Vol 49, No 6
Journal of the American Association for Laboratory Animal Science
by the American Association for Laboratory Animal Science
Pinworms continue to beset modern rodent facilities,4,18,19,22
likely because the eggs of 2 species (Aspiculuris tetraptera and
Syphacia muris) are environmentally persistent25 and resistant
to common disinfectants8 while infections for all species can
be difficult to detect due to intermittent shedding of eggs and
reduction in parasite load with age.20,26 Although often thought
to be merely nuisances2 (older reports of rectal prolapse11 did not
rule out Helicobacter spp. or Citrobacter rodentium), pinworms can
affect immunologic parameters,1,23 and their presence can inhibit
interinstitutional transfer of mice for research collaborations.
Once pinworms are detected, attempts at eradication are
common. Of the available anthelminthics, fenbendazole in-
corporated in the feed at 150 to 450 mg/kg, intermittently or
continuously for as long as 8 wk, is a popular choice because
of its ease of use and perceived safety, efficacy, and lack of
interference with ongoing research.5,9,12,21 Fenbendazole is a
benzimidazole derivative that is absorbed from the gastroin-
testinal tract and metabolized in the liver to its active form,
fenbendazole sulfoxide.24 The drug is a microtubule polymeriza-
tion inhibitor and binds to the structural protein β tubulin.15 Its
efficacy as an anthelminthic results from a much greater affinity
for helminth tubulin than for mammalian tubulin at 37 °C.15
Benzimidazole-resistant helminths have a greater proportion
of low-affinity tubulin.16
Previous reports suggest that fenbendazole is not entirely
lacking in research effects. For instance, fenbendazole inhibited
tumor xenograft growth in immunodeficient mice when fed in
combination with high vitamin levels,10 and the drug may have
curtailed reproduction in Sprague–Dawley rats.13 Reported ef-
fects on the immune system range from no perceived effects7
to reduced activity in activated precursor B lymphocytes of
BALB/cN mice.17 However, reports of effects on behavioral
tests are scarce and limited to rats. For example, the offspring
of fenbendazole-treated Sprague–Dawley rats demonstrated
delayed righting reflex and reduced locomotor activity when
a running wheel was first introduced.3 In contrast, no effects of
fenbendazole on conditioned behavior (overall response rates,
temporal patterning, interresponse time, drinking behavior)
were detected in Sprague–Dawley rats.14 To our knowledge,
effects of fenbendazole on behavior in mice have not been
reported. Therefore we were surprised to receive anecdotal
reports from a neuroscience laboratory at our institution of
adverse effects on behavioral testing in vendor-supplied mice
during a routine fenbendazole treatment.
In the absence of published data, anecdotes regarding del-
eterious effects of treatments or concerns that treatments may
interfere with results can cause research to be delayed or can-
celled unnecessarily. Conversely, research that proceeds during
treatment may be confounded. Therefore, we undertook this
prospective controlled study to provide additional data on the
effect of fenbendazole treatment in male C57BL/6N mice on 3
common behavioral tests (open field, elevated plus maze, and
rotarod). C57BL/6N mice were selected because the C57BL
Effect of Fenbendazole on Three Behavioral Tests
in Male C57BL/6N Mice
Bharathi S Gadad,1,† João P L Daher,1,5 Eric K Hutchinson,3 Cory F Brayton,3 Ted M Dawson,1,4
Mikhail V Pletnikov,2,3,4 and Julie Watson3,*
Pinworms are highly contagious parasites of laboratory rodents that often are treated with fenbendazole. To our knowl-
edge, the effect of fenbendazole at therapeutic dosages on behavioral tests in mice has not been evaluated. Here we studied
6-wk-old male C57BL/6N mice. We compared the behavior of control mice (fed regular diet) with 3 groups of mice treated
with dietary fenbendazole. Treatment groups were 4 wk of fenbendazole, 2 wk of fenbendazole followed by 2 wk of regular
diet, and 2 wk of regular diet followed by 2 wk of fenbendazole. At the end of dietary treatment all groups were tested by
open field for central, peripheral and vertical activity; elevated plus maze for anxiety; and rotarod for motor ability and then
evaluated by clinical pathology and selected histopathology. Treated and control groups showed no differences in open field
or elevated plus maze testing, histopathology, or clinical pathology. However mice treated for 4 wk with fenbendazole or 2 wk
of fenbendazole followed by 2 wk regular diet stayed on the rotarod for shorter periods than did controls, and mice treated
with 2 wk of regular diet followed by 2 wk fenbendazole showed a trend toward shorter rotarod times. In light of this study,
we suggest that open field and elevated plus maze testing is unlikely to be affected by 4 wk fenbendazole treatment in male
C57BL/6 mice; however, behavioral tests of motor ability such as rotarod tests may be affected during and for at least 2 wk
after fenbendazole treatment.
