containing cold fetal bovine serum (FBS), pelleted by centrifu-
gation, resuspended in a small volume of FBS and spread onto a
glass slide (2/animal). The slides were air dried for 24–72 h, fixed in
methanol and stained with acridine orange for microscopic evalua-
tion. Coded slides were scored by fluorescent microscopy at 1000×
magnification for the presence of micronuclei (2000 PCEs/animal;
12 000 per group), and for the proportion of PCEs to total eryth-
rocytes (PCE/EC ratio) as a measure of bone marrow cytotoxicity
(1000 total erythrocytes/animal).
Ex vivo PBL CAb assay
The ex vivo PBL CAb assay was performed and scored as pre-
viously reported (35). Briefly, whole blood was washed by cen-
trifugation using phosphate-buffered saline containing ~2% (v/v)
FBS and resuspended in a 10-fold volume of RPMI 1640 medium
containing 25-mM HEPES, 10% (v/v) heat inactivated FBS, 100-
units/ml penicillin, 100-μg/ml streptomycin, 2-mM L-glutamine
and 20-μg/ml phytohemagglutinin-P to stimulate lymphocyte
proliferation. Single cultures were established in 25-cm2
flasks
(one per animal) and incubated at 37 ± 2°C in a humidified atmos-
phere of ~5% CO2
in air. Cultures were grown for ~48 h, with
Colcemid®
(0.2 μg/ml) being added for the last ~2.5 h before har-
vest. Duplicate slides were prepared from each culture, stained
with Giemsa, air dried and coded, and 100 cells with 2n ± 2 chro-
mosomes were scored per animal for structural aberrations and
numerical aberrations, whereas 500 cells/ animal were evaluated
for mitotic index.
Comet assay
The alkaline comet assay was used to analyze all tissues using the
latest JaCVAM protocol (36,37). Peripheral blood samples were
centrifuged and resuspended in phosphate-buffered saline to pre-
pare comet slides. Liver, kidney, lung, brain and spleen were placed
in cold mincing buffer and finely minced with a pair of fine scis-
sors to release the cells. Each cell suspension was strained through
a 40-µm sieve into a 50-ml-conical tube and stored on wet ice until
the slides were prepared. Approximately 2.5–5 µl of cell suspension
were mixed with 75-µl 0.5% (w/v) low melting agarose and applied
to glass microscope slides (four slides/animal/tissue) that were previ-
ously coated with 1% (w/v) normal melting agarose (to minimize
sample degradation, dosing and necropsy was staggered and highly
choreographed, such that processing up to the application to the
slides was accomplished in ~15 min or less). The slides were kept at
2–8°C for at least 10 to 15 min to solidify.
Following solidification, the slides were submerged in cold lysis
solution (100-mM Na2
EDTA, 2.5-M NaCl, 10-mM Tris, 1% Triton
X-100 and 10% dimethyl sulfoxide; pH 10) for at least overnight
at 2–8°C. After lysis, slides were neutralized in 0.4-M Tris (pH
~7.5), transferred to the electrophoresis chamber which was slowly
filled with cold electrophoresis buffer (300-mM NaOH and 1-mM
Na2
EDTA; pH 13), and maintained for 20 min at 2–10°C, protected
from light, to allow the DNA to unwind. Electrophoresis was con-
ducted for 30 min at 0.7 V/cm, at 2–10°C and protected from light.
After electrophoresis, the slides were removed and washed with neu-
tralization buffer, dehydrated with 200-proof ethanol, air dried and
stored at room temperature with desiccant. Slides were stained with
Sybr-gold™ in Tris-boric acid- EDTA buffer.
Up to 50 randomly selected, nonoverlapping cells were scored
per slide, for a total of 100 cells evaluated per animal and tissue,
using the comet Assay IV scoring system (version 4.11; Perceptive
Instruments Ltd.; Bury St. Edmunds, UK). DNA damage was
assessed based upon %Tail DNA. Each slide was also examined for
indications of possible cytotoxicity (i.e. the percentage of “clouds” in
100 cells/slide, where possible).
Data analyses
Pig-a analyses were performed on log(10) transformed mutant
frequencies (RETCD59−
and RBCCD59−
), and untransformed %RETs,
using the individual animal as the unit of measure. An offset of 0.1
was also added to each mutant RET value prior to transformation
to avoid zero values. One-way analysis of variance was performed
(for mutant frequencies and %RETs), followed by Dunnett’s post
hoc analysis to compare each dose to the vehicle control. A pair-
wise t-test was performed to compare the saline and EMS PCs.
