Mouse Liver Tumours and their Relevance to Humans
Sciences © The Author . Published by Oxford University Press [on behalf of
the Society of Toxicology]. February 16, 2005
Trevor Green* (email@example.com)
Alison Toghill* (firstname.lastname@example.org)
Robert Lee* (email@example.com)
Felix Waechter* (firstname.lastname@example.org)
James Noakes* (email@example.com)
* Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire,
** DSM Nutritional Products AG, Bau 205 / 315, Postfach 3255, CH-4002 Basel,
Thiamethoxam, a neonicotinoid insecticide, which is not mutagenic either in
vitro or in vivo,
caused an increased incidence of liver tumours in mice when fed in the diet for
18 months at
concentrations in the range 500 to 2500 ppm. A number of dietary studies of up
to 50 weeks
duration have been conducted in order to identify the mode of action for the
the liver tumours seen at the end of the cancer bioassay. Both thiamethoxam and
metabolites have been tested in these studies. Over the duration of a 50-week
dietary feeding study in mice, the earliest change, within one week, is a marked
up to 40%) in plasma cholesterol. This was followed 10 weeks later by evidence
toxicity including single cell necrosis and an increase in apoptosis. After 20
weeks there was
a significant increase in hepatic cell replication rates. All of these changes
persisted from the
time they were first observed until the end of the study at 50 weeks. They
occurred in a dose
dependent manner and were only observed at doses (500, 1250, 2500 ppm) where
tumours were increased in the cancer bioassay. There was a clear no-effect level
of 200 ppm.
The changes seen in this study are consistent with the development of liver
cancer in mice
and form the basis of the mode of action. When the major metabolites of
CGA322704, CGA265307 and CGA330050 were tested in dietary feeding studies of up
weeks duration, only metabolite CGA330050 induced the same changes as those seen
liver in the thiamethoxam feeding study. It was concluded that thiamethoxam is
and hepatocarcinogenic as a result of its metabolism to CGA330050. Metabolite
CGA265307 was also shown to be an inhibitor of inducible nitric oxide synthase
increase the hepatotoxicity of carbon tetrachloride. It is proposed that
its effects on nitric oxide synthase, exacerbates the toxicity of CGA330050 in
Increases in the incidences of mouse liver tumours are a common finding in
studies with a wide range of agrochemicals (Carmichael et al., 1997). It is also
uncommon to find that the increases occur solely in the mouse liver and are not
seen in other
organs or in rats in comparable studies. It is equally true that the vast
majority of these
chemicals are devoid of mutagenic activity and induce their effects by
of action. In some cases the modes of action are known and they give a clear
the likely human hazard; in others, data are lacking or incomplete resulting in
conservative approach towards human hazard and risk assessment.
In recent years a common set of guidelines has emerged that outline the types of
to establish a mode of action in laboratory animals in order for the animal data
to be used as
the basis of human hazard and risk assessments. The guidelines are based on the
Hill criteria (Hill, 1965) for establishing causality and have been developed by
and the US-EPA (EPA, 2003). They provide a rational scientific basis for
changes measured in animals in the short term are causally linked to the
cancer in the long term. In this paper we have used these guidelines to evaluate
associated with an insecticide, thiamethoxam, which is a mouse liver specific
Thiamethoxam is a neonicotinoid insecticide active against a broad range of
important sucking and chewing pests. A comprehensive genotoxicity assessment (including
bacterial mutagenicity, gene mutation, cytogenetic, unscheduled DNA synthesis &
micronucleus tests) demonstrated that thiamethoxam was not genotoxic. It did,
cause an increased incidence of liver tumours in male and female Tif:MAGf mice
in the diet for 18 months at concentrations up to 2500 ppm (see supplementary
total liver adenoma + adenocarcinoma incidence at dose levels of 0, 5, 20, 500,
2500ppm was 12, 7, 12, 19, 27 & 45 out of 50 in male mice, and 0, 0, 0, 5, 9 &
32 out of 50
in female mice respectively. In marked contrast, there were no increases in
either in the liver, or at any other site, in rats fed on diets containing up to
thiamethoxam for 2 years (see supplementary data). A series of feeding studies,
of up to 50
weeks duration have been conducted in mice in order to establish the early
changes, or key
events, which lead to liver cancer in mice. Dose responses for these changes
compared with the tumour responses, temporal relationships have been established
changes have been shown to be reproducible in several studies and in two strains
The major metabolites of thiamethoxam (Figure 1) have been fed to mice and the
responsible for the hepatic changes which precede the development of tumours has
identified. One of the metabolites, CGA322704, has previously been tested for
carcinogenicity in CD-1 mice (Federal Register, 2003) and found not to be a
Comparisons of the effects of this metabolite in Tif:MAGf and CD-1 mice with
thiamethoxam and its other metabolites were used to give a further insight into
the mode of
action of thiamethoxam. Metabolite CGA265307 was found to be structurally
known inhibitors of inducible nitric oxide synthase (Figure 2). In view of the
known role of
this enzyme in the development of liver toxicity (Taylor et al. 1998; Kim et al.
and Chakraborty, 2001; Brennan and Moncada, 2002; Wang et al. 2002), the
CGA265307 to inhibit inducible nitric oxide synthase has been investigated in
vivo and in
vitro. Other possible modes of action have been evaluated in experimental
in order to assess whether infants and children are potentially more susceptible
following exposure to thiamethoxam, the sensitivity of young and adult mice to
thiamethoxam treatment has been compared.