Abbreviations: FF, fenbendazole diet only; RF, regular diet followed by fenbendazole diet; FR, fenbendazole diet followed by
Received: 25 Feb 2010. Revision requested: 12 Apr 2010. Accepted: 26 Apr 2010.
1Neuroregeneration Program, Institute for Cell Engineering and the Department of Neu-
rology, 2Department of Psychiatry and Behavioral Sciences, 3Department of Molecular
and Comparative Pathobiology, and 4Solomon H Snyder, Department of Neuroscience,
Johns Hopkins University School of Medicine, Baltimore, Maryland; and 5Department
of Pathology, School of Medicine, Fluminense Federal University, Niterói, Rio de Janeiro,
*Corresponding author. Email: email@example.com
†Current affiliation: Department of Psychiatry, UT Southwestern Medical Center at
Dallas, Dallas, Texas.
Vol 49, No 6
Journal of the American Association for Laboratory Animal Science
numbers of entries into the dark and bright arms over a 5-min
period were scored for each mouse.
Rotarod test. Rotarod tests are used to measure motor coor-
dination and balance. Mice must continuously walk forward
to keep from falling off a rotating cylinder.6 Mice were tested
for their ability to maintain balance on an accelerating rotating
rod (rotarod; Rotamex 4/8, Columbus Instruments, Columbus,
OH). The rotarod accelerated from 4 to 40 rpm over 5 min. The
time elapsed before falling and the speed at that time were
recorded for each mouse. Immediately before testing, mice
were trained until their fall latency reached a plateau. For the
experiments, the average of 3 consecutive runs per mouse was
used for statistical analysis.
Clinical and anatomic pathology. Histologic examination. The
liver, kidneys, salivary gland, and tongue were fixed in 10%
nonbuffered formalin, and the dissected head was fixed and
decalcified (Formical 4, Decal Chemical, Tallman, NY). Tissues
were embedded in paraffin for sectioning and then stained with
hematoxylin and eosin. The tissues were examined in a blinded
fashion by a veterinary pathologist, who assigned a numerical
score according to the number or severity of lesions present: 0,
no significant lesions; 1, few or mild lesions only; and 2, numer-
ous or moderate to severe lesions.
Hematologic and clinical chemistry analysis. After carbon
dioxide euthanasia, 500 to 600 μL blood was collected from
each mouse by intracardiac aspiration with a 25-gauge needle
and 1-mL syringe. Blood was placed in a 600-μL centrifuge tube
coated with lithium heparin to prevent clotting. Total WBC
count, segmented neutrophils, lymphocytes, RBC, hemoglobin,
hematocrit, and platelets were analyzed by automated hemocy-
tometer (Hemavet HV950FS, Drew Scientific, Oxford, CT). The
remainder of the blood was centrifuged and the plasma drawn
off and analyzed with an automated clinical chemistry analyzer
(VeTACE, Alfa Wassermann, West Caldwell, NJ) for cholesterol,
triglyceride, creatine kinase, ALT, AST, lactate dehydrogenase,
amylase, ALP, glucose, total protein, total calcium, BUN, creati-
nine, albumin, high-density lipoprotein, and uric acid.
Statistical analysis. The effects of fenbendazole in the
open-field performance were evaluated by using 2-way
repeated-measures ANOVA. The effects of fenbendazole in the
elevated plus maze and rotarod performances were evaluated
by using one-way ANOVA. Significant effects were explored
further with post hoc comparisons. Data were analyzed and
plotted by using SigmaPlot 2000 (SPSS, Chicago, IL). Statistics
were performed by using SigmaStat 2.0 (SPSS), and values are
presented as mean ± SEM; a P value of less than 0.05 was used
to define significance. Animal weights were compared by using
the Student t test.
Behavioral tests. Open field test. We conducted open field
tests to assess the effects of fenbendazole on novelty-induced
locomotor activity in mice. The drug had no significant effects
on horizontal or vertical activities, specifically including absence
of group effects on central activity (F3,287 = 0.932, P = 0.433;
Figure 1 A), peripheral activity (F3,287 = 2.496, P = 0.072; Figure
1 B), or rearing (F3,287 = 0.677, P = 0.571; Figure 1 C). Two-way
repeated-measures ANOVA during successive time intervals
revealed a significant effect for all groups (including control)
for central activity (F3,287 = 32.628, P < 0.001), peripheral activity
(F3,287 = 21.387, P < 0.001), and rearing (F3,287 = 4.236, P < 0.001).
