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A Novel Mode-of-Action Mediated by the Fetal Muscle Nicotinic Acetylcholine

Receptor Resulting in Developmental Toxicity in Rats

Article  in  Toxicological Sciences · March 2012

DOI: 10.1093/toxsci/kfs118 · Source: PubMed

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TOXICOLOGICAL SCIENCES 127(2), 522–534 (2012)
doi:10.1093/toxsci/kfs118
Advance Access publication March 29, 2012

A Novel Mode-of-Action Mediated by the Fetal Muscle Nicotinic

Acetylcholine Receptor Resulting in Developmental Toxicity in Rats
Reza J. Rasoulpour,*,1 Robert G. Ellis-Hutchings,* Claire Terry,† Neil S. Millar,‡ Carol L. Zablotny,* Alasdair Gibb,‡
Valerie Marshall,* Toby Collins,‡ Edward W. Carney,* and Richard Billington†
*Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI 48674; †Human Health Assessment, Dow AgroSciences,
LLC, Abingdon, U.K.; and ‡Department of Neuroscience, Physiology & Pharmacology, University College London, London, U.K.

1
To whom correspondence should be addressed at Toxicology & Environmental Research and Consulting, The Dow Chemical Company, 1803 Building, Midland,
MI 48674. Fax: (989) 638-9863. E-mail: rrasoulpour@dow.com.

Received December 21, 2011; accepted March 6, 2012

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Sulfoxaflor (X11422208), a novel agricultural molecule, induced
fetal effects (forelimb flexure, hindlimb rotation, and bent clavicle)
and neonatal death in rats at high doses (‡ 400 ppm in diet);
however, no such effects occurred in rabbit dietary studies despite
achieving similar maternal and fetal plasma exposure levels. Mode-
of-action (MoA) studies were conducted to test the hypothesis that
the effects in rats had a single MoA induced by sulfoxaflor agonism
on the fetal rat muscle nicotinic acetylcholine receptor (nAChR).
The studies included cross-fostering and critical windows of
exposure studies in rats, fetal ((a1)2b1gd) and adult ((a1)2b1de)
rat and human muscle nAChR in vitro agonism experiments, and
neonatal rat phrenic nerve-hemidiaphragm contracture studies. The
weight of evidence from these studies supported a novel MoA where
sulfoxaflor is an agonist to the fetal, but not adult, rat muscle
nAChR and that prolonged agonism on this receptor in fetal/
neonatal rats causes sustained striated muscle contracture resulting
in concomitant reduction in muscle responsiveness to physiological
nerve stimulation. Fetal effects were inducible with as little as 1 day
of exposure at the end of gestation, but were rapidly reversible after
birth, consistent with a pharmacological MoA. With respect to
human relevance, sulfoxaflor was shown to have no agonism on
human fetal or adult muscle nAChRs. Taken together, the data
support the hypothesis that the developmental effects of sulfoxaflor
in rats are mediated via sustained agonism on the fetal muscle
nAChR during late fetal development and are considered not
relevant to humans.
Key Words: developmental toxicity; safety evaluation; in vitro
and alternatives; mechanisms.
Sulfoxaflor (X11422208; CAS 946578-00–3) is a novel
agricultural molecule that targets sap-feeding pests via agonism
on the insect nicotinic acetylcholine receptor (nAChR). The
effects of sulfoxaflor on the insects include excitatory
symptoms such as tremors, followed by paralysis and
mortality, which suggested that the molecule acted on the
insect nervous system (Zhu et al., 2011). Sulfoxaflor was found