MATERIALS AND METHODS
ylidene-N-nitroamine, 98.6%), metabolite CGA265307
nitroguanidine, 99%) and metabolite CGA322704, (N-(2-chloro-thiazol-5-ylmethyl)-N’methyl-
N’’-nitroguanidine, 99%) were supplied by Syngenta Crop Protection AG, Basle,
Switzerland. Metabolite CGA330050
N-nitroamine) was synthesized as described by Maienfisch et al, (2001). The
structure of the product was confirmed by NMR and mass spectrometry and had a
There was no evidence of a sex difference in the outcome of the cancer study in
consequently only male animals were used for the mode of action studies. The
male mice used in these studies were of the same strain and were obtained from
the same supplier (RCC Ltd, Biotechnology and Animal Breeding Division,
Fullinsdorf, Switzerland) as those used in the cancer study. Male CD-1 mice were
supplied by Charles River, Manston Kent, UK. The mice were housed singly in a
room with 16-20 air-changes per hour, a temperature of 22..2°C, relative
humidity of 55..10%, and a 12-hour light/dark cycle. The animals were acclimated
to laboratory conditions for 14 days prior to dosing. Food (see below) and tap
water were available throughout the studies ad libitum. The animals were not
fasted overnight prior to sacrifice the following morning.
Dietary feeding studies
A number of dietary feeding studies were conducted as follows:
A 50-week hepatotoxicity study with thiamethoxam at six concentrations from
A study of up to 20 weeks duration with thiamethoxam (2500 ppm) and metabolites
CGA322704 (2000 ppm) and CGA265307 (500 ppm).
A study of 10 weeks duration with metabolite CGA330050 at dietary concentrations
and 1000 ppm.
The 50 week study was conducted at Syngenta’s laboratory in Switzerland (as was
biassay) and the remaining studies at Syngenta’s laboratory in the UK. For the
study the test material was admixed with a standard rodent chow, NAFAG 8900 FOR
(Nafag, Gossau SG, Switzerland), the same diet that was used in the cancer study.
studies conducted in the UK the test materials were admixed with CT1 diet
Special Diet Services Limited, Stepfield, Witham, Essex, UK.
The hepatotoxicity of thiamethoxam over a 50-week feeding study
Young adult male mice with a starting body weight of between 30 and 43
used for the study. 525 mice were randomly assigned to 35 groups via a computer
randomization program. Groups of 15 mice each received thiamethoxam at dietary
concentrations of 0, 50, 200, 500, 1250, 2500, or 5000 ppm for 10, 20, 30, 40,
or 50 weeks.
The dose levels included all of those at which tumour incidences were increased
in the long
term study (500, 1250, 2500 ppm) together with an additional higher dose and two
dose levels. Clinical observations were made daily and bodyweights and food
Three days before sacrifice, each animal was fitted with an osmotic mini-pump (Alzet,
1003D, 100..l), filled with 5 mg bromodeoxyuridine (BrdU), dissolved in 0.5 M
bicarbonate at a concentration of 50 mg/ml. The release rate of the mini-pumps
was 1.0 ..l/h.
The mini-pumps were implanted subcutaneously in the back under slight ether
At sacrifice, blood was collected by cardiac puncture and analysed for alanine
aminotransferase, aspartate aminotransferase, alkaline phosphatase and
standard automated methods. Livers were removed, weighed, and processed for
histopathology, cell proliferation measurements and for assessment of apoptosis.
A testis was
taken as a control for the cell proliferation studies.
The liver and testis samples were processed for paraffin embedding and mounted
paraffin block (containing three liver and one testis sample). Serial sections
from paraffin blocks, stained with haematoxylin & eosin and examined by light
Cell proliferation studies
Replicative DNA synthesis was assessed by immunohistochemical staining of liver
for nuclear incorporated BrdU, a diagnostic parameter for cell proliferation (Dolbeare
1995b, 1996). A combined staining for Feulgen and BrdU-immunohistochemistry was
performed on liver paraffin sections (including testis) after deparaffinization.
assessment of BrdU-labelling of hepatocyte nuclei was performed by image
(analySIS Pro, Soft Imaging System GmbH, Münster, Germany). Uniform dark brown
nuclear staining for incorporated BrdU identified cells in S-phase of the cell
cycle. The total
number of hepatocyte nuclei and the number of BrdU-labelled hepatocyte nuclei
counted on Feulgen/BrdU-immunohistochemistry stained paraffin sections. The
index (LI) for BrdU-positive hepatocytes was calculated as the percentage of
over the total number of nuclei.