Repeated-measures during successive time intervals are used
to evaluate habituation to the open field. These data showed
strains are the most frequently used background strains for
genetically manipulated mice.
Materials and Methods
Male C57BL/6N mice (age, 6 wk; n = 48; Harlan Teklad, Madi-
son, WI) were randomly assigned on arrival from the vendor to
1 of 4 dietary groups of 12 mice, which received either regular
diet (2018S, Harlan Teklad) or fenbendazole diet (2018S plus 150
mg/kg fenbendazole, Harlan Teklad). Control group mice were
fed regular diet for 4 wk. Treatment groups were FF (4 wk of
fenbendazole diet), FR (2 wk of fenbendazole diet followed by
2 wk of regular diet), and RF (2 wk of regular diet followed by
2 wk of fenbendazole diet). Mice were housed 2 per cage in in-
dividually ventilated racks (Allentown Caging, Allentown, PA),
on autoclaved corncob bedding (Bed O’Cobs, The Andersons,
Maumee, OH) with reverse-osmosis–treated hyperchlorinated
water. Cages were sanitized every 2 wk by using aseptic proce-
dures. The colony was routinely tested by sentinel surveillance
and remained free of common mouse pathogens, including
Sendai virus, pneumonia virus of mice, mouse hepatitis virus,
mouse minute virus, mouse parvovirus types 1 and 2, Theiler
mouse encephalomyelitis virus, reovirus, epizootic diarrhea
of infant mice, lymphocytic choriomeningitis virus, ectromelia
virus, murine adenovirus types 1 and 2, murine cytomegalovi-
rus, Mycoplasma pulmonis, fur mites, and pinworms. Mice were
weighed on arrival (day 0), after 2 wk on the diet (day 15), and
just before euthanasia (day 30). After 4 wk of dietary treatment,
mice were tested sequentially in 3 behavioral tests and then
euthanized by carbon dioxide overdose. Blood was collected
by cardiocentesis for clinical pathology, and liver, kidney, and
head tissues were collected and preserved in 10% nonbuffered
formalin for routine histopathology. Mice were examined prior
to behavioral testing for physical abnormalities that could affect
behavioral testing, such as fight wounds or whisker loss. All
procedures were approved by the Johns Hopkins IACUC, and
Johns Hopkins animal facilities are AAALAC-accredited.
Behavioral tests. Mice were tested sequentially, first in the
open field, then in the elevated plus maze, and finally on the
rotarod. Mice were tested over a 3-d period, and all mice were
tested in the same sequence. To eliminate tester variability, only
one person conducted each test, and the tester was blinded to
treatment group. Mice were tested in random order, and all
mice were tested between the hours of 1800 and 2100 during
the light cycle. The last-used diet was continued throughout
the testing period.
Open field test. Open field tests reflect multiple underlying
traits, including locomotor activity, exploratory activity, olfac-
tion and vision, as well as fear and anxiety,6 therefore this test is
used frequently for initial evaluation of many mouse behavioral
characteristics. The open field consisted of a square acrylic box
incorporating an automated activity monitor (Cage Rack Flex-
Field Photobeam Activity System, San Diego Instruments, San
Diego, CA), which provides horizontal and vertical grids of 16
× 16 infrared beams. The total number of beam breaks in both
horizontal and vertical planes over a period of 30 min was
recorded and analyzed.
Elevated plus maze test. The elevated plus maze test rests on
the innate conflict between the tendency of mice to explore a
novel environment and the aversive properties of a brightly-lit
open area.6 This test is used widely as a model of anxiety-like
behavior. Each mouse was placed in the center of a plus-shaped
runway6 elevated 1 m above the floor and containing 2 dark
enclosed arms and 2 open arms (San Diego Instruments). The
Fenbendazole and behavior in C57BL/6N mice
no significant effect of the drug between groups (F3, 47 = 1.658,
P = 0.190; Figure 2 B).
Rotarod test. We used the rotarod test to analyze the effects
of fenbendazole on motor coordination and balance skills. We
found that mice in the FF (4 wk of fenbendazole-containing
diet) and FR (2 wk of fenbendazole-containing diet followed
by 2 wk of regular diet) groups stayed on the accelerating ro-
tarod for significantly shorter periods than did controls (control
compared with FF, P = 0.003; control compared with FR, P =
0.009; Figure 3). Mice in the RF group showed a trend for staying
on the rod for shorter periods than controls (P = 0.070; Figure
3). Fenbendazole had a significant effect on time spent on the
rotarod (one-way ANOVA, F3, 47 = 3.743, P = 0.018).