Significance for all statistical analyses was established at the P  0.05
or 0.01 levels using a two-tailed test.
The percent MN-RET and %RET per group were tested for nor-
mal distribution (Levene’s test, P  0.05), and log transformed if not
normally distributed. Urethane data were analysed using one-way
analysis of variance followed by Dunnett’s post hoc analysis. A pair-
wise t-test was performed to compare saline versus the EMS PC.
MN-PCE frequencies were tested for normal distribution
(Levene’s test, P  0.05; and log transformed if not normally distrib-
uted) and analysed using the binomial distribution (Kastenbaum–
Bowman tables). CAb data were analysed using Fisher’s exact test,
while the comet assay results (animal mean calculated from the
median %Tail DNA, 50 cells/slide per animal) were analysed as
described for MN-RET.
Body weight, food consumption and organ weight data were
analysed by Dunnett’s test.
Results
Survival and clinical observations
All animals survived to Day 29 and none exhibited overt signs of
toxicity due to urethane or EMS treatment (data not shown). The
dose level for the EMS PC is the same as we normally use in an
acute, stand-alone comet assay, or in a combination comet/micro-
nucleus study using three daily dose administrations. However, the
cumulative dose and bone marrow toxicity induced by the eight
administrations used here was clearly cytotoxic to the bone mar-
row, as evidenced by the %RET, PCE/EC, and CAb results below.
In contrast, there were no significant changes in Clin Path for the
Integration of Pig-a, micronucleus, chromosome aberration and comet assay endpoints, 2015, Vol. 30, No. 3 337

urethane-treated animals except for an increase in % monocytes at
a dose of 250 mg/kg/day.
Body weight, body weight change and food
consumption
Significant differences in body weight were noted for the 250-mg/kg/
day urethane treatment group, as well as the EMS PC group, starting
on Day 8 (P  0.05; not shown). In addition, significant differences
in body weight gain were noted during the first 2 weeks of the study,
for the same two groups. Absolute mean body weight gains over the
course of the study, as compared to the control animals, were 22.9%
and 51.4% lower, respectively (not shown). Group mean food con-
sumption values were lower in the 250-mg/kg/day urethane group,
as compared to the control animals, but the differences were not
significant (P 0.05; food consumption data were not collected for
the EMS PC group).
Pig-a assay
Statistically significant, dose-dependent increases in the frequency of
RETCD59−
were observed at doses of 100 and 250 mg/kg/day on Days
15 and 29 (P  0.05; Figure 1, Table 3). The maximum observed
RETCD59−
frequencies were ~74- and 24-fold vehicle control values
on Days 15 and 29, respectively (the relatively smaller increase
on Day 29 was due to the higher mutant frequency in the vehicle
control animals at the later time point). Significant increases in
RBC CD59−
also were observed at a dose of 250 mg/kg/day on Day
15, and at doses of 100 and 250 mg/kg/day on Day 29 (P  0.05;
Figure 2, Table 3). These increases reached maximums of ~4.2- and
8.1-fold control values on Days 15 and 29, respectively.
MN-RET assay
%RET, measured concurrently with the MN-RET endpoint, indi-
cated urethane and the EMS PC reached the bone marrow and
induced significant toxicity. Statistically significant decreases in
%RET were observed for urethane at a dose of 250 mg/kg/day on
Days 4 and 29 (but not Day 15), and for EMS on Days 15 and 29
(Figure 3, Table 3). The apparent lack of bone marrow toxicity for
urethane on Day 15 is curious and may reflect an initial compensa-
tory increase in erythropoiesis.
Statistically significant, dose-dependent increases in the frequency
of MN-RET, to ~4.0- to 6.6-fold control values, were observed
for urethane at all doses on Days 4 and 29, and doses of 100 and
250 mg/kg/day on Day 15 (P  0.05 or 0.01; Figure 4, Table 3). EMS
also induced significant increases in MN-RET at all time points;
these increases were larger at the later two time points, likely due
to the change in dose regimen (two vs. three daily doses before the
scheduled collection times).
MN-PCE assay
Significant, dose-dependent increases in MN-PCE were observed for
urethane-treated animals at terminal sacrifice at a dose of 250 mg/
kg/day (Figure 5; Table 3). EMS induced a much larger increase in
MN-PCE. In contrast to measurements in the peripheral blood, sig-
nificant decreases in PCE/EC ratios were only noted for EMS (not
shown).