Hepatocellular apoptosis was
assessed by TUNEL, i.e. terminal deoxynucleotidyl transferase
mediated dUTP nick end labelling histochemistry (Gavrieli et al. 1992).
assessment of apoptosis was performed by image analysis (analySIS Pro, Soft
System GmbH, Münster, Germany). Measurements included counting and area
determination of hepatocellular apoptotic figures (apoptotic hepatocyte nuclei
and clusters of
apoptotic fragments). The total hepatic tissue area was used as the reference
area. As a
measure of apoptotic activity, the area fraction of apoptotic events was
The comparative hepatotoxicity of thiamethoxam and metabolites CGA322704 and
Male and male CD-1 mice (22 – 30 g bodyweight) were fed on diets containing
either 2500 ppm thiamethoxam, 2000 ppm metabolite CGA322704 or 500 ppm
CGA265307 for 1, 10 or 20 weeks. There were 12 animals per group per time point
with an equal number of controls for each test material. The dose levels were
chosen on the
following basis. 2500 ppm was the highest dose level used in the thiamethoxam
bioassay; similarly 2000 ppm was the highest dose tested for metabolite
(Federal Register, 2003). The dose of CGA265307 was selected from dose setting
which showed that 500 ppm of this material in the diet gave comparable blood
levels to those
seen in mice fed on diets containing 2500 ppm thiamethoxam. The two strains of
reflect the fact that the carcinogenicity bioassay of thiamethoxam was conducted
Tif:MAGf mice and that of CGA322704 in CD-1 mice. Clinical observations were
twice daily and bodyweights and food consumption measured weekly.
After 1, 10 and 20 weeks 12 mice from each dose group, and from the control
killed with an overdose of anaesthetic (halothane). Three days prior to
sacrifice the mice
were fitted with minipumps containing BrdU as described above. At sacrifice
removed by cardiac puncture and the livers removed and weighed. A testis was
taken as a
control for the cell proliferation studies.
Blood samples were analysed for glucose, urea, creatinine, albumin, total
albumin/globulin ratio, total bilirubin, alkaline, phosphatase, alanine
aminotransferase, aspartate aminotransferase, creatine kinase, gamma-glutamyl
transferase, sodium, potassium,
chloride, phosphorus, calcium, cholesterol and triglycerides by standard
Livers were processed for histopathological examination (H&E sections, cell
apoptosis by TUNEL) as described above.
The hepatotoxicity of metabolite CGA330050
The hepatotoxicity of metabolite CGA330050 was assessed in male Tif:MAGf mice
animals per group per time point) after 1, 4 and 10 weeks feeding on diets
containing 0, 500
and 1000 ppm CGA330050. The protocol and study design were as given above for
Arithmetic means with standard deviations were used for descriptive statistics
if the data
were of normal distribution. Otherwise, medians with 95% confidence intervals
For the blood chemistry, cell proliferation and apoptosis (TUNEL) data, one-way
variance (ANOVA) was applied (Gad and Weil, 1986) if the data were of normal
and equal variance. Otherwise, a Log10 transformation was performed. If
homoscedasticity were still not given after transformation, a non-parametric
test was used (Kruskal and Wallis, 1952). Treated groups were compared to
by Dunnett’s test (Dunnett, 1955) if the ANOVA was significant and by Dunn’s
1964) in case of significant Kruskal-Wallis test.
For the macropathology and histopathology data, incidences of macroscopic or
findings were submitted to Fisher Exact Tests (Gad and Weil, 1986) if the sum of
observations <100 or to Chi-Square Tests if sum of observations >100. The
comparisons were performed by a sequential step down procedure with respect to
All tests were performed using SigmaStat for Windows, Version 2.03, Build 2.03.0
Inc.). P-values < 0.05 were considered to be significant.
Plasma Metabolite analysis
Blood samples collected at each of the time points in the 10, 20 and 50 week
described above, and liver samples from mice fed on thiamethoxam diets for 10
analysed for thiamethoxam and its three major metabolites, CGA322704, CGA330050
Plasma was separated from red blood cells by centrifugation at 1000g for 15
minutes at 4..C.
Plasma or red blood cells (75 ..l) were deproteinated by the addition of an
equal volume of
ice cold methanol / acetonitrile (4:1 v/v), mixing, and leaving on ice for 60
samples were then centrifuged at 14000g for 15 minutes at room temperature and
25 or 50 ..l
of the supernatant analysed by HPLC as described below.
Liver samples were homogenised in Tris/HCl buffer, pH 7.5, containing 250 mM
give a 10% w/v homogenate which was centrifuged at 100 g for 15 min. An aliquot
of 0.7 ml
of the supernatant was diluted with water to a final volume of 1 ml and loaded
OASIS HLB (10 mg) SPE cartridge which was equilibrated with 1 ml methanol and 1
water. The cartridge was rinsed with 1 ml water followed by 1 ml 10% aqueous
Thiamethoxam and its metabolites were eluted with 1 ml 70% aqueous methanol.