The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For permissions, please email: journals.permissions@oup.com
to be a nAChR agonist, as evidenced by its ability to activate
Drosophila melanogaster DR2/b2 receptors expressed in
Xenopus laevis oocytes (Zhu et al., 2011). Due to its efficacy
on sap-feeding insects, this molecule was developed for
production as a pesticide, which required a definitive toxico-
logical assessment.
Product safety assessment for new agricultural molecules
involves comprehensive hazard identification in model organ-
isms and human exposure assessments under acute to chronic
scenarios, which are brought together within the risk
assessment. Toxicity testing in new product development
incorporates in silico, in vitro, and in vivo models to evaluate
the potential for genotoxicity, short to long-term systemic
toxicity, target organ identification, carcinogenicity, toxicoki-
netics, biotransformation, neurotoxicity, as well as develop-
mental and reproductive toxicity studies. The developmental
and reproductive toxicity studies typically performed as part
of the toxicity testing package include prenatal developmen-
tal toxicity studies in two species (rodent and nonrodent)
under the Organisation for Economic Co-operation and
Development (OECD) 414 guideline and a two-generation
reproductive toxicity study in rats under the OECD 416
guideline.
During the conduct of developmental and reproductive safety
assessment studies on this novel molecule, developmental effects
were observed in rats following exposure to a relatively high
dose of
400 ppm (~26.4 mg/kg/day) sulfoxaflor in the diet.
These effects primarily included neonatal offspring death, limb
abnormalities (forelimb flexure and hindlimb rotation), and bent
(misshapen) clavicles. The rat limb abnormalities appeared as
part of a generalized muscle contracture and were without
associated changes in the limb skeletal bone structure, whereas
the offspring deaths occurred to postnatal day (PND) 4.
This paper describes the series of follow-up studies
conducted to test the hypothesis that the developmental effects
of sulfoxaflor in rats are mediated via sustained agonism on
 NOVEL FETAL MUSCLE nAChR MoA IN RATS
the fetal muscle nAChR during late fetal development. Key
events were identified in this mode-of-action (MoA), and the
relevance of the effects to humans was investigated.
MATERIALS AND METHODS
Test Material
Sulfoxaflor (X11422208; CAS 946578-00-3; purity 95.6%) was provided by
Dow AgroSciences, LLC (Indianapolis, Indiana). For rabbit studies, sulfoxaflor
was mixed with ground feed and 0.5% apple flavoring, then formed into pellets
by PMI Nutritional International, LLC (St Louis, MO). For rat studies,
sulfoxaflor was mixed with ground feed and fed in meal form. All diets were
prepared as fixed percent of test material in the feed. The stability and
homogeneity of sulfoxaflor in the feed were confirmed by analytical methods.
Animals
All animal studies described in this paper were reviewed and approved by
the Institutional Animal Care and Use Committee. Male and female
Crl:CD(SD) rats and time-mated female Crl:CD(SD) rats were obtained from
Charles River Breeding Laboratories (Portage, MI), and time-mated New
Zealand white (NZW) rabbits were obtained from Covance Research Products
(Kalamazoo, MI). Nonmated rats were 6–8 weeks of age, and time-mated
females were 10–11 weeks of age at study start. Rabbits were 5–6 months of
age at study start for the developmental toxicity study and were 8–12 months of
age at study start for the neonatal survival study. Animals were acclimated to
the laboratory for up to 1 week prior to the start of dosing.
Rats were housed in rooms designed to maintain environmental conditions
at 22 ± 3C, 40–70% relative humidity, 12-h light/dark photocycle, and 12–15
air changes per hour. Rats were singly or doubly housed (during breeding) in
stainless steel cages with wire mesh floors. Dams were housed one per cage
(with their litter) in plastic cages containing ground corncob nesting throughout
the lactation phase. Feed and water were provided ad libitum.
Rabbits were housed one per cage in stainless steel cages in rooms designed
to maintain environmental conditions at 19 ± 4C, 40–70% relative humidity,
12-h light/dark photocycle, and 10–15 air changes per hour. Cages were
suspended above catch pans with absorbent noncontact bedding. During the
littering/lactation phase, a nesting box containing bedding was placed in the
cages. Dams and offspring were housed in the nesting boxes with nesting
material until PND 4. Water was provided ad libitum, and feed was offered in
gradual increments to avoid gastrointestinal disturbances during the acclimation
period with a final provision of approximately 150 g of feed given daily
through the treatment period and approximately 200 g given during the
lactation phase.
In Vivo Study Designs
Developmental and reproduction studies. The prenatal developmental
toxicity studies (OECD 414) involved dietary administration of sulfoxaflor on
gestation day (GD) 6–21 (n ¼ 26 rats; dose ¼ 0, 25, 150, or 1000 ppm) or GD
7–28 (n ¼ 26 rabbits; dose ¼ 0, 30, 150, 750 ppm) followed by evaluation of
the fetuses on GD 21 or 28, respectively. In the reproductive/developmental
toxicity screening study (OECD 421), rats (n ¼ 12; dose ¼ 0, 100, 500, or 1000
ppm) were given sulfoxaflor in the diet for at least 2 weeks prebreeding
and during breeding, with females continuing on treated diets through gestation
and lactation. As part of these studies, systemic toxicity, reproductive, and
developmental parameters were evaluated. In the reproduction study, rats (n ¼
27; dose ¼ 0, 25, 100, or 400 ppm) were given sulfoxaflor in the diet for
approximately 10 weeks and during breeding, gestation, and lactation for two
generations. A comprehensive array of systemic toxicity, reproductive, and
offspring developmental parameters was evaluated. All four studies followed
the relevant test guidelines and were compliant with good laboratory practice
regulations.
523
Rat cross-fostering study. In the cross-fostering study, groups of 32
female rats were given diets supplying 0 or 1000 ppm sulfoxaflor. The females
were treated for 2 weeks prior to breeding, continuing through breeding,
gestation, and lactation. Mated females were subdivided into two groups,
namely foster and donor dams. The control or sulfoxaflor-treated foster dams
were allowed to deliver live offspring, which were removed as soon as possible
after completion of parturition. Concurrently, control or sulfoxaflor-treated
donor dams underwent cesarean section on GD 21. The newly delivered pups
were tattooed to identify litter of origin and immediately transferred to the
appropriate foster dams.
Two male and two female fetuses from each donor litter obtained via
cesarean section were cross-fostered to a control dam and an sulfoxaflor-treated
dam, both of which had delivered previously and were lactating. Thus, after
cross-fostering was completed, each control and sulfoxaflor-treated lactating
foster dam had mixed litters comprised of two male and two female pups that
originated from control and two male and two female pups that originated from
sulfoxaflor-treated foster dams (total foster litter size of eight). This design
controlled for litter of origin effects and enabled comparison of the survival of
pups exposed to sulfoxaflor during gestation alone or during lactation alone
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with unexposed control pups and pups exposed during both gestation and
lactation.
Rat critical window I and II studies. In the critical window of exposure
studies, sulfoxaflor was given to time-mated pregnant dams at particular
intervals during gestation. In critical window I, groups of 12 time-mated female
rats were given 0 or 1000 ppm sulfoxaflor in the diet. Treated groups were
given 1000 ppm from GD 6–16 or GD 16 parturition. In critical window II,
groups of 10 time-mated female rats were given 0 or 1000 ppm sulfoxaflor in
the diet. Treated groups were given 1000 ppm from GD 16–18, GD 18–20, or
GD 20–22. For both studies, in the offspring, effects on litter size, survival, sex,
body weight, and the presence of gross external morphological alterations were
carefully assessed.
Rabbit neonatal survival study. In the neonatal survival study, groups of
12 rabbits were given 0 or 750 ppm sulfoxaflor in the diet from GD 7 through
parturition. Kits were observed through PND 4 for general appearance,
behavior, and survival.
Receptor Agonism Study Designs
In the receptor agonism assays, sulfoxaflor was incubated with recombinant
rat or human nAChRs on Xenopus oocytes. Mature oocytes were isolated from
adult female X. laevis and their follicular cell layer removed by treatment with
collagenase type I (6 mg/ml) in calcium-free Barth’s solution for 4 h at room
temperature, followed by several washes in calcium-free Barth’s solution. If
necessary, collagenase treatment was followed by manual defolliculation.
Oocytes were then injected with either complementary DNA (cDNA) (to
express human nAChRs) or complementary RNA (cRNA) (to express rat
nAChR subunits) using a Drummond variable volume microinjector in order to
form functional receptors. After injection, oocytes were incubated at 18C in
Barth’s solution for 1–5 days. The choice of cDNA or cRNA was dictated by
the type of plasmid expression vector into which the human and rat nAChR
subunits were cloned. Human nAChRs were cloned into plasmid pcDNA3
(Croxen et al., 2001) downstream from a cytomegalovirus promoter that
permits messenger RNA (mRNA) transcription when injected into the Xenopus
oocyte nucleus (Bertrand et al., 1991; Millar and Lansdell, 2009). The rat
nAChR subunits were cloned into plasmid pSPOoD (Witzemann et al., 1990),
which were then used for in vitro synthesis of mRNA (cRNA). The cRNA was
injected into the oocyte cell cytoplasm where it was translated into protein
(Millar and Lansdell, 2009; Soreq, 1985).
In vitro synthesis of cRNA. Plasmid expression vector constructs
encoding nAChR subunits were linearized by restriction enzyme digestion.
In vitro transcription, to generate cRNA, was then performed using the
mMessage mMachine SP6 transcription kit (Ambion, Huntington, U.K.).
Reactions were carried out according to the manufacturer’s protocol.
Transcripts were recovered by precipitation with propan-2-ol, dissolved in
524 RASOULPOUR ET AL.
nuclease-free water at a final concentration of 0.5 mg/ml and stored at 80C
prior to use.
Functional expression of rat and human nAChRs in Xenopus
oocytes. Two-electrode voltage-clamp recording from Xenopus oocytes was
performed 1–5 days after microinjection with cDNA or cRNA. Oocytes were
placed in a recording chamber and continuously perfused with saline solution
(115mM NaCl, 2.5mM KCl, 1.8mM CaCl2, 10mM 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid, pH 7.3 with NaOH, 235 mOsm). Known
agonists or sulfoxaflor were applied using a computer-controlled perfusion
system (BPS-8; ALA Scientific Inc., Westbury, NY). Two-electrode voltage-
clamp recording was performed using two microelectrodes filled with 3M KCl,
and oocytes voltage clamped at 60 mV using an Axon Geneclamp 500B
amplifier (Molecular Devices, Winnersh, U.K.). Membrane currents were
digitized and stored on computer disk using pClamp software (Molecular
Devices).
Neonatal Rat Hemidiaphragm Contracture Assays
In this assay, sulfoxaflor was evaluated for its ability to induce skeletal
muscle contracture of the diaphragm. Isolated phrenic nerve-hemidiaphragm
preparations from newborn (PND 0) Sprague Dawley rats (supplier, UCL
Biological Services) were used. All experiments were carried out in accordance
with the Animals (Scientific Procedures) Act of 1986 and in accordance with
local ethical approvals. Preparations were constantly superfused at 1.5 ml/min
in a total bath volume of 0.5 ml with physiological salt solution containing (in
mM): NaCl 125, KCl 3, NaHCO3 25, NaH2PO4 1.0, CaCl2 2.5, MgCl2 1.0,
glucose 25 of pH 7.4 when saturated with 95% O2/5% CO2. The muscle was
fixed at the ribs in a recording chamber on an upright microscope, viewed under
low magnification (312.5) via a CCD camera and video monitor, and
a myograph wire attached to the muscle tendon was connected to a Harvard
isometric strain gauge transducer to record muscle tension. The transducer
output was amplified and filtered before digitizing and stored on a computer.
The y-axis gain was varied in order to show twitch tension from different
preparations on a similar scale and so has arbitrary scaling. The phrenic nerve
was stimulated using a bipolar stimulating electrode placed at the nerve entry
point to the muscle with supramaximal rectangular voltage pulses of 0.2 ms
duration at a frequency of 0.5 Hz. Strain gauge transducer and stimulating
electrode were mounted on micromanipulators in order to allow accurate
positioning relative to the muscle. Acetylcholine (ACh) (ICN Biomedicals; Lot
no. 45682), tubocurarine (ICN Biomedicals; Lot no. 85291), and sulfoxaflor
(Dow AgroSciences; Lot no. E2162-34) were applied by manually switching
taps controlling solutions flowing to the inflow manifold of the recording
chamber.
Statistical Analyses
A least squares regression analysis was run with the plasma concentrations
of sulfoxaflor regressed against dose and the quadratic term (squared value of
dose). If there was a significant quadratic term (a £ 0.025), the dataset was
reevaluated with removal of the high dose level. Parental body weights,
gestation and lactation body weight gains, litter mean body weights, feed
consumption, anogenital distance (absolute and relative to the cubed root of
body weight), sperm count, follicle count, percent total and progressively
motile sperm, mean estrous cycle length, and organ weights (absolute and
relative) were evaluated by Bartlett’s test (alpha ¼ 0.01) for equality of
variances. Based upon the outcome of Bartlett’s test, either a parametric or
nonparametric ANOVA was performed and if significant was followed by
a Dunnett’s test or the Wilcoxon Rank-Sum test with Bonferroni’s correction,
respectively. Feed consumption values were excluded from analysis if the feed
was spilled or scratched. In diet studies, statistical outliers (alpha ¼ 0.02) were
identified by the sequential method and were routinely excluded from feed
consumption. Gestation length, age at vaginal opening (females), age at
preputial separation (males), average time to mating, litter size, number of
corpora lutea, and number of implants were analyzed using a nonparametric
ANOVA, and if significant, the Wilcoxon Rank-Sum test with Bonferroni’s
correction was performed. The mating, conception, fertility and gestation
indices, and pregnancy rate were analyzed by the Fisher exact probability test
with Bonferroni’s correction. Survival index, pre- and post-implantation loss,
and fetal alterations were analyzed using the litter as the experimental unit by
the censored Wilcoxon test as modified by Haseman and Hoel with
Bonferroni’s correction. Gestation survival index ¼ % delivered pups alive
at birth. Nonpregnant females, females with resorptions only, females found to
be pregnant after staining of their uteri, females lacking visible implantations,
or females not delivering a litter were excluded from the appropriate analyses.
All analyses were two sided with alpha ¼ 0.05 unless stated otherwise.
RESULTS
Results of the developmental and reproductive toxicity
studies are summarized in Table 1. In the rat reproduction/
developmental toxicity probe study (according to the OECD
421 guideline), dams given 1000 ppm (78 mg/kg/day)
sulfoxaflor lost 93% of their offspring between birth and
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PND 4, whereas dams in the 500 ppm (40 mg/kg/day) group
lost 19% of their offspring by PND 4. These findings were in
the presence of, but not a direct consequence of, decreased
maternal body weights. There were no treatment-related effects
in the 100 ppm (8 mg/kg/day) group.
In order to determine if the offspring deaths were due to
gestation or lactation exposure, a cross-fostering study was
performed. In this study, all offspring from dams given 1000
ppm sulfoxaflor in gestation died by PND 4, irrespective of
whether they were cross-fostered during lactation to control or
treated foster dams (Table 1). Consistent with reduced pup
survival, some offspring were cold to the touch, had bluish skin
(cyanosis), were autolyzed or cannibalized, and had stomachs
void of milk. Conversely, there was no effect on neonatal
survival for pups exposed to sulfoxaflor after birth only. In
addition to pup survival, PND 1 pup body weights from dams
exposed during gestation were significantly lower than
controls. These data demonstrated unequivocally that the effect
of sulfoxaflor on pup survival was due to in utero, not
lactational, exposure.
Two-Generation Reproductive Toxicity Study in the Rat
A two-generation reproductive toxicity study (according to
the OECD 416 guideline) was performed to evaluate the
potential effects of sulfoxaflor (25, 100, or 400 ppm in diet,
corresponding to 1.6, 6.63, or 26.4 mg/kg/day) on male and
female reproductive function, as well as the survival, growth,
and development of the offspring. Parental toxicity at 400 ppm
consisted of increased absolute and relative liver weights in the
P1 (12.8 and 10.9%, respectively) and P2 (6.5 and 7.8%,
respectively) males. This effect on liver weight correlated with
histopathological findings of very slight to slight centrilobular
hepatocyte hypertrophy, often with a very slight increase in
individual cell necrosis of centrilobular hepatocytes. No other
systemic effects were noted at 400 ppm, and there were no
treatment-related effects on P1 or P2 parameters in male or
female rats at 25 or 100 ppm.
 TABLE 1
Summary of Findings From Developmental and Reproductive Toxicity Studies on Sulfoxaflor (X11422208)