Figure 1. Frequency of RETCD59−
induced by urethane or the EMS PC (all n = 6;
±1 SD). Statistically significant increases are indicated at P  0.05 and 0.01
levels (* and **, respectively). The 95% upper control limit for historical
negative control RETCD59−
data was 2.90 × 10−6
.
Table 3. Summary of results
Endpoint Maximum fold-increasea
Day 4 Day 15 Day 29
RETCD59−
nd 74 24
RBCCD59−
nd 4.2 8.1
MN-RET 6.6 4.0 5.3
MN-PCE nd nd 5.0
PBL cab – – –
Comet
PBL – 3.0 –
Brain nd nd –
Kidney nd nd –
Liver nd nd 1.5
Lung nd nd –
Spleen nd nd –
nd = not determined.
– = negative response.
a
Only the greatest observed increase, at any dose level or time point, is
reported; any numeric entry represents a statistically significant, positive re-
sponse (P  0.05).
Figure 2. Frequency of RBCCD59−
induced by urethane or the EMS PC (all
n = 6; ±1 SD). Statistically significant increases are indicated at P  0.05 and
0.01 levels (* and **, respectively).The 95% upper control limit for historical
negative control RBCCD59−
data was 1.39 × 10−6
.

Ex vivo PBL CAb assay
No significant increases were observed in structural or numerical
aberrations at any dose or sample time for the urethane-treated ani-
mals (results not shown, but summarized in Table 3). This lack of
response was clearly not due to a lack of exposure, since relative
mitotic indices decreased in a dose dependent manner and reached
relative mitotic indices of ~69, 47 and 53% at a dose of 250 mg/kg/
day on Days 4, 15 and 29, respectively. Cultures from EMS-treated
animals were unable to be scored due to insufficient metaphases,
likely as the result of excessive toxicity (relative mitotic indices were
~17, 10 and 9% of control values on Days 4, 15 and 29, respectively).
Comet assay
Statistically significant, dose-dependent increases in median %Tail
DNA were observed for urethane only in PBL at Day 15, and in liver
at terminal sacrifice (P  0.05; Figures 6 and 7; Table 3). Urethane
was considered to be negative in all other tissues and time points
analysed.The EMS PC induced significant increases in median %Tail
DNA in all tissues and at all sampling times (P  0.05). The percent-
age of clouds observed in the urethane-treated animals was compa-
rable to, or less than, that observed in the vehicle control animals,
for all tissues and time points (not shown). In contrast, increases in
the percentage of clouds in the EMS-treated animals ranged from
~2- to 4-fold (blood on Day 4 and brain, lung and spleen) to ~8-fold
or greater in the other tissues, and blood at the later time points (not
shown).
Discussion
One goal of this study was to validate the integration of the in
vivo Pig-a assay, as well as other genetic toxicology endpoints, into
a typical 28-day rodent toxicity assay in compliance with Good
Laboratory Practice regulations (33). As such, all of the typical body
and organ weight, cage-side and detailed observations, food con-
sumption and Clin Path procedures/analyses were performed (tissues
were collected for histopathology but not analysed due to the time
and expense involved). Only the results relevant to interpretation of
the effects of urethane are reported here.
Based on data in the literature, urethane was positive in most in
vivo cytogenetic and mutation studies in vivo (24,25,28). Results
from our study compare favorably with these previous in vivo obser-
vations as discussed below. Urethane induced statistically significant,
dose-dependent increases in the frequencies of MN-RET at all time
points analysed, and in MN-PCE at terminal sacrifice. The maximal
fold-increases observed were similar (~4- to 6-fold for all observa-
tions), even though the absolute frequencies were quite different for
MN-RET and MN-PCE. It should be noted that positive (significant)
responses were observed for MN-RET at doses ≥100 mg/kg/day, but
only at 250 mg/kg/day for the MN-PCE endpoint. This difference
is likely due to the improved sensitivity and power afforded by the
ability to routinely score many more cells using the automated flow
cytometric method (38). Also, the apparent “disappearance” of the
positive MN-RET response at a dose of 25.0 mg/kg/day on Day 15
may reflect that the dose/response was at or near the limit of detec-
tion for the assay.