Between 10 and 50 ..l of each sample was analysed by HPLC (Schimadzu LC10) using
mm x 4.6 mm Hypersil ODS 5µm column, with 10 mm x 4.6 mm Hypersil ODS 5 µm guard
column. The initial mobile phase consisted of 90% water and 10% methanol/acetonitrile
v/v). The gradient rose linearly to 45% methanol/acetonitrile (4:1 v/v) over 25
then rose linearly to 100% methanol/acetonitrile (4:1 v/v) over the next 5
concentration was held for 5 minutes, before returning to the starting
conditions over a
further 5 minutes. The column was allowed to re-equilibrate for 10 minutes prior
injection of the next sample. The flow rate of the mobile phase was 0.75 ml/min,
column eluent was monitored with a UV detector set at 254 nm. Approximate
for thiamethoxam, CGA265307, CGA322704 and CGA330050, were 21.0, 23.5, 25.5 and
27.5 minutes respectively. The samples were quantified against standard curves
using a range of concentrations of thiamethoxam or each of its metabolites from
ng/ml. The limits of detection were 20 ng/ml for thiamethoxam, CGA322704 and
CGA265307 and 50 ng/ml for CGA330050.
The recovery of the test materials from biological samples was determined by
thiamethoxam and its metabolites to control whole blood, to plasma and to the
100 g liver
supernatant to give concentrations of each component of 5 ug/ml. These samples
extracted and analysed as described above.
Metabolite CGA265307 and inducible nitric oxide synthase (iNOS) inhibition
Inhibition of nitric oxide synthase in vitro
The method used to measure inducible iNOS activity was that described by Rendon
(1997) using purified iNOS. The ability of CGA265307 to inhibit iNOS activity
determined and compared with that of N-nitro-L-arginine methyl ester (L-NAME)
range of substrate concentrations from 0-0.5 mM. These experiments were repeated
thiamethoxam and metabolites CGA322704 and CGA330050, at 1 mM concentrations.
Inhibition of nitric oxide synthase in vivo
The hepatotoxicity of carbon tetrachloride is known to be enhanced in mice
inhibitors of iNOS (L-NAME) and in iNOS knock-out mice (Morio et al. 2001).
Consequently, the effect of dietary administration of CGA265307 on carbon
hepatotoxicity has been investigated in a study in which 2 groups of male Tif:MAG
per group) were placed on a diet containing 2000 ppm CGA265307 for 7 days. At 16
before termination all of the mice in one group were given a single
of 10 µl/kg carbon tetrachloride in corn oil (10 ml/kg). The other group was
of corn oil alone. Two further groups of mice (5 per group) were given control
diet for 7
days. At 16 hours before termination, one group was given single
of 10 µl/kg carbon tetrachloride in corn oil (10 ml/kg), the other group was
given single intraperitoneal
injections of corn oil alone. The mice were killed with an overdose of halothane
and blood collected by cardiac puncture in lithium/heparin tubes. Livers were
part of each of the three main lobes placed in formol saline. The livers were
embedded in paraffin wax, sectioned and stained with haematoxylin and eosin
being examined by light microscopy. Blood samples were centrifuged to separate
alanine aminotransferase and aspartate aminotransferase activities determined by
The comparative sensitivity of young and adult mice
The sensitivity of adult (15-17 weeks old) and weanling mice (21 days old) has
compared in a study in which thiamethoxam was fed in the diet at concentrations
of 0, 500,
1250 and 2500 ppm for 7 days.
Plug positive pregnant female Tif:MAG mice were supplied by RCC Ltd,
Animal Breeding Division, Fullinsdorf, Switzerland. The animals were housed in
plastic cages under the same environmental conditions as the adults. They
diet and mains water ad libitum. The day of littering (day 1) was noted together
with the size
of the litters. The pups were sexed on day 7 and remained with the dams until
day 18. When
the dams were removed, the pups were randomly housed in groups of 6 until the
start of the
study on day 21 (body weight approx. 8 g). Only male mice were used for the
Groups of 6 male adult or 6 male weanling mice were fed on diets containing 0,
and 2500 ppm thiamethoxam for 7 days. The adult animals were housed singly and
weanling mice together by group. Clinical observations and body weights were
daily. At the end of the treatment period, all of the mice were killed by
terminal anaesthesia induced by halothane vapour. Blood was collected by cardiac
and transferred to lithium heparin tubes. Livers were removed and weighed.
separated from red blood cells by centrifugation at 1000g for 15 minutes at
cholesterol, alanine aminotransferase and aspartate aminotransferase were
standard automated procedures. Plasma samples were also analysed for
its major metabolites as described above. Livers were fixed in 10% (w/v) neutral
formol saline, dehydrated through an ascending ethanol series and embedded in
Sections (5-7..m) were cut and stained with haematoxylin and eosin.