Study type (guideline) Doses NOAEL LOAEL Target organ/principal effects at LOAEL

Reproduction/developmental probe in rats
(OECD 421)
0, 100, 500, 1000 ppm Reproduction: 100 ppm (8.1 mkd)
500 ppm (39.5 mkd)
Parental: 100 ppm (8.1 mkd)
Reproduction: Reproduction: 500 ppm-Decreased neonatal
survival. PND 4 survival 81.2% (95.4% in
controls). 1000 ppm-PND 4 pup survival
7.3%.
Parental: 500 ppm Parental toxicity: Decreased body weight
(39.5 mkd) gain at 500 and 1000 ppm in males and
females. Increased liver weight in males at
500 and 1000 ppm. Hepatocellular
hypertrophy in males at 500 and 1000 ppm
and females at 1000 ppm.

NOVEL FETAL MUSCLE nAChR MoA IN RATS

Offspring: 100 ppm (8.1 mkd) Offspring: 500 ppm Offspring: 500 and 1000 ppm-Decreased
(39.5 mkd) neonatal survival (see above). 1000 ppm-
PND 1 pup body weight 25% lower than
controls.
Cross-fostering study in rats Offspring dose Litter: Groups 1 and 2 Litter: Groups 3 and 4 Dam: Reduced feed consumption and body
(gestation/lactation); (negative control and (gestation exposure weight gain when dams given 1000 ppm
Group 1: 0/0 ppm, lactation exposure only) only and positive Litter: All offspring exposed prior to birth
Group 2: 0/1000 ppm, had no effects control) had decreased died by PND 4. No effect on neonatal
Group 3: 1000/0 ppm, neonatal survival survival with lactation-only exposure.
Group 4: 1000/1000 ppm Therefore, pup survival effect due to in utero
exposure.
Two-generation reproduction in rats 0, 25, 100, 400 ppm Reproduction: 100 ppm (6.63 mkd) Reproduction: 400 ppm Reproduction: Decreased neonatal survival
(OECD 416) (26.4 mkd) (~2–5%)
Parental: 100 ppm (6.63 mkd) Parental: 400 ppm Parental toxicity: Increased liver weight in
(26.4 mkd) males at 400 ppm with correlating
histopathologic changes
Offspring: 100 ppm (6.63 mkd) Offspring: 400 ppm Offspring: Decreased neonatal survival and
(26.4 mkd) a slight delay in preputial separation (puberty
onset) in F1 males
Developmental toxicity in rats (OECD 414) 0, 25, 150, 1000 ppm Dam: 150 ppm (11.5 mkd) Dam: 1000 ppm Dam: 1000 ppm-reduced feed consumption
(70.2 mkd) and body weight gain; increased liver weight
Litter: 150 ppm (11.5 mkd) Litter: 1000 ppm Litter: Decreased fetal body weight; fetal
(70.2 mkd) abnormalities (forelimb flexure, bent clavicle,
hindlimb rotation, convoluted/hydroureter)
Developmental toxicity in rabbits 0, 30, 150, 750 ppm Dam: 150 ppm (6.6 mkd) Dam: 750 ppm Dam: Decreased feed consumption, body
(OECD 414) (31.9 mkd) weight gain, and fecal output
Litter: 750 ppm (31.9 mkd) Litter: > 750 ppm Litter: No treatment-related effects
Neonatal survival study in rabbits 0, 750 ppm Dam: N/A Dam: 750 ppm Dam: Decreased feed consumption and body
weight gain.
Litter: 750 ppm Litter: None Litter: None

Note. NOAEL, No-Observed-Adverse-Effect Level. LOAEL, Lowest-Observed-Adverse-Effect Level.

525
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526 RASOULPOUR ET AL.

TABLE 2
Neonatal Offspring Limb and Clavicle Abnormalities Resulting From Sulfoxaflor (X11422208) Exposure

Critical window 2 Critical window 1 Developmental toxicity

Treatment period GD 20–22/LD 0 GD 16–birth GD 6–21

Applied dose (ppm)
Avg. TMI (mg/kg/day)
Internal dose (lg/g)
Forelimb flexureb
Hindlimb rotation
Bent clavicle
1000 1000 1000
35.7 38.6 70.2
D 5.41–16.1a D 32.1–43.2 D 35.25 ± 5.4; F 30.00 ± 5.3
P 7/96 (7.3%)c; L 4/8 (50.0%)c P 50/143 (35.0%)c; L 11/12 (91.7%)c F 122/295 (41.4%); L 23/24 (95.8%)
P 11/96 (11.5%)c; L 6/8 (75.0%)c P 19/143 (13.3%)c; L 8/12 (66.7%)c F 12/295 (4.1%); L 7/24 (29.2%)
P 0/86 (0%)d; L 0/8 (0%)d P 0/49 (0%)d; L 0/7 (0%)d F 40/133 (30.1%); L 23/24 (70.8%)

Notes. D, dam; F, fetus; L, litter; N/A, not applicable; P, pup; TMI, test material intake; LD, lactation day.
a
Three of the four sampled rats had undergone parturition prior to blood collection.
b
A severe, > 90 persistent flexure at the wrist or any flexure that cannot straighten.
c
Evaluated in surviving pups on PND 0.
d
Evaluated in surviving pups on PND 4.

