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Following the outbreak of pet food-induced nephrotoxicity in March 2007, a voluntary online survey of all AAVLD-accredited laboratories, commercial laboratories, and veterinary clinics across North America was conducted. There was no information on toxicity of melamine or factors affecting the disease outcome following exposure to melamine in pets. Data were collected from affected pets to learn about the disease outcome and the affected pet population. The web-based electronic survey used the online tool, Survey Monkey™. Data were collected between April 5 and October 31, 2007. Four hundred fifty-one cases of 586 reported cases met the criteria for inclusion in the study. Most reported cases were from California, Texas, Michigan, Florida, and Ontario. Of the 451 cases, 424 were reported as affected. Of these, 278 cases (65.6%) were cats and 146 (34.4%) were dogs. A total of 278 pets (171 cats and 107 dogs) were reported to have died (a ratio of 1.6:1). However, within species, there was a higher percentage of deceased dogs (73.3%) than cats (61.5%). Of the affected pet population, older male cats with preexisting disease conditions were more likely to be deceased. Analysis of the pets in this large database of naturally affected pets yielded interesting findings. It showed that more cats than dogs were affected and also that preexisting renal diseases and old age predicted the most severe outcome (death or euthanasia) than any other factors.
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Analysis of a Survey Database of Pet Food-Induced
Poisoning in North America
Wilson K. Rumbeiha &Dalen Agnew &Grant Maxie &
Brent Hoff &Connie Page &Paul Curran &
Barbara Powers
Published online: 15 April 2010
#American College of Medical Toxicology 2010
Abstract Following the outbreak of pet food-induced
nephrotoxicity in March 2007, a voluntary online survey
of all AAVLD-accredited laboratories, commercial labo-
ratories, and veterinary clinics across North America was
conducted. There was no information on toxicity of
melamine or factors affecting the disease outcome
following exposure to melamine in pets. Data were
collected from affected pets to learn about the disease
outcome and the affected pet population. The web-based
electronic survey used the online tool, SurveyMonkey.
Data were collected between April 5 and October 31,
2007. Four hundred fifty-one cases of 586 reported cases
met the criteria for inclusion in the study. Most reported
cases were from California, Texas, Michigan, Florida,
and Ontario. Of the 451 cases, 424 were reported as
affected. Of these, 278 cases (65.6%) were cats and 146
(34.4%) were dogs. A total of 278 pets (171 cats and
107 dogs) were reported to have died (a ratio of 1.6:1).
However, within species, there was a higher percentage
of deceased dogs (73.3%) than cats (61.5%). Of the
affected pet population, older male cats with preexisting
disease conditions were more likely to be deceased.
Analysis of the pets in this large database of naturally
affected pets yielded interesting findings. It showed that
more cats than dogs were affected and also that
preexisting renal diseases and old age predicted the most
severe outcome (death or euthanasia) than any other
Keywords Melamine .Pet food-induced toxicity .
Melamine-induced nephrotoxicity
In March and April of 2007, a massive recall of
melamine-contaminated pet food products occurred
across North America, prompted by reports of acute
renal failure among pets that had consumed tainted pet
food containing ingredients imported from China. Ill-
nesses and deaths among pets were first noticed in early
March 2007 and were initially associated with feeding
pet foods manufactured by Menu Foods, Inc. The first
nationwide recall of pet foods was issued on March 16,
2007 [1]. Investigations to detect possible food conta-
minants focusing on common nephrotoxicants (ethylene
W. K. Rumbeiha (*):D. Agnew
Diagnostic Center for Population and Animal Health,
College of Veterinary Medicine, Michigan State University,
4125 Beaumont Road,
Lansing, MI 48910-8104, USA
G. Maxie :B. Hoff
Animal Health Laboratory, Laboratory Services Division,
University of Guelph,
P. O. Box 3612, Guelph, ON, Canada, N1H 6R8
C. Page :P. Curran
Department of Statistics and Probability,
Michigan State University,
East Lansing, MI 48823, USA
B. Powers
Veterinary Diagnostic Laboratory, Colorado State University,
Fort Collins, CO 80523, USA
J. Med. Toxicol. (2010) 6:172184
DOI 10.1007/s13181-010-0022-9
glycol, soluble oxalates, mycotoxins such as ochratoxins,
and heavy metals such as arsenic and mercury) were
fruitless. Despite intensive efforts, it was not until March
30, 2007 that the first contaminant, melamine, was
officially identified in pet foods sickening and killing
pets. Reports surfaced that two Chinese companies had
sold wheat gluten and rice gluten purposefully contami-
nated with melamine, an industrial chemical, in order to
artificially inflate their apparent protein concentration.