Weight, physical examination, and histopathology. All mice
remained physically normal, with intact whiskers and absence
of fight wounds. Mice in the FF, FR, and RF groups were signifi-
cantly (P = 0.04, P = 0.004, and P = 0.003, respectively) heavier
than were controls at day 15, and the RF and FR groups were
significantly (P = 0.02 and P = 0.01, respectively) heavier than
were controls at day 30. Although significant, numeric differ-
ences in weight were small (Figure 4). The liver, kidney, and
middle and inner ear tissues were examined histologically.
Mild hepatic anisocytosis (variation in cell size) was present in
9 of the control mice, 9 FF mice, 7 FR mice, and 10 RF mice (n =
12 per group). Mild centrilobular hypertrophy (slightly larger
relative size of centrilobular hepatocytes) was identified in 3
control mice, 2 FF mice, 2 FR mice, and 4 RF mice. These liver
changes did not correlate with increased liver enzyme activi-
ties or other findings and could not be attributed to treatment
effect. Mild, focal infiltrates of few inflammatory cells in the
that mice showed reduced horizontal activity, but not vertical
activity (rearing), over time.
Elevated plus maze test. We used the elevated plus maze to
assess the effects of fenbendazole treatment on anxiety in mice.
We found no significant effects of the drug on the exploratory
behavior. One-way ANOVA for the percentage of entries into
open arms showed no significant effect of the drug between
groups (F3, 47 = 0.110, P = 0.954; Figure 2 A). Likewise, one-way
ANOVA for the percentage of time spent in open arms showed
Figure 1. Open field test behavior of male C57BL/6N mice did not
differ between treatment and control groups. (A) Central activity.
(B) Peripheral activity. (C) Vertical activity. Data are expressed as mean
± SEM (n = 12) per group. Data are reported as total number of infrared
beams broken in a 5-min period.
Figure 2. Elevated plus maze behavior of male C57BL/6N mice did
not differ between treatment and control groups. (A) Percentage of en-
tries into open arm. (B) Percentage of time spent in the open arm. Data
are expressed as mean ± SEM (n = 12 per group). Data are reported as
total number of infrared beams broken in a 5-min period.
Vol 49, No 6
Journal of the American Association for Laboratory Animal Science
This study was initiated because of anecdotal reports that
fenbendazole treatment had affected behavioral testing in a
neuroscience laboratory at our institution. We tested open field,
rotarod, and elevated plus maze in the current study because
these tests are used frequently in neuroscience laboratories.
Spontaneous activity in the open field is a standardized meas-
ure of motor function, rotarod testing is rigorous test of motor
coordination and balance, and the elevated plus maze is a test
of anxiety.6 Reduced open field activity in concert with reduced
rotarod persistence would have indicated a problem with motor
function. Reduced open field activity (particularly entries into
the central open area) together with reduced entries into the
open arms of the elevated plus maze would have revealed an
increase in anxiety. Our finding that only rotarod ability was
affected, primarily in the FF and FR treatment groups, suggests
that fenbendazole has no effect on general motor activity but
exerts a subtle effect on motor coordination and balance that
takes more than 2 wk to develop and persists at least 2 wk
after treatment cessation. The only prior published evidence
for an effect of fenbendazole on behavioral tests in rodents was
reduced motor ability in the offspring of fenbendazole-treated
Sprague–Dawley rats.3 The authors of that study speculated
that the reduced running wheel activity may have been due to
a difference in reaction to novelty because the effect occurred
only during initial introduction of the running wheel. Mice in
our study were acclimated to the rotarod before testing, and
results were averaged over 3 trials, suggesting that the effect
in our mice is not related to novelty.
Another potential cause for decreased motor activity is diso-
rientation due to systemic toxicity; however, liver and kidney
function tests and hepatic and renal histopathology were all
within normal limits and revealed no significant differences
between test and control groups. After initial behavioral results
became known, we conducted histopathology of ear tissues to
discover whether middle ear abnormalities were responsible for
loss of balance. However, the minor accumulations of proteina-
ceous material noted in the middle ears of several mice were
unlikely to have been responsible for reduced rotarod ability.
Other causes, such as weakness due to inappetance, also were
discounted because mice in treated groups were at least as heavy
as controls; in fact RF and FR groups were significantly heavier
at day 30, although actual weight differences were small and
most likely attributable to differences in dietary hardness—the
regular feed, but not the fenbendazole feed, was autoclaved. The
lack of anorexia among our mice was consistent with a study in
rats,27 which showed no difference in food intake or body weight
in rats fed diets with and without added fenbendazole.