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Reproductive effects were limited to 400 ppm and consisted cyanosis. Administration of 150 or 25 ppm sulfoxaflor in
of a marginal decrease in neonatal survival (~2–5%) in both rodent feed produced no treatment-related maternal toxicity
generations; this in turn led to a 2–3% lower incidence of live and no indications of embryo/fetal toxicity or developmental
pups prior to culling on PND 4 relative to controls (Table 1). toxicity.
Taken together, administration of greater than, or equal to, 400
ppm sulfoxaflor in the diet of rats during gestation led to Developmental Toxicity Studies in the NZW Rabbit
decreased neonatal survival with robust two-generation reproduc-
Interestingly, the fetal abnormalities and decreased off-
tive toxicity data indicating the lower end of the dose
spring survival noted in rats were not observed in the NZW
response at 400 ppm (26.4 mg/kg/day) sulfoxaflor, which
rabbit despite similar achieved maternal and fetal plasma
induced a marginal increase in neonatal pup loss in both
levels of sulfoxaflor between the species. In the rabbit, fetal
generations and a no-observed-effect level (NOEL) of 100 ppm
(6.63 mg/kg/day). sulfoxaflor plasma concentrations of does given 750 ppm
(31.9 mg/kg/day) of test material had an average of 21.2 lg/g
(range: 16.8–25.8 lg/g) compared with 24.8 lg/g (range:
Prenatal Developmental Effects in the Rat
21.7–27.5 lg/g) and 30 lg/g (range: 24.0–35.5 lg/g) in rats
In the rat dietary prenatal developmental toxicity study (25, given 1000 ppm sulfoxaflor in the cross-fostering (75 mg/kg/
150, or 1000 ppm in diet, corresponding to 1.95, 11.5, or 70.2
mg/kg/day; according to the OECD 414 guideline), adminis-
tration of 1000 ppm sulfoxaflor in rodent feed resulted in
maternal and developmental toxicity (Tables 1 and 2). Maternal
toxicity consisted of decreases in body weight and body weight
gains, relative to controls, with concomitant decreased feed
consumption and increased relative liver weights.
Developmental toxicity was indicated by decreases in fetal
body weight and gravid uterine weight. In addition, fetuses
from dams given 1000 ppm sulfoxaflor had external and
visceral abnormalities in the form of forelimb flexure (a greater
than 90 bend in the wrist, shown in Fig. 1) and hindlimb
rotation, as well as convoluted/hydroureters with no associated
dilatation of the renal pelvis. Convoluted/hydroureters are
regarded in the literature as a minor variant known to be
reversible (Solecki et al., 2003). Skeletal examination revealed
no structural changes associated with the external limb FIG. 1. Photomicrographs of representative limb contracture (e.g., forelimb
abnormalities, but there was an additional finding of bent flexure) (B) and bent clavicles (D) in neonatal offspring of rat dams exposed
(misshapen) clavicle. Consistent with neonatal pup loss, fetuses to 1000 ppm sulfoxaflor (X11422208) relative to controls (A and C). In the
guideline developmental toxicity study, dams given 1000 ppm sulfoxaflor in
from this study were easily distinguishable from other dose the diet from GD 6–21 had offspring with an incidence of forelimb flexure of
groups due to contracted posture of limbs as well as decreased 42% of fetuses in 96% of litters and incidence of bent clavicle of 30% of
activity with some fetuses observed with labored breathing and fetuses in 71% of litters.
 NOVEL FETAL MUSCLE nAChR MoA IN RATS 527

day) and prenatal developmental (78 mg/kg/day) toxicity

studies, respectively. In the NZW rabbit definitive prenatal
developmental toxicity study (OECD 414), groups of 26 time-
mated female rabbits received sulfoxaflor at dietary concen-
trations of 0, 30, 150, or 750 ppm (1.3, 6.6, or 31.9 mg/kg/
day) on GD 7–28. Animals in the 750-ppm dose group
exhibited treatment-related maternal toxicity in the form of
decreased feces in 7 of 26 animals, decreased mean body
weight gain (55%) from GD 7–13, decreased mean body
weight gain (12%) throughout treatment (GD 7–28), and
decreased mean feed consumption (8–21%) from GD 7–17.
There was no treatment-related maternal toxicity for animals
in the 30- or 150-ppm dose groups. There was no treatment-
related developmental toxicity in any dose group (Table 1).
The daily systemic dose of sulfoxaflor on GD 27–28 was FIG. 2. Dose-response curves with nonlinear regression analyses for the
percent of neonatal death or limb contracture in rats. Study type abbreviations:
dose proportional as indicated by near identical mean dose– 1st gen and 2nd gen, 1st and 2nd generations of the two-generation

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corrected AUC24h values. Levels of sulfoxaflor in maternal
and fetal blood were similar.
Neonatal Survival Study in Rabbits
In order to determine if the reduced neonatal survival
observed in rats would occur in rabbits, a neonatal survival
study was performed where groups of 12 litter-experienced
time-mated female NZW rabbits were given 0 or 750 ppm
sulfoxaflor from GD 7 through the initiation of parturition (GD
31–32; 25–26 consecutive days). Actual test material intake in
the 750-ppm group was 29 mg/kg/day during GD 7–28. All F0
females were allowed to deliver and rear their offspring to LD
4 (Table 1). No test substance–related effects were observed on
the mean number of offspring born, offspring survival, or the
general physical condition of the offspring. In conclusion, there
were no effects in the rabbit developmental toxicity study or in
the neonatal survival study at the high-dose level of 750 ppm
(29 mg/kg/day).
MoA Studies
As sulfoxaflor revealed significant developmental effects
in the form of fetal abnormalities and neonatal offspring death
in rats (Table 1), a series of studies was conducted to identify
the MoA for these developmental effects. Given that the
insecticidal target for sulfoxaflor is the insect nAChR, and the
fetal limbs had a contracted appearance in the absence of
underlying skeletal changes, it was hypothesized that the MoA
for sulfoxaflor-induced developmental toxicity in the rat was
mediated by the muscle nAChRs within the limbs and
diaphragm. Specifically, sulfoxaflor agonism on the fetal
muscle nAChR in the limb or diaphragm leading to limb
abnormalities and death, respectively.
As the fetal abnormalities and neonatal death occurred at the
same dose (1000 ppm) with similar incidences (75.6 and
93.0%, respectively), it was considered that one MoA was most
likely to be responsible for both effects. In order to evaluate the
plausibility of a single MoA, data from all sulfoxaflor studies
reproductive toxicity study; 421-like, reproductive toxicity screening study;
X-foster, cross-foster study; CW 1, critical window study phase 1; CW 2,
critical window study phase 2; DNT, developmental neurotoxicity study; DT,
prenatal developmental toxicity study.
with observations of the developmental toxicity effects were
plotted as a percentage of affected offspring with respect to
received mg/kg/day dose to the dam (Fig. 2). Nonlinear
regression dose-response curves were created for the neonatal
death and limb abnormality data with R2 values of 0.93 and
0.91, respectively. As shown in Figure 2, these fitted curves for
the two effects are nearly identical, consistent with a single
MoA for these effects.
Agonism of sulfoxaflor on the rat muscle-type nAChR. In
mammalian muscle tissue, the pentameric nAChRs expressed
at the neuromuscular junction are composed of a b1 subunit,
a d subunit, two a1 subunits, and either a c (fetal) or e (adult)
subunit (Fig. 3). In rats, the switch from the fetal to adult
subtype occurs during the first 2 weeks of postnatal life
(Yampolsky et al., 2008). To determine if sulfoxaflor is an
agonist on the muscle nAChR, fetal rat nAChR subunits (fetal
subtype (a1)2b1cd or adult subtype (a1)2b1de) were recombi-
nantly expressed in Xenopus oocytes, which, following
injection, combine to form functional nAChRs. This experi-
mental method has been used in many hundreds of research
FIG. 3. Models of the subunit composition of fetal- versus adult-type
muscle nAChRs. Note the switch of the fetal-specific c subunit to the adult e
subunit, which occurs in the early postnatal rat.
528 RASOULPOUR ET AL.

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FIG. 4. Agonist activation of nAChRs expressed in Xenopus oocytes. Data
are shown for the rat fetal (a1)2b1cd nAChR (A) and rat adult (a1)2b1de
nAChR (B). AChRs were expressed by microinjection of cRNA in Xenopus
oocytes. Dose-response curves are shown in which agonist-evoked responses
are normalized to the maximal response detected with the endogenous agonist,
acetylcholine (ACh). Data points are means of 3–7 responses.
FIG. 5. Critical window of exposure experiments (phase 1 and phase 2). In
these experiments, groups of time-mated rats were given 1000 ppm sulfoxaflor
(X11422208) from: GD 6–16 (group 1), GD 16–birth (group 2), GD 16–18
(group 3), GD 18–20 (group 4), or GD 20–22 (group 5). Offspring were
evaluated for limb abnormalities and postnatal survival until PND 4, then
evaluated for hydro/convoluted ureter and bent clavicles postmortem. Only
groups given sulfoxaflor near the time of birth (groups 2 and 5) had the
developmental effects, indicating a pharmacologic MoA. No offspring had any
indication of bent clavicles at PND 4.