Urethane also induced statistically significant, dose-dependent
increases in the comet assay (median %Tail DNA) in PBL on Day
15, and in liver at terminal sacrifice. However, the relative increases
(1.5- to 3.0-fold control values) were even less than observed for
MN-RET and MN-PCE. In addition, no significant increases were
observed for the comet endpoint in blood on Day 4 or 29. Thus, the
Figure 3. Percent RET in peripheral blood, following treatment with urethane
or the EMS PC, as assessed by flow cytometry (all n = 6; ±1 SD).
Figure 4. Frequency of MN-RET induced by urethane or the EMS PC (all
negative control MN-RET data was 0.175%.
Figure 5. Frequency of MN-PCE induced by urethane or the EMS PC (all
negative control MN-PCE data was 0.0947%.

“positive” comet response, especially in blood, is considered to be
suspect. An apparent dose-dependent increase in %Tail DNA was
observed in lung, but the individual values did not reach statisti-
cal significance (P 0.05). Although dose-dependent decreases in
median %Tail DNA were observed in the brain, the assay condi-
tions were not optimized for detection of crosslinkers nor was that
intended for this study. The lack of any appreciable increases in
clouds and the lack of any significant deleterious changes in Clin
Path parameters suggest that any significant increases in comet
response were not due to cytotoxicity.
Also to our knowledge, this study represents the first reported
in vivo comet assay with urethane. The negative comet results in
lung are perhaps unexpected, since it is one of the principal sites for
tumor formation (24,25), and positive results have been observed in
transgenic rodent mutation (TRM) assays, in multiple tissues includ-
ing forestomach, liver and lung, following oral dosing [reviewed in
refs. (39–41)]. However, in the cases of earlier positive TRM results,
the cumulative dose levels generally were much higher, and/or the
sampling time was much later, than studied here (see additional
discussion below regarding cumulative dose and sampling times).
Likewise, the sampling time was our standard, in compliance with
OECD recommendations (22), but not necessarily optimal based
upon uptake and metabolism kinetics or DNA repair.
No significant increases were observed for urethane in the CAb
assay at any dose or sampling time. In searching the literature, we
found no reports of structural CAb induced by urethane when
scored by metaphase analysis; rather, the induction of CAb was
inferred using micronuclei as a surrogate for chromosome breakage.
Although the micronucleus test should be considered the in vivo test
of choice for CAb (21), little additional effort was required to add
the CAb endpoint to this study, as has been suggested elsewhere (42).
Since a daily dosing regimen was used, and the 48-h culture time
after stimulation has proved reliable in this laboratory, the recom-
mended sampling time for the comet assay that was used here (22)
is likely not responsible for the lack of a positive CAb response for
urethane. However, we also note the PCs for this assay failed, but
likely due to excessive toxicity.
Finally, and most importantly for this study, urethane induced
statistically significant, dose-dependent increases in the frequen-
cies of RETCD59−
and RBCCD59−
at both time points analysed. Larger
responses were observed in RETCD59−
, which is consistent with earlier
observations for other agents and is due to slower turnover in the
RBC population (18). The maximum mutant frequencies observed
at a dose of 250 mg/kg/day were ~20 × 10−6
for RETCD59−
on Day 15,
and ~6 × 10−6
for RBCCD59−
on Day 29. These numbers compare very
favorably to those observed for urethane used under identical con-
ditions as a PC in a study of methyl carbamate (43). The relatively
larger fold-increase in RETCD59−
on Day 15 versus Day 29 (Table 3)
is the result of an approximate 3-fold difference in negative con-
trol mutant frequencies at those time points (2.82 vs. 0.678 × 10−6
,
respectively).
The 24- and 74-fold increases in RETCD59−
frequencies on Day 29
and 15 far exceeded those observed in the other positive endpoints.
Based upon the biology and expression time for the RETCD59−
and
RBCCD59−
to appear in the peripheral blood, and as seen for a num-
ber of chemicals, at least some additional increases in mutant fre-
quencies could be expected (5,6,18,44,45). Given sufficient time, it
is anticipated that the fold-increases in RBCCD59−
frequencies would
reach levels similar to those observed for RETCD59−
. These results,
in comparison to the other endpoints studied, further reinforce the
dramatic benefits of repeat dosing, and accumulation of induced
mutants, that are associated with the Pig-a assay [as has been
reported previously and reviewed by Gollapudi et al. (18)].