The hepatotoxicity of thiamethoxam in mice over a 50-week study
Clinical signs and mortality were not affected by treatment. The mean daily food
consumption was consistently below control level from week 40 at 1250 ppm and
9 at 2500 and 5000 ppm. The overall mean food consumption (weeks 1 to 49)
97, 96, and 95 % of control at these dose levels, respectively.
Organ and Body weights
The mean body weight was consistently below control level at the 2500 and 5000
levels (by 8% at 2500 and by 14% at 5000 ppm at week 50). The mean relative
was increased at 2500 ppm (weeks 20 and 40: 111%, and 116% of control,
at 5000 ppm (weeks 10, 20, 30, 40, 50: 113%, 114%, 117%, 124%, 129% of control,
The median aspartate aminotransferase activity was increased at 2500 ppm (weeks
20 and 40:
122% and 131% of control, respectively) and at 5000 ppm (all time points; 148 –
control). After combining all time points, increased values were noted at 1250,
5000 ppm (116%, 122% and 169% of control, respectively). Alanine
activities were increased in a similar manner. After combining all time points,
were noted at 1250, 2500 and 5000 ppm (139%, 207% and 256% of control,
The alkaline phosphatase activity was not affected by treatment.
A significant dose dependent reduction in plasma cholesterol levels, at 500 ppm
was seen at the earliest time point of 10 weeks and was sustained throughout the
1). The cumulative data for all time points is shown against dose in Figure 3
and against time
for the four highest dose levels in Figure 4.
Increases in hepatocellular hypertrophy, single cell necrosis, apoptosis,
infiltration, pigmentation and fatty change were seen in a dose and time
dependent manner at
dose levels of 500 ppm and above.
Hypertrophy was characterized by enlarged centrilobular/midzonal hepatocytes
increased amounts of cytoplasmic glycogen, fat, and smooth endoplasmic reticulum
seen in the 2500 ppm dose group at weeks 30, 40 and 50 and in the 5000 ppm dose
all time points (Figure 5). Hepatocellular necroses affected single cells or
small groups of
cells with mainly centrilobular localization and were often accompanied by
cells. After combining all time points, increased necroses were seen at 500,
1250, 2500 and
5000 ppm (Figure 6). The pattern of inflammatory cell increases largely followed
necroses (Figure 5).
Hepatocellular apoptosis, first seen at week 10, affected single cells or small
groups of cells,
again with mainly centrilobular localization. After combining all time points,
increased incidences were observed at 500, 1250, 2500 and 5000 ppm (Figure 6).
Pigmentation, which was characterized as lipofuschin (yellow/brown pigment
occurred in the cytoplasm of centrilobular hepatocytes, and was increased at the
dose level and above (Figure 5). Occasionally, pigmented Kupffer cells were
Significantly increased incidences (p<0.05) of fatty change over control (40%)
at 500 (72%), 1250 (82%) and 2500 (79%) ppm but a reduced incidence was observed
5000 ppm (17%).
An increased median BrdU labelling index was observed at 1250 ppm (week 40: 246%
control), at 2500 ppm (weeks 30, 40 and 50: 356%, 422% and 311% of control,
and at 5000 ppm (weeks 10, 30, 40, 50: 211%, 484%, 933%, 485% of control,
These data combined for all time points are shown in Figure 6.
The histopathological examination of the liver described above revealed that the
necroses and apoptosis were largely confined to the centrilobular region.
Examination of the
BrdU labelling index in this region of the livers of mice fed on the 500 ppm
diet for 40 weeks
revealed a statistically significant increase in the labelling index compared to
the same region
in control liver (control, 0.15..0.10, 500 ppm 0.36..0.31 p<0.05). This increase
apparent when comparisons were made across the whole liver. The labelling index
increased in the centrilobular region at the 200 ppm dose level (0.10..0.07).
An increased median TUNEL area density was observed at dose levels of 500 ppm
above. The densities increased with increasing dose and with increasing duration
After combining all time points, increased median TUNEL area densities were
500 ppm (156% of control), at 1250 ppm (188% of control), at 2500 ppm (219% of
and at 5000 ppm (316% of control).
The comparative hepatotoxicity of thiamethoxam and its metabolites
The hepatotoxicity of metabolites CGA322704 and CGA265307 was compared with that
thiamethoxam in two strains of mouse in a study of up to 20 weeks duration. The
reported above up to 20 weeks (in the 50-week study) were essentially replicated
in the mice
fed on a diet containing 2500 ppm thiamethoxam. There was no evidence of a
strain difference in response in the mice used in this study. Metabolites
CGA265307 induced none of the clinical or histopathological changes seen in the
thiamethoxam treated mice. The histopathological data from this study are shown
in Table 2.
In contrast, Tif:MAG mice treated with metabolite CGA330050 for up to 10 weeks,
essentially the same changes in the liver as those seen with thiamethoxam at
this time point.