We selected 6-wk-old male C57BL/6N mice for this study
because C57BL strains are the most common background strains
used for genetic mouse models. Male mice are used frequently
for behavioral testing because of the potentially confounding
effect of the female reproductive cycle. Additional studies are
needed to determine whether the effect is present in female
C57BL/6 mice and in other common background strains.
The fenbendazole dosage (150 ppm) and treatment periods
(4 wk or 2 wk on–2 wk off) were selected in light of common
treatment paradigms: fenbendazole is often used continuously
for 4 wk or more or cycled for 2 wk on and 2 wk off for several
treatment cycles.21 Further studies are needed to determine
whether longer treatment duration exacerbates the effect on
rotarod performance and to determine whether the effect per-
sists permanently or disappears with a washout period longer
than 2 wk.
liver or gall bladder (or both) were identified in 0 to 2 mice
in each group. Decalcified head sections were examined from
all mice. None had otitis media or otitis interna that might be
expected to affect behavioral results. Sporadic findings in the
head and neck region of 0 to 2 mice per group included: focal
retinal degeneration; mild otitis externa; mild, focal inflamma-
tion of the submandibular salivary gland; mild accumulations
of proteinaceous material in the middle ear, and a single case
of neoplasia of the submandibular salivary gland. In summary,
histologic changes were very mild or sporadic and could not be
attributed to treatment effect. ANOVA indicated no significant
difference in lesion score between treatment groups (P = 0.8126).
Hematology and clinical chemistry. All results were within
normal limits. Analysis of variance showed no significant dif-
ferences between treatment groups (P = 0.9995).
This study showed no significant effects of fenbendazole
treatment on open field or elevated plus behavioral tests in
young male C57BL/6N mice compared with controls. However,
mice treated with 4 wk of fenbendazole diet (FF mice) or 2 wk
of fenbendazole diet followed by 2 wk regular diet (FR mice)
showed significantly reduced ability to stay on the accelerating
rotarod, and mice on 2 wk of regular diet followed by 2 wk of
fenbendazole diet (FR mice) showed a trend toward shorter
Figure 3. Rotarod performance of male C57BL/6N mice expressed as
time spent on the accelerating rotarod. Mice in the FF and FR groups,
but not the RF group, stayed on the accelerating rotarod for signifi-
cantly (P = 0.003, P = 0.009, and P = 0.07, respectively) shorter peri-
ods than did controls. Data are expressed as mean ± SEM (n = 12 per
Figure 4. Weights of male C56BL/6N mice were significantly greater
in FF, FR, and RF groups at day 15 (P = 0.04, P = 0.004, P = 0.003,
respectively) and in RF and FR groups at day 30 (P = 0.02, P = 0.01,
respectively) compared with those of controls. Data are expressed as
mean ± SEM (n = 12 per group).
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Fenbendazole and behavior in C57BL/6N mice
This study showed a significantly reduced ability of fenbenda-
zole-treated male C57BL/6N mice to persist on an accelerating
rotarod compared with mice on regular diet. The effect was
significant in mice treated for 4 wk or for 2 wk then returned
to regular diet for 2 wk prior to testing but not in mice treated
for 2 wk and then tested immediately, suggesting that the effect
took more than 2 wk to develop but persisted at least 2 wk after
treatment cessation. Because open field tests were not affected,
motor effects were subtle. Additional tests of motor ability,
such as grip strength and balance beam testing, are needed to
further describe the effect. The cause of the rotarod effects is
unknown and deserves further study. Whether fenbendazole’s
inhibitory effect on microtubule polymerization is responsible
for the subtle motor coordination effects we observed despite
the drug’s low affinity for mammalian tubulin15 remains to be
Given our results, we suggest that, for male C57BL/6 mice,
fenbendazole is not a factor in open field and elevated plus maze
testing but should be used with caution during tests of motor
function, such as rotarod, because treatment may confound
The authors thank Dr Myriam Dumas Hahn (Department of Pathol-
ogy, Fluminense Federal University) for support of João P L Daher’s
training program, Ms Nadine Forbes for technical assistance, Dr Robert
J Adams for departmental support, and Harlan Teklad for donation of
the mice. This work was supported in part by Johns Hopkins Research
Animal Resources, NIH grants 5R01MH083728-02 (MVP) and NINDS
NS38377 (TMD), and the Herbert Friedberg Postdoctoral Fellowship
in Parkinson Disease (BSG).
Harlan Teklad had no input into this study apart from donation of
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