Urethane has been reported to be one of the weakest mutagens
examined to date in the TRM assays using the currently recom-
mended study design (46–48). Only half of the urethane treatments
previously reviewed (28/56) produced positive findings [by any
route; results compiled in refs. (39–41)]. Although mutant frequen-
cies in the lung and liver reached ~5- to 19-fold control values in
some of these TRM studies (representing induced mutant frequen-
cies of ~800–1300 × 10‒6
), these were 6- to 8-month drinking water
or feed studies performed over much longer intervals (105–237 days)
at ~2- to 10-fold higher cumulative doses (13 650–69 678 mg/kg)
than in our study (7250 mg/kg). Most other oral dosing TRM studies
(gavage or diet) reported in the literature using dose regimens similar
to our study (1400–5600 mg/kg by oral gavage) resulted in approxi-
mately only 2-fold increases in mutant frequencies in the lung or
liver (representing induced mutant frequencies of ~60–110 × 10−6
)
or negative results (39–41). Similar TRM studies (cumulative doses
Figure 7. Comet analysis in various tissues following 29 daily doses of
urethane (median %Tail DNA ±1 SD; n = 6, except as noted). EMS PC
responses were off scale (~30–34%). Statistically significant increase
indicated at P 0.05 level (*). Lung results from one animal were excluded
as an extreme outlier (13.1%). The 95% upper control limits for historical
negative control median %Tail DNA data were: 2.48% (liver), 22.3% (brain),
4.20% (spleen), 4.14% (kidney) and 11.6% (lung).
Figure 6. Comet analysis in PBL following 29 daily doses of urethane (median
%Tail DNA; all n = 6; ±1 SD). EMS PC responses were off scale (~16–26%).
Statistically significant increases are indicated at P 0.01 level (**).The 95%
upper control limit for historical negative control median %Tail DNA data
was 0.750%.

of 700 to 5600 mg/kg by oral gavage) produced ~1.7- to 2.8-fold
increases in mutant frequencies in haematopoietic tissues (represent-
ing induced mutant frequencies of ~35–100 × 10−6
). Although the
net increases in Pig-a mutant frequencies reported here were smaller
than some observed in the TRM assays, the fold-increases were
larger due to the lower spontaneous mutant frequencies for Pig-a.
Also as previously stated, the Pig-a mutant frequencies would be
expected to increase with a longer phenotypic expression time.
These results demonstrate the utility and sensitivity of the Pig-a
in vivo gene mutation assay. This endpoint, as well as the other
genetic toxicology endpoints, can be integrated into repeat dose tox-
icity studies easily and economically, resulting in a comprehensive
analysis of genotoxicity with reduced animal use. The relative sensi-
tivity of the various endpoints is likely due in part to the statistical
power and sample sizes of the various assays, and the ability of Pig-a
mutant cells to persist and accumulate during repeated dosing.
The negative and/or weak CAb and comet responses are simi-
lar to our previous observations for 4-nitroquinoline oxide using a
29-day dose regimen (16). Those results, and the weak MN-PCE and
comet responses here, further demonstrate that not all genotoxicity
endpoints respond similarly. Although it is certainly possible to inte-
grate multiple (or all of these) endpoints into a single study, it is not
necessarily prudent or scientifically justified. As previously discussed
(45), the Pig-a and MN-RET are easily integrated, complimentary
endpoints that offer the ability to detect gene mutation and cytoge-
netic damage, sometimes arising from two distinct modes of action,
that cannot be universally detected for all agents by a single assay.
Where limitations exist (e.g. questions over target tissue exposure),
then the comet assay would be a valuable addition. Thus, care must
be taken in dose, endpoint and tissue selection when incorporating
genotoxicity endpoints into routine toxicity studies as has been rec-
ommended, for example, by the ICH (19), the OECD (20–22) and
the US Food and Drug Administration (42).
Funding
This work was supported by BioReliance Corporation and also sup-
ported in part by a grant to Litron Laboratories (National Institute
of Health/National Institute of Environmental Health Sciences,
R44ES018017).
Acknowledgements
The authors thank Sarah Miller for her expert technical assistance, and
Stephen Dertinger for invaluable advice and support. The contents herein are
solely the responsibility of the authors, and do not necessarily represent the
official views of the institutions involved.
Conflict of interest statement: All of the authors are employees of
BioReliance Corporation and M.J.A. also serves as a consultant to
BioReliance. BioReliance is a contract research organization offering
these and other studies for sale.
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