Plasma cholesterol levels were significantly reduced at both dose levels (Figure
histopathological changes in the liver included increases in hepatocellular
cell necrosis, apoptosis and a significant increase in cell replication rates in
Plasma metabolites were measured in all of the studies. Thiamethoxam and
CGA322704, CGA330050 and CGA265307 were detected at all time points in the
thiamethoxam study. Data for 10 and 50 weeks are shown in Figure 8. The
thiamethoxam was linear with dose over the range 500 to 2500 ppm. The
metabolites in the liver was comparable to that in plasma (data not shown). In
with CGA322704 the only components detected in plasma over the duration of the
1, 10 and 20 weeks) were CGA322704 itself (4.8 – 11.6 ug/ml) and CGA265307 ( 3.0
ug/ml). In CGA265307 dosed mice (1, 10 and 20 weeks), only the starting material
detected in plasma (3.0 – 4.1 ug/ml), and in mice dosed with CGA330050, the
material (4.2 ug/ml) and CGA265307 (7.2 ug/ml) were present (measured after 1
Metabolite CGA265307 and nitric oxide synthase (NOS) inhibition
The kinetics for the inhibition of iNOS with CGA265307 and L-NAME are shown in
9. The data show that both CGA265307 and L-NAME are competitive inhibitors of
with the inhibition constants (Ki) of 0.79 and 0.43 mM respectively.
metabolites CGA322704 and CGA330050 did not inhibit iNOS.
When mice were fed on a diet containing 2000 ppm CGA265307 for 7 days and then
single intraperitoneal injection of 10 µl/kg carbon tetrachloride there was an
increase in liver
damage compared to mice given carbon tetrachloride alone. Liver damage was
aminotransferase activities (Figure 10) and histopathology (Table 4). There was
of hepatotoxicity in mice fed on the CGA265307 diet alone.
The comparative sensitivity of young and adult mice
There was no evidence of liver toxicity in either weanling or adult mice fed on
containing thiamethoxam. Liver weights and ALT and AST values from mice in all
groups were comparable to those in the control groups (data not shown). Plasma
levels were reduced in adult mice to approximately 70% of control values at the
2500 ppm dose levels, and to 78% of control at the 500 ppm dose level.
were also reduced in weanling mice but to a lesser extent, to 85% of control at
the 1250 ppm
dose level and 79% at 2500 ppm. The 500 ppm dose level was a clear no effect
Histopathological examination of the livers of adult mice found a clear
effect in mice fed on the 2500 ppm thiamethoxam diet for 7 days. The changes
increased centrilobular vacuolation and/or decreased eosinophilia. Changes at
the lower dose
levels were less defined, with a possible weak effect at 1250 ppm and no effect
at 500 ppm.
In weanling mice, there was also a clear effect at the 2500 ppm dose level with
similar to those observed in adults but less severe.
The pattern of metabolites in plasma at the end of the study was essentially
similar in adult
and weanling mice. The actual concentrations of thiamethoxam and its major
the plasma of weanling mice were up to double those in adult animals (Figure
Thiamethoxam is not a mutagen, yet it significantly increased the incidences of
tumours in mice in an 18 month feeding study. By contrast, thiamethoxam did not
tumour incidences at any site in a 2-year feeding study in rats. This phenomenon
of a nongenotoxic mouse liver specific carcinogen is not uncommon (Carmichael et
al. 1997), yet it
remains one of the most difficult areas of rodent toxicology to extrapolate to
humans. In the
absence of other data the default assumption by some regulatory authorities is
to assume that
these tumours indicate a hazard to human health from the chemical in question.
Nevertheless, the very nature of the species specific, organ specific response
such an assumption may be ultra-conservative. In recent years it has been
this issue can only be resolved by first gaining an understanding of the reasons
tumours develop in the sensitive species. With this insight the lack of response
species can be resolved and a framework developed to determine the likely
hazard to humans. The principle of understanding mode of action in order to
hazard has been developed by ILSI (2003) and now forms part of the US-EPA Cancer
Assessment Guidelines (EPA, 2003). These mode of action guidelines have been
design a series of studies with thiamethoxam to understand the mode of action of
chemical as a mouse liver carcinogen, and, as described in subsequent papers, to
the lack of response in rats, and to determine how humans will respond to
thiamethoxam (Green et al., 2005; Pastoor et al., 2005).