study at a dose level of 1000 ppm given in the diet to dams on

publications to examine the functional properties of ion
GD 6–21 (Table 2). This clear effect dose level was used in
channels such as the nAChR (Dascal, 1987). As sulfoxaflor
a two-phase critical window of exposure study. In the first
is not metabolized in tested laboratory animals, there were no
phase of this study, sulfoxaflor treatment was divided into two
concerns of proximate metabolites directly causing the de-
periods: GD 6–16 and GD 16–birth (Fig. 5) to determine if the
velopmental toxicity. In this experiment, ligand-induced
effects occurred during primary organogenesis. These divisions
activation on the rat fetal muscle nAChR (a1)2b1de was
were based on the fact that in the rat, primary organogenesis is
measured by ion flux through the channel following agonist
complete by GD 15, and the nAChR develops fetal muscle-
binding and receptor activation. Acetylcholine (ACh) is the
type subunit expression between GD 15 and 17 (Kues et al.,
native agonist and was used as a positive control ligand. The
1995) resulting in the onset of synchronized fetal limb
results (Fig. 4A) clearly show that sulfoxaflor is a partial
movements (Robinson and Smotherman, 1988) and diaphrag-
agonist to the rat fetal muscle nAChR, thus supporting the
matic responsiveness (Bennett and Pettigrew, 1974) between
proposed hypothesis. Also presented on this graph is a soil
GD 16 and 17, the latter being important for the transition to
metabolite of sulfoxaflor (X11719474) that, despite being only
extrauterine respiration. Dams given 1000 ppm sulfoxaflor
one functional group different from the parent molecule, did
from GD 16–birth (group 2) had offspring effects of neonatal
not show agonism. Interestingly, this soil metabolite also did
death and limb abnormalities, whereas exposure from GD 6–16
not cause fetal abnormalities or neonatal offspring death when
(group 1) did not induce offspring effects. These data indicated
given to rats in guideline studies at dose levels much higher
that the critical window of exposure for sulfoxaflor-induced
than the parent (data not shown). In addition to evaluating the
offspring effects was between GD 16 and birth, consistent with
fetal subtype, the adult form of the rat muscle nAChR was
the time course of functional expression of the fetal muscle
tested, but no agonist action of sulfoxaflor was detected (Fig.
nAChR in rodents (Yampolsky et al., 2008). In the second
4B); thereby, ruling out the possibility that the fetal effects
phase of this experiment, the GD 16–birth window (group 2
could be mediated by the muscle nAChR in the maternal
used in phase 1) was further divided into three 48-h windows
uterus or that the adult nAChR subtype led to the postnatal
of exposure starting on GD 16 (group 3), GD 18 (group 4), and
effects.
GD 20 (group 5). Results from phase 2 of this experiment
Critical window for sulfoxaflor-induced effects. Sulfoxaflor- revealed that dams given 1000 ppm sulfoxaflor in group 5 (GD
induced effects occurred in the prenatal developmental toxicity 20–22/LD 0) had offspring effects of neonatal death and limb
 NOVEL FETAL MUSCLE nAChR MoA IN RATS 529

TABLE 3
Neonatal Offspring Death Resulting From Sulfoxaflor (X11422208) Exposure

Applied Avg. TMI Internal dose Incidence of

Study type Treatment period dose (ppm) (mg/kg/day) (lg/g) pup death (%)

Two-generation reproduction 10 weeks prior to breeding-PND 21 400 29.2 15.9 (LD 4) 4.5
(two generations)
Reproduction screening 2 weeks prior to breeding-PND 21 500 30.3 N/A 18.8
Critical window 2 GD 20–22/LD 0 1000 35.7 5.41–16.1a 10.4
Critical window 1 GD 16–birth 1000 38.6 32.1–43.2 (GD 21) 53.1
Reproduction screening (OECD 421) 2 weeks prior to breeding-PND 21 1000 62.0 14.3–41.9 (LD 4) 92.7

Note. TMI, test material intake.

a
Three of the four sampled rats had undergone parturition prior to blood collection.

abnormalities, whereas groups 3 and 4 had no observed effects other test compounds such as caffeine (Collins et al., 1987)

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(Tables 2 and 3). These data demonstrated that the critical
period of developmental susceptibility to sulfoxaflor-induced
fetal abnormalities and reduced neonatal survival effects
occurred shortly before birth. In summary, the effects did not
occur due to sulfoxaflor exposure during organogenesis, but
instead resulted from exposure at the end of pregnancy, which
is consistent with onset of fetal limb movement and
development of functional nerve receptor-muscle networks
(Bennett and Pettigrew, 1974).
Reversibility of effects. Within the critical window of
exposure studies in the rat, it was noted that surviving
offspring from the affected groups (groups 2 and 5; GD 16–
birth and GD20–birth, respectively) born with limb abnor-
malities fully reversed shortly after withdrawal of maternal
dietary exposure to sulfoxaflor (Table 4). Full reversal of
the limb abnormalities occurred the day after birth in some
cases and was evident for all remaining surviving animals by
PND 4; reversal also occurred in some animals that sub-
sequently died before PND 4. Likewise, the skeletal finding
of bent clavicle, which had a high incidence (30.1% of
fetuses in 70.8% of litters) in the definitive developmental
toxicity study, was not present (0% of fetuses) in this
experiment at necropsy on PND 4 (Table 2). Postnatal
remodeling of skeletal abnormalities has been shown with
TABLE 4
Reversibility of Limb Abnormalities
Critical window Critical window
Study type 1 study
Treatment period GD 16–birth GD 20–22/LD 0
Reversals between Reversed limb effects/total reversed
PND 0 to 1 11/21
PND 1 to 2 6/21
PND 2 to 3 3/21
PND 3 to 4 1/21
Total reversals 21/21
and ethylene glycol (Marr et al., 1992). This provides further
evidence that bent clavicles have the ability to reverse during
the early postnatal period, which suggests that they are
transient alterations consistent with a pharmacological MoA
of sulfoxaflor.
Sulfoxaflor-induced diaphragm muscle contracture ex
vivo. To determine whether sulfoxaflor could cause contrac-
tion of the diaphragm by activation of the muscle nAChR,
experiments using the rat phrenic nerve-diaphragm muscle
preparation from PND 0 neonates were conducted. In testing
the model system, the ex vivo neonatal rat diaphragm muscle
was shown to maintain its physiological function as it was
responsive to phrenic nerve stimulation and to acetylcholine
(ACh, 100lM), and these responses could be specifically
blocked by the selective muscle nAChR antagonist, tubocura-
rine (10lM; data not shown). In a series of experiments,
sulfoxaflor (1mM) consistently produced robust contractures
of the neonatal rat diaphragm muscle with a corresponding
decrease in contracture-related muscle twitch tension evoked
by phrenic nerve stimulation (Fig. 6). In five of five pre-
parations tested, the muscle twitch response (contracture
twitch tension) decreased to 34 ± 3.2% of control during
responses to 1mM sulfoxaflor, similar to ACh. As with ACh,
the sulfoxaflor-induced muscle contraction was blocked by
a selective muscle nAChR antagonist (tubocurarine; 10lM),
demonstrating the specificity of this effect for agonism on the
postjunctional neuromuscular junction nAChR. In addition,
prolonged application of sulfoxaflor (7 min) resulted in sus-
tained contracture of the diaphragm muscle and a reduction of
2 study the contracture-related twitch tension by 82% of the baseline
twitch.
Agonism experiments with sulfoxaflor on the human muscle-
5/7 type nAChR. In order to address the question of human
2/7 relevance, the Xenopus oocyte nAChR agonism experiments
0/7 were repeated with recombinant human fetal muscle nAChR
0/7
(Fig. 7). In addition to testing the recombinant human fetal
7/7
muscle nAChR, the recombinant human adult muscle nAChR
530 RASOULPOUR ET AL.
FIG. 6. (A) Preapplication of 10lM tubocurarine (Tubo) effectively blocks
the neonatal rat diaphragm muscle twitches and antagonizes responses to
100lM (not shown) or 1mM sulfoxaflor (X11422208). (B) Prolonged
application (7 min) of 1mM sulfoxaflor shows a sustained contracture by the
diaphragm muscle and decreased muscle twitch response.
was tested because, unlike rats (Yampolsky et al., 2008), the
switch from the ‘‘fetal’’ to ‘‘adult’’ subtype occurs during
gestation in humans (Hesselmans et al., 1993).
These experiments demonstrated that although the native
ligand, acetylcholine, is able to induce an agonist response with
expression of human fetal or human adult nAChR subtypes,
sulfoxaflor is not an agonist to the human fetal receptor
(Fig. 7). In summary, these experiments demonstrated that
sulfoxaflor is a partial agonist at the rat fetal muscle nAChR but
not to the corresponding human receptors or the adult nAChR
subtype in rats.
DISCUSSION
When developing the MoA to investigate the fetal effects
(forelimb flexure, hindlimb rotation, and bent clavicle) and
neonatal death seen in rats treated with sulfoxaflor, the first
challenge was to identify a biologically plausible MoA
hypothesis for the effects in rats. As the sulfoxaflor
insecticidal mechanism is agonism on the insect nAChR,
attention was drawn to the mammalian nAChR as a potential
target and cause for these effects. Interestingly, sulfoxaflor’s
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FIG. 7. Agonism on recombinant human fetal (a1)2b1cd and adult
(a1)2b1ed nAChR for endogenous ligand (acetylcholine) and sulfoxaflor
(X11422208). AChRs were expressed by microinjection of cDNA in Xenopus
oocytes. Dose-response curves are shown in which agonist-evoked responses
are normalized to the maximal response detected with the endogenous agonist,
acetylcholine (ACh). Note that unlike the rat nAChR, sulfoxaflor is not an
agonist for the human fetal or adult muscle nAChR subtype. Data points are
means of 3–7 responses.
primary environmental metabolite (X11719474) was known
not to bind to the insect nAChR (Watson and Young, 2010)
and did not produce neonatal pup loss or developmental
effects at dose levels 5–10 times higher than the sulfoxaflor
effect levels (data not shown). In both invertebrate and
vertebrate species, nAChRs are important neurotransmitter
receptors (Millar and Gotti, 2009; Sattelle et al., 1979) and
comprise a diverse family of oligomeric cell surface receptors
assembled from five (of many) subunits that coassemble in
a doughnut-shaped arrangement (Millar and Denholm, 2007;
Millar and Gotti, 2009) (Fig. 3). In the center of the pen-
tameric arrangement of subunits is a cation-selective ion
channel, whereby binding of the endogenous neurotransmit-
ter, acetylcholine, or other agonists stabilizes the open
conformation allowing the influx of cations (mainly Naþ
and Ca2þ) into the cell (agonism). There are two major types
of mammalian nAChR, neuronal and muscle. In mammalian
muscle cells, nAChRs are expressed at the neuromuscular
junction and are composed of five nAChR subunits (two a1,
 NOVEL FETAL MUSCLE nAChR MoA IN RATS
FIG. 8. Proposed key events within the MoA for sulfoxaflor (X11422208)-
induced developmental toxicity effects. Sulfoxaflor binds (key event 1) and is
an agonist (key event 2) to the rat fetal muscle nAChR. This persistent agonism
leads to sustained skeletal muscle contracture (key event 3), which in turn leads
to the developmental toxicity effects.
b1, d, and either c or e subunits). Transcription of the c and e
subunits is differentially regulated during development, with
the c subunit expressed in fetal muscle and the e subunit
expressed in adult muscle (Mishina et al., 1986) (Fig. 8).
Muscle nAChRs contain two agonist-binding sites, one at the
interface of the a1 and d subunits and another at the interface
of the a1 and c (or e) subunits (Arias, 2000).
Findings Consistent With Agonism on Fetal Muscle-Type
nAChR
In the muscle, there is a single fetal and a single adult
nAChR subtype. In humans, the switch from fetal to adult
receptor subtype occurs prior to birth (Hesselmans et al., 1993)
versus shortly after birth (Yampolsky et al., 2008) in rodents.
Evidence indicates that the fetal and adult muscle-type
nAChRs each represent a separate single homogeneous
population containing the same subunit combinations assem-
bled in a fixed stoichiometry and order around the central ion
channel pore (Karlin, 2002). Fetal examination at cesarean
section in the rat developmental toxicity study indicated
involvement of muscle as the limb abnormalities, notably
forelimb flexure, appeared to reflect contracture of the skeletal
muscle. Subsequently, misshapen (bent) clavicle was detected
at skeletal examination, and this was also considered
consistent with the evident forelimb muscle contracture.
Regarding the neonatal death, the diaphragm is the most
important muscle responsible for initiation and maintenance of
breathing at birth and hence sustained contracture and
associated compromised breathing movements of this muscle
would also be a consistent and plausible explanation for the
pup loss and cyanosis at birth (Greer et al., 2006). The critical
window of exposure experiment data is consistent with
a pharmacological action of sulfoxaflor at the muscle receptor
and is also consistent with neonatal death as the diaphragm
muscle is a critical component for normal neonatal respiration,
a system which sulfoxaflor agonism on this muscle receptor
would adversely affect. In addition, fetuses ‘‘practice’’
breathing at the end of gestation (Kobayashi et al., 2001);
therefore, it is plausible that functional alterations in the
531
diaphragm in utero could also delay this practice and adversely
affect breathing at birth.
Agonism on the Rat Fetal-Type Muscle nAChR
Rat fetal-type nAChR subunits ((a1)2b1dc) or adult-type
nAChR subunits ((a1)2b1de) were expressed as functional
recombinant receptors by microinjection of cRNA in Xenopus
oocytes. Electrophysiological data obtained from such recep-
tors have been demonstrated to be similar to data obtained from
native nAChRs expressed in muscle tissue (Mishina et al.,
1986). Due to its correlation with in vivo functionality, the
method has been used in hundreds of research publications to
examine the functional properties of ion channels such as the
nAChR (Dascal, 1987). It has also been previously demon-
strated to confirm agonism of nAChR ligands (Cooper et al.,
1996), some of which have been demonstrated to cause limb
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contracture abnormalities (Forsyth et al., 1996). In the current
studies, functional nAChR responses (membrane currents)
were confirmed via application of the endogenous agonist