The primary experiment in these studies was a 50 week dietary feeding study in
strain of mouse that was used in the carcinogenicity study. The carcinogenic
dose levels of
500, 1250 and 2500 ppm were used, but the lower dose levels used in the study of
5 and 20
ppm were replaced with dose levels of 50 and 200 ppm in order to give a better
curve and a more accurate definition of any no-effect level. A higher dose of
5000 ppm was
also used for dose response reasons. The results of this study gave a clear
indication of the
mode of action of thiamethoxam as a mouse liver carcinogen. Essentially,
exposure to thiamethoxam results in cell death, mainly as single cells dying
either by necrosis
or apoptosis, which is followed by increased cell replication. The timescale for
is extended, cell death occurring only after 10 weeks of feeding and increased
from 20 weeks onwards. Thereafter, the cycle of cell death and cell replication
the remainder of the 50 week study. The rates of cell death and replication
appeared to be in
balance and did not result in a significant increase in liver weights. The small
liver weight that did occur were attributed to hypertrophy resulting from
of cytoplasmic glycogen, fat, and smooth endoplasmic reticulum. Other
changes included lymphocytic infiltration and pigmentation of hepatocytes and
Thus, the livers of thiamethoxam treated mice undergo a continuous insult, which
cell death and increased cell replication for at least 30 weeks. Such changes
form a well
established and accepted mode of action for the development of liver tumours in
2003). The dose response for these changes followed that for the tumour
significant changes were only seen at carcinogenic dose levels of 500 ppm and
12). In addition, the changes had a logical temporal relationship, the
including depletion of cholesterol occurred with the first few weeks of the
study to be
followed by cell death which, in turn, was followed by an increase in reparative
A question arises as to the cause of the cell death that ultimately leads to the
liver cancer in mice. Some of the changes in liver biochemistry such as
aminotransferase activities are indicative of the subsequent histopathological
most significant and earliest change was a marked reduction in plasma
cholesterol, which, as
with the histopathological changes, only occurred at doses of 500 ppm and above
and had a
dose response that was comparable to the tumour dose response. Furthermore,
levels in treated animals remained lower than those in controls throughout the
The correlation between reduced plasma cholesterol, the histopathological
events), and the subsequent development of cancer was absolute in these studies,
with thiamethoxam and CGA330050 but not with CGA322704 and CGA265307, nor with
thiamethoxam in rats. These data suggest that the changes in cholesterol levels
causally linked to the subsequent histopathological changes. However, at this
mechanistic link has been established and cholesterol change must therefore be
viewed as an
associative event. It is interesting to note the strong correlation that also
changes in lipid metabolism in rodents and increases in liver tumours with other
A large number of drugs have been developed in recent years for the control of
(triglycerides and cholesterol) and the prevention of coronary heart disease.
These fall into
two main classes, the hypolipidemic drugs (triglycerides), and the more recent
(cholesterol). Both classes have been evaluated in rodent carcinogenicity
studies during their
development, and the majority of drugs in each class cause liver cancer,
particularly in the
mouse (MacDonald et al. 1988; Gerson et al. 1989; Newman and Hulley, 1996; von
and Schulter, 1998). As with thiamethoxam, a causal link has not been
the effects of these drugs on lipid metabolism and the subsequent development of
In order to understand the lack of response in rats, and to provide a possible
extrapolating the animal data to humans, an understanding of the role of
metabolites in the development of liver cancer in mice is required. To that end,
the 3 major
metabolites of thiamethoxam were fed to mice in the diet for periods of up to 20
their hepatotoxicity compared with that of thiamethoxam itself. These studies
also gave an
opportunity to further test the mode of action indicated by the 50-week study.
CGA322704 has also been tested for carcinogenicity and shown not to be a liver
(Federal Register, 2003). Thus, the changes seen in thiamethoxam treated animals
occur in mice treated with this close structural analogue. The CGA322704
study also used a different strain of mice (CD-1) to that used in the
(Tif:MAGf). Both strains were used in the metabolite studies in order to
possible strain differences in response.
The outcome of the metabolite studies was clear and consistent with the known
profiles of thiamethoxam and CGA322704. The hepatic changes seen with the
hepatocarcinogen thiamethoxam were not seen with CGA322704, which is known not
cause liver tumours in mice (Federal Register, 2003). Metabolite CGA265307 also
induce hepatotoxicity in mice in these studies. Consistent with this, the plasma
concentrations of CGA265307 were comparable in both CGA322704 and thiamethoxam
treated mice providing further evidence that this metabolite alone is not
responsible for the
liver tumours. By contrast, metabolite CGA330050 did induce the same changes in
livers of mice as thiamethoxam itself. Again this is consistent with the total
CGA330050 is not formed from CGA322704. Metabolism studies and comparisons of
plasma thiamethoxam concentrations in mice and rats have shown that the blood
thiamethoxam are higher in the rat than the mouse at the highest dietary dose
used in the respective cancer bioassays and, vice versa, those of CGA330050 are
in the rat than the mouse (Green et al. 2005). Consequently, it is highly
thiamethoxam itself plays a role in the development of liver cancer in mice and
it can be
concluded that metabolite CGA330050 is responsible for the hepatic changes which
liver cancer in thiamethoxam treated mice. In the studies which used both
strains of mouse,
the responses in the livers were identical, as were the metabolite profiles in
plasma. Thus, it
is reasonable to conclude that the responses seen in mice are not a consequence
of the strain
used in the cancer studies.