ACh. Clear agonist-evoked responses were observed with
sulfoxaflor at the rat fetal-type nAChR. Consistent with the
dose-response data from the sulfoxaflor binding experiments,
thresholded, dose-dependent agonism on the fetal-type muscle
nAChR was demonstrated; the lowest tested concentration
having no agonism, whereas incubation of the oocytes with
higher concentrations of sulfoxaflor showed increasing
agonism.
Evidence for the specificity of sulfoxaflor’s agonism on the
fetal-type muscle nAChR comes from experiments, which
showed that despite normal ACh agonist responses, no
agonism occurred at the rat adult-type muscle nAChR up to
the limit of solubility for sulfoxaflor. Furthermore, an
sulfoxaflor soil metabolite, X11719474, which is known to
be inactive at the insect nAChR (Watson and Young, 2010),
did not induce agonism on the fetal-type muscle nAChR.
Consistent with this lack of in vitro agonism, X11719474 was
previously demonstrated to produce no neonatal pup loss or
developmental effects at dose levels 5–10 times higher than the
sulfoxaflor effect levels.
There are several precedents for species-selective agonist
activity of nAChR ligands. For example, nicotine is an agonist
of neuronal a3b2 nAChRs from rat but is an antagonist of
a3b2 nAChRs from chick (Hussy et al., 1994). Indeed, a single
amino acid difference in the a3 subunit of rat and chick can
account for the selective agonist effect of nicotine on rat and
chick a3b2 nAChRs (Hussy et al., 1994). A similar situation
has been reported for TMAQ, a nicotinic agonist that is
selective for neuronal nAChRs containing a b4 subunit (Young
et al., 2007). TMAQ binds to both human and rat a3b4
nAChRs but acts as an agonist only on human a3b4 nAChRs
(Young et al., 2007). The selective agonist activity of TMAQ
for human a3b4 nAChRs can be explained by two amino acids
differences between the human and rat b4 subunit (Young
532 RASOULPOUR ET AL.
et al., 2007). Comparison of the amino acid sequence of the rat
and human c subunit revealed that although the two subunits
are similar (approximately 90% identical), they contain 53
amino acid differences (Supplementary fig. 1). Given the
precedent that one or two amino acid differences can confer
species-selective agonist activity upon nicotinic ligands, it
seems entirely plausible that the differences in agonist activity
of sulfoxaflor can be explained by differences in the amino acid
sequence of the rat and human nAChR c subunits. The c and e
subunits show even greater sequence differences that the
human and rat c subunit (even from the same species).
Supplementary figure 2 shows an alignment of the c and e
subunits from rat. These subunits share only about 50%
identity in amino acid sequence. To date, there have been no
reports of the molecular cloning of nAChR subunits from
rabbit; therefore, sequence alignment in this species is not
possible.
Sustained Muscle Contracture
Toxicokinetic blood analyses across in vivo study types
strongly suggest that blood concentrations of sulfoxaflor are
maintained at steady-state levels due to continuous exposure
via the diet. As sulfoxaflor is not metabolized in mammals and
readily diffuses into muscle from the blood (data not shown), it
is predicted that muscle sulfoxaflor concentrations would also
be maintained at a steady state as long as dietary treatment
continued. At the location of the neuromuscular junction
nAChR, unlike ACh, which undergoes tightly regulated
synaptic vesicle release followed by rapid hydrolysis by
acetylcholinesterase (AChE), sulfoxaflor would remain at the
nAChR in the synaptic cleft due to its lack of hydrolysis by
AChE, and thus, receptor occupancy of sulfoxaflor would only
be limited by association/dissociation kinetics of the molecule
and not by removal from the receptor endplate region (as with
ACh). Thus, upon fetal-type muscle nAChR activation, an
sulfoxaflor-induced muscle contracture would be sustained for
as long as sufficient sulfoxaflor molecules remain available for
receptor binding, which is consistent with the observed
experimental evidence.
To directly assess muscle contracture at the neonatal
diaphragm, sulfoxaflor was tested for agonist action on isolated
phrenic nerve-hemidiaphragm preparations (Bulbring, 1946)
from newborn rats. Since its introduction, the isolated phrenic
nerve-hemidiaphragm preparation has become established as
the standard nerve-muscle preparation for mechanistic inves-
tigations of drug action at the mammalian neuromuscular
junction (see for example, Fortier et al., 2001; Gibb and
Marshall, 1984, 1986, 1987; Hubbard and Wilson, 1973;
Liley and North, 1953; Wareham et al., 1994). The value of
the preparation rests with the fact that it is amenable to both
muscle tension and electrophysiological measurements and, as
the main muscle involved in breathing (Vander et al., 2001),
the preparation is routinely used to investigate responses of
respiratory muscle (including impairment) to pharmacological
test materials (e.g., muscle relaxant drugs used in surgery)
(Bowman, 1990; Fortier et al., 2001; Gibb and Marshall,
1986, 1987). The phrenic nerve-hemidiaphragm experiments
conducted with sulfoxaflor demonstrated a consistent concen-
tration-dependent contracture of the fetal-type diaphragm
muscle with prolonged application of sulfoxaflor causing
a sustained muscle contracture. These experiments additionally
demonstrated clear specificity as the contracture was com-
pletely reversible upon removal of sulfoxaflor. In addition, the
sulfoxaflor-induced contracture was blocked by coexposure
with the highly selective muscle-type nAChR antagonist,
tubocurarine (Wenningmann and Dilger, 2001), showing that
the contracture induced by sulfoxaflor is mediated via muscle
nAChR activation, rather than a separate mechanism (more
specifically, a postreceptor mechanism).
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Based on these results, the contracture-related decrease in
muscle twitch tension could account for the breathing
difficulties observed in some neonatal rats after birth because
breathing requires brief nerve-evoked contractions of the
diaphragm and there is a well established direct correlation
between muscle twitch block and inhibition of mammalian
muscles responsible for breathing (Kobayashi et al., 2001). The
proposed mechanism behind the muscle contracture is through
sustained agonism of the fetal muscle nAChR by sulfoxaflor.
Pharmacologically, sustained agonism without receptor de-
sensitization would lead to the observed contracture due to
buildup of calcium ions in the sarcoplasm following failure of
the Caþþ/ATPase membrane-bound pump (Harper, 2003).
Findings Inconsistent With Neuronal nAChR Agonism
One of the considered alternative MoAs for the sulfoxaflor-
mediated effects was whether or not agonism at central neuronal
nAChRs, and not direct action at the muscle nAChR, could be
the initiating key event. Through a weight of evidence approach,
which is briefly outlined below, this possibility was discounted.
Based on the short duration of prenatal exposure necessary
and sufficient to cause these effects, the target for develop-
mental toxicity with sulfoxaflor is the fetus, which has a single
muscle-type nAChR. Interestingly, there are upward of 25
known neuronal nAChR, which are comprised of different
subtypes that can have different responses to the same ligand.
Of particular importance is that in contrast to the muscle-type
nAChR with fetal and adult type, there is no postnatal switch in
the subunit composition of neuronal nAChRs. Therefore, if
neuronal nAChR agonism led to neonatal death via an effect on
respiration, or limb abnormalities via a central mechanism,
effects on respiration or muscle contracture in adults would
also be expected. For sulfoxaflor, at various life stages in the rat
(during lactation, weaning, adolescence, and in adults), there
was never an effect on respiration or muscle contracture at
dietary levels exceeding a maximum tolerated dose (e.g., up to
11,000 ppm, which is almost 303 above the neonatal effect
 NOVEL FETAL MUSCLE nAChR MoA IN RATS
lowest-observed-effect level [LOEL]). Nor were there any
neuronal nAChR–mediated clinical signs in dietary neurotox-
icity studies.
In considering a possible neuronal nAChR agonist effect, the
profile of toxicity with studies using agonists to these receptors
was examined. Nicotine, the archetypal neuronal nAChR
agonist, is a full agonist of the most widespread neuronal
nAChR (a4b2) and does not cause the same neonatal effects as
sulfoxaflor. In addition, sulfoxaflor does not cause effects on
the fetal lung, which is a known outcome of neuronal nAChR
activation (Kobayashi et al., 2001). Taken together, although
there is no evidence for sulfoxaflor causing neonatal death or
limb abnormalities via neuronal nAChR agonism, there is
consistent evidence for a single MoA for all developmental
effects that is mediated by agonism at the fetal muscle-type
nAChR.
Weight of Evidence Integration
During the conduct of the mammalian developmental and
reproductive toxicology studies to support the safety assess-
ment of a new agricultural molecule sulfoxaflor, developmental
effects were observed in rats following exposure to doses
greater than, or equal to, 400 ppm sulfoxaflor in the diet. These
effects fell into categories of fetal abnormalities (primarily
forelimb flexure and hindlimb rotation) and reduced neonatal
offspring survival.
Fetal limb abnormalities and bent clavicle suggested muscle
contraction, which is a process mediated by the muscle-type
nAChR. Neonatal offspring death is consistent with agonism
on the muscle-type nAChR, most notably the diaphragm. The
most plausible cause of both effects was sustained mammalian
fetal muscle-type nAChR agonism causing muscle contracture.
Around birth, the rat diaphragm muscle fiber endplate region
is multiply innervated, and this gradually reduces during the
first 2 weeks after birth to achieve the mature focal innervations
(Bennett and Pettigrew, 1974). In contrast, during the period
beginning about 1 week before birth until a week after birth, rat
skeletal muscle fibers express both neuromuscular junction and
extrajunctional nAChRs spread along the whole extent of the
muscle fiber surface (Sanes and Lichtman, 1999; Yampolsky
et al., 2008). These transient physiological conditions also
make the near-term and neonatal rat muscle more likely to
respond to nAChR agonists with a sustained contracture
(Maclagan and Vrbova, 1966).
The lowest NOEL for the developmental effects was 100
ppm (6.63 mg/kg/day) from the two-generation reproduction
study in the CD rat based on marginally reduced (2–5%)
neonatal survival at 26.4 mg/kg/day. Despite similar achieved
internal doses between rats and rabbits (i.e., similar maternal/
fetal plasma concentrations of sulfoxaflor in both species), the
developmental effects in the rat were not observed in the rabbit
studies. Throughout the course of the subsequent studies
conducted to elucidate the MoA for these findings, it was
533
revealed that (1) sulfoxaflor exposure in rats just prior to birth
was sufficient to induce the effects, (2) exposure throughout
organogenesis (GD 6–15) did not cause the effects, (3) the
effects are consistent with a pharmacological MoA, which is
coincident with establishment of a functional fetal muscle
nAChR and fetal limb movement, (4) sulfoxaflor is an agonist
to neonatal rat diaphragm muscle via the fetal-type muscle
nAChR, (5) the rat fetal abnormalities are reversible transient
alterations that completely resolve in surviving pups by PND
4, and (6) that sulfoxaflor is an agonist at the rat fetal muscle-
type nAChR but not human fetal or adult-type muscle
nAChRs.
In summary, these studies demonstrated that the develop-
mental target for sulfoxaflor is the fetal rat muscle nAChR.
Prolonged activity (agonism) at this receptor in rats causes
sustained striated muscle contracture and reduced muscle
Downloaded from http://toxsci.oxfordjournals.org/ by guest on November 15, 2015
responsiveness, considered responsible for the fetal abnormal-
ities and neonatal death in the rat (Fig. 8). All morphological
effects in fetal rats were shown either directly or indirectly, to
be reversible after birth, and sulfoxaflor was also shown not to
be an agonist to the corresponding human receptors. Therefore,
these were novel pharmacological effects mediated via in utero
exposure from the dam at the end of gestation. Because
sulfoxaflor does not cause agonism at the human muscle
nAChRs, these effects are considered not relevant to humans.
SUPPLEMENTARY DATA
Supplementary data are available online at http://toxsci.
oxfordjournals.org/.
FUNDING
This work was supported by The Dow Chemical Company.
ACKNOWLEDGMENTS
The authors thank the Developmental and Reproductive
Toxicology (DART) study team for all of their hard work in
generating these data, Amanda Andrus and Keith Brooks for
lending their expertise in conducting several DART studies, Dr
Bhaskar Gollapudi for his thoughtful review of the manuscript,
Dr Shakil Saghir for lending his toxicokinetic expertise, and
Lisa McFadden for statistical assistance.
REFERENCES
Arias, H. R. (2000). Localization of agonist and competitive antagonist binding
sites on nicotinic acetylcholine receptors. Neurochem. Int. 36, 595–645.
Bennett, M. R., and Pettigrew, A. G. (1974). The formation of synapses in
striated muscle during development. J. Physiol. 241, 515–545.
534 RASOULPOUR ET AL.
Bertrand, D., Cooper, E., Valera, S., Rungger, D., and Ballivet, M. (1991).
Electrophysiology of neuronal nicotinic acetylcholine receptors expressed in
Xenopus oocytes following nuclear injection of genes or cDNAs. In Methods
in Neurosciences (P. M. Conn, Ed.), Vol. 4, pp. 174–193. Academic Press,
San Diego, CA.
Bowman, W. (1990). In Pharmacology of Neuromuscular Function, 2nd ed.,
John Wright, London, U.K.
Bulbring, E. (1946). Observations on the isolated phrenic nerve diaphragm
preparation of the rat. Br. J. Pharmacol. Chemother. 1, 38–61.
Collins, T. F., Welsh, J. J., Black, T. N., Whitby, K. E., and O’Donnell, M. W.,
Jr (1987). Potential reversibility of skeletal effects in rats exposed in utero to
caffeine. Food Chem. Toxicol. 25, 647–662.
Cooper, J. C., Gutbrod, O., Witzemann, V., and Methfessel, C. (1996).
Pharmacology of the nicotinic acetylcholine receptor from fetal rat muscle
expressed in Xenopus oocytes. Eur. J. Pharmacol. 309, 287–298.
Croxen, R., Young, C., Slater, C., Haslam, S., Brydson, M., Vincent, A., and
Beeson, D. (2001). End-plate c- and e-subunit mRNA levels in AChR
deficient syndrome due to e-subunit null mutations. Brain 124, 1362–1372.
Dascal, N. (1987). The use of Xenopus oocytes for the study of ion channels.
CRC Crit. Rev. Biochem. 22, 317–387.
Forsyth, C. S., Speth, R. C., Wecker, L., Galey, F. D., and Frank, A. A. (1996).
Comparison of nicotinic receptor binding and biotransformation of coniine in
the rat and chick. Toxicol. Lett. 89, 175–183.
Fortier, L. P., Robitaille, R., and Donati, F. (2001). Increased sensitivity to
depolarization and nondepolarizing neuromuscular blocking agents in young
rat hemidiaphragms. Anesthesiology 95, 478–484.
Gibb, A. J., and Marshall, I. G. (1984). Pre-and post-junctional effects of
tubocurarine and other nicotinic antagonists during repetitive stimulation in
the rat. J. Physiol. 351, 275–297.
Gibb, A. J., and Marshall, I. G. (1986). Nicotinic antagonists produce differing
amounts of tetanic fade in the isolated diaphragm of the rat. Br. J.
Pharmacol. 89, 619–624.
Gibb, A. J., and Marshall, I. G. (1987). Examination of the mechanisms
involved in tetanic fade produced by vecuronium and related analogues in
the rat diaphragm. Br. J. Pharmacol. 90, 511–521.
Greer, J. J., Funk, G. D., and Ballanyi, K. (2006). Preparing for the first breath:
Prenatal maturation of respiratory neural control. J. Physiol. 570, 437–444.
Harper (2003). Harper’s Illustrated Biochemistry, Lange Medical Books/
McGraw-Hill, New York, NY.
Hesselmans, L. F., Jennekens, F. G., Van den Oord, C. J., Veldman, H., and
Vincent, A. (1993). Development of innervation of skeletal muscle fibers in
man: Relation to acetylcholine receptors. Anat. Rec. 236, 553–562.
Hubbard, J. I., and Wilson, D. F. (1973). Neuromuscular transmission in
a mammalian preparation in the absence of blocking drugs and the effect of
D-tubocurarine. J. Physiol. 228, 307–325.
Hussy, N., Ballivet, M., and Bertrand, D. (1994). Agonist and antagonist effects
of nicotine on chick neuronal nicotinic receptors are defined by alpha and
beta subunits. J. Neurophysiol. 72, 1317–1326.
Karlin, A. (2002). Emerging structure of the nicotinic acetylcholine receptors.
Nat. Rev. Neurosci. 3, 102–114.
Kobayashi, K., Lemke, R. P., and Greer, J. J. (2001). Ultrasound measurements
of fetal breathing movements in the rat. J. Appl. Physiol. 91, 316–320.
Kues, W. A., Sakmann, B., and Witzemann, V. (1995). Differential expression
patterns of five acetylcholine receptor subunit genes in rat muscle during
development. Eur. J. Neurosci. 7, 1376–1385.
Liley, A. W., and North, K. A. (1953). An electrical investigation of effects of
repetitive stimulation on mammalian neuromuscular junction. J. Neuro-
physiol. 16, 509–527.
View publication stats
Maclagan, J., and Vrbova, G. (1966). A study of the increased sensitivity of
denervated and re-innervated muscle to depolarizing drugs. J. Physiol. 182,
131–143.
Marr, M. C., Price, C. J., Myers, C. B., and Morrissey, R. E. (1992).
Developmental stages of the CD (Sprague-Dawley) rat skeleton after
maternal exposure to ethylene glycol. Teratology 46, 169–181.
Millar, N. S., and Denholm, I. (2007). Nicotinic acetylcholine receptors:
Targets for commercially important insecticides. Invert. Neurosci. 7, 53–66.
Millar, N. S., and Gotti, C. (2009). Diversity of vertebrate nicotinic
acetylcholine receptors. Neuropharmacology 56, 237–246.
Millar, N. S., and Lansdell, S. J. (2009). Characterisation of insect nicotinic
acetylcholine receptors by heterologous expression. Adv. Exp. Med. Biol.
683, 65–73.
Mishina, M., Takai, T., Imoto, K., Noda, M., Takahashi, T., Numa, S.,
Methfessel, C., and Sakmann, B. (1986). Molecular distinction between fetal
and adult forms of muscle acetylcholine receptor. Nature 321, 406–411.
Robinson, S. R., and Smotherman, W. P. (1988). Behavior of the Fetus. The
Telford Press, Caldwell, NJ.
Downloaded from http://toxsci.oxfordjournals.org/ by guest on November 15, 2015
Sanes, J. R., and Lichtman, J. W. (1999). Development of the vertebrate
neuromuscular junction. Annu. Rev. Neurosci. 22, 389–442.
Sattelle, D. B., Pelhate, M., and Hue, B. (1979). Pharmacological properties of
axonal sodium channels in the cockroach Periplaneta americana L. I.
Selective block by synthetic saxitoxin. J. Exp. Biol. 83, 41–48.
Solecki, R., Bergmann, B., Burgin, H., Buschmann, J., Clark, R., Druga, A., Van
Duijnhoven, E. A., Duverger, M., Edwards, J., Freudenberger, H., et al. (2003).
Harmonization of rat fetal external and visceral terminology and classification.
Report of the fourth workshop on the terminology in developmental
toxicology, Berlin, 18–20 April 2002. Reprod. Toxicol. 17, 625–637.
Soreq, H. (1985). The biosynthesis of biologically active proteins in mRNA-
microinjected Xenopus oocytes. CRC Crit. Rev. Biochem. 18, 199–238.
Vander, A., Sherman, J., and Luciano, D. (2001). In Human Physiology: The
Mechanisms of Body Function. McGraw-Hill, London, U.K.
Wareham, A. C., Morton, R. H., and Meakin, G. H. (1994). Low quantal content
of the endplate potential reduces safety factor for neuromuscular transmission
in the diaphragm of the newborn rat. Br. J. Anaesth. 72, 205–209.
Watson, G. B., and Young, C. D. (2010). Lack of affinity of the green peach
aphid [3H] imidacloprid binding site by the sulfoxaflor soil metabolites
X11519540 and X11719474 (DAI 0977). Report of the Dow AgroSciences,
Indianapolis, IN.
Wenningmann, I., and Dilger, J. P. (2001). The kinetics of inhibition of
nicotinic acetylcholine receptors by (þ)-tubocurarine and pancuronium. Mol.
Pharmacol. 60, 790–796.
Witzemann, V., Stein, E., Barg, B., Konno, T., Koenen, M., Kues, W.,
Criado, M., Hofmann, M., and Sakmann, B. (1990). Primary structure and
functional expression of the a-, b-, c-, d- and e-subunits of the acetylcholine
receptor from rat muscle. Eur. J. Biochem. 194, 437–448.
Yampolsky, P., Gensler, S., McArdle, J., and Witzemann, V. (2008). AChR
channel conversion and AChR-adjusted neuronal survival during embryonic
development. Mol. Cell. Neurosci. 37, 634–645.
Young, G. T., Broad, L. M., Zwart, R., Astles, P. C., Bodkin, M., Sher, E., and
Millar, N. S. (2007). Species selectivity of a nicotinic acetylcholine receptor
agonist is conferred by two adjacent extracellular beta4 amino acids that are
implicated in the coupling of binding to channel gating. Mol. Pharmacol. 71,
389–397.
Zhu, Y., Loso, M. R., Watson, G. B., Sparks, T. C., Rogers, R. B.,
Huang, J. X., Gerwick, B. C., Babcock, J. M., Kelley, D., Hegde, V. B., et al.
(2011). Discovery and characterization of sulfoxaflor, a novel insecticide
targeting sap-feeding pests. J. Agric. Food Chem. 59, 2950–2957.