Another possible factor in the development of hepatotoxicity in thiamethoxam
treated mice is
the role of metabolite CGA265307 and the inhibition of inducible nitric oxide
vivo, nitric oxide, produced from arginine by the nitric oxide synthases, has
been shown to
have a regulatory role in the development of hepatotoxicity and apoptosis. For
chemical inhibition of iNOS, or the use of iNOS knock out mice, has been shown
exacerbate chemically induced hepatotoxicity (Morio et al. 2001). Nitric oxide
is believed to
regulate hepatotoxicity and apoptosis by modulating the adverse effects of TNF..
endothelial cells in response to a toxic challenge (Bradham et al. 1998; Taylor
Luster et al. 1999). CGA265307 is identical to the iNOS inhibitor L-NAME in the
part of the molecule, the amino acid function not being a structural component
potency or selectivity of iNOS inhibitors (Garvey et al. 1994, 1997). In the
studies CGA265307 was shown to inhibit iNOS in vitro and to enhance the toxicity
tetrachloride in vivo. It seems likely, therefore, that CGA265307, although not
could enhance the hepatotoxicity of metabolite CGA330050.
As part of the risk assessment process, the US-EPA are required to assess the
risks to infants
and children whenever it appears that their risks might be greater than those of
2003). Although there are no reasons to suspect that infants and children would
susceptible than adults to the proposed mode of action of thiamethoxam, the
addressed experimentally. Such an assessment is problematical in terms of study
even using experimental animals. It is particularly so for thiamethoxam because
histopathological changes are not seen in the liver until 10 weeks after the
start of the
experiment. Young mice reach maturity at 6-7 weeks, well before the first
changes are seen
in the liver, and hence any differences between young and adult animals may no
apparent in a 10-20 week study. The earliest change, within one week, seen in
mice fed on
diets containing thiamethoxam was a reduction in plasma cholesterol levels. The
between reductions in plasma cholesterol, subsequent changes in liver
histopathology and the
incidences of liver cancer were absolute, both quantitatively and qualitatively,
over a wide
range of studies with thiamethoxam and its metabolites in two species. Changes
cholesterol were, therefore, used as a short-term marker for the mode of action
thiamethoxam and a means of comparing the sensitivity of young and adult animals.
cholesterol levels were lowered in adults at all three dose levels, but only at
1250 and 2500
ppm in weanling mice. The magnitude of the response in weanlings at the two
levels was also less than that in adults. Plasma metabolite concentrations were
approximately 2-fold higher in weanling mice reflecting the increased dietary
intake in young
animals. Overall, the study showed that young mice, despite a significantly
intake, were at least 2-fold less sensitive than adult mice to the earliest key
event in the mode
of action of thiamethoxam. It is concluded, based on the results of this study,
that infants and
children would not be more susceptible than adults following exposure to
Other possible modes of action have been investigated in the course of these
studies. There is
no plausible sequence of events for liver tumour formation by thiamethoxam where
interference with nicotinic acetylcholine receptors, the target for
neonicotinoids in insects,
would represent a key event. As a class, the neonicotinoids have not been found
oncogenic in rats and mice. Thiamethoxam is not genotoxic in bacteria,
eukaryotic cells and
mammalian systems. There was no evidence of hepatic peroxisome proliferation (by
microscopy or from increases in peroxisomal beta-oxidation) in mice fed on diets
up to 2500 ppm thiamethoxam for 14 days (data not shown) nor was there any
based on hepatic 8-isoprostane F2.., glutathione or ..-tocopherol concentrations,
stress in mice fed on diets containing up to 5000 ppm thiamethoxam for periods
up to 50
weeks (data not shown). Thiamethoxam did induce several cytochrome P-450
but the magnitude of the increases (max 11-fold, CYP2B) were considered
to be causally related to the development of liver cancer (data not shown). For
level of enzyme induction with thiamethoxam was much lower than that reported
phenobarbital, a known rodent liver carcinogen (Honkakoski et al., 1992a, 1992b,
1990; Whysner et al., 1996).
In summary, a mode of action has been identified for the development of liver
thiamethoxam treated mice which includes marked and sustained cholesterol
followed by cell death, both as necrosis and apoptosis, and increased cell
replication over a
30 week period. These changes are believed to lead to the tumours seen at 18
key metabolite inducing these changes has been identified as CGA330050. The
of hepatotoxicity is believed to be enhanced by inhibition of inducible nitric
by metabolite CGA265307. The studies described fulfil the criteria identified
acceptable mode of action, including dose response, temporal relationships,
consistency and reproducibility. Table 5 illustrates the strength of the
correlation between the
early events and the development of tumours. The responses seen with
been reproduced in studies of 50 and 20 weeks duration, the latter in two
strains of mouse.
The metabolite studies were internally consistent in that CGA330050 is only
thiamethoxam and not from the non-carcinogenic metabolite CGA322704. In all of
studies the dose responses follow that of the tumour response and the temporal
follow a logical sequence of biochemical change (starting with plasma
leading to cell death followed by increased cell replication followed by the
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