Melamine, which has no nutritional value but a high
nitrogen content, is used for the manufacture of plastic
resins used in kitchenware, for manufacture of glues and
adhesives, and in Asia, as a fertilizer [2]. Wheat and rice
gluten are used in the manufacture of wet foods as
thickening agents. In 2008, thousands if infants in China
were poisoned by melamine-contaminated milk food
products. As such, the toxicity of melamine and other
related triazine compounds is of interest to both medical
and veterinary professionals [3].
Melamine is known to be of low toxicity in mammals
[2,4]. The oral LD
in the rat is 3,161 mg/kg body
weight [2]. In dogs and rats, melamine given in high acute
oral doses causes diuresis [5]. Chronic exposure of rats to
melamine is associated with tumors of the urinary bladder
and not the acute renal failure that was observed in pets
[68]. The concentration of melamine found in pet food
was relatively low (50400 ppm). Based on all this, it
became clear that other contaminants could be present in
the pet food.
Further analysis of contaminated food in April 2007
revealed the presence cyanuric acid, ammeline, and amme-
lide; still later ureidomelamine and N-methylmelamine were
also found [9]. Of these contaminants, melamine was the
most abundant followed by cyanuric acid [9]. The wheat
gluten was found to contain melamine at 8.4%, cyanuric
acid at 5.3%, ammelide at 2.3%, ammeline at 1.7%, and
ureidomelamine and methyl melamine at <1% each [9].
All these contaminants are triazine compounds that have
wide industrial applications. Cyanuric acid, ammelide, and
ammeline appear to be by-products generated in the
process of manufacturing melamine.
Cyanuric acid by itself, like melamine, has very low
toxicity [10]. However, when melamine and cyanuric acid
are ingested together, they interact in the renal tubules to
form stable melamine-cyanurate crystals [11]. These
crystals have very low water solubility, and renal failure
may result from physical blockage of the uriniferous
tubules and tubular epithelial cell necrosis [11].
Affected pets suffered mainly from acute renal failure
characterized by polyuria, isosthenuria, polydipsia, oliguria,
hyperphosphatemia, and azotemia [8]. The melamine-
cyanurate crystals found in the urine of these animals have
been characterized recently [12].
At the time of the disease outbreak, very little had
been published regarding the toxicity of triazine com-
pounds in pets, and there was no information on the
toxicity of a mixture of these compounds. The volume of
unsubstantiated reports of pets having been affected and/
or having died as result of consuming melamine-
contaminated food was exacerbated by confusion sur-
rounding the case definition of the pet food-induced
illness or death [13]. Through its national organizations, the
veterinary professional community worked to develop
information about this new disease in order to assist affected
pet owners and veterinarians in its proper identification and
appropriate treatment.
This survey was posted online on April 5, 2007, 1 week
after melamine was identified in the pet foods. The major
objectives of the survey were to collect data from veterinary
professionals across North America to determine (a) the
minimum number of confirmed cases of pets affected by
melamine-contaminated food, (b) the geographic distribu-
tion of cases across North America, (c) the common
signalment among affected animals, and (d) to determine
the disease outcome among confirmed cases in this
Materials and Methods
The web-based electronic survey used the online tool,
SurveyMonkey. The initial target audience was vete-
rinarians in AAVLD-accredited laboratories, but the
scope rapidly expanded to include those in commercial
laboratories and veterinary clinics across North America.
The online survey was widely publicized through the
American Veterinary Medical Association (AVMA), the
American College of Veterinary Pathologists (ACVP),
and the American College of Veterinary Internal
Medicine (ACVIM), and links were created on the
AAVLD, AVMA, and ACVP websites. More publicity
was generated by direct e-mails through the veterinary
toxicology list serve, the news media, and conference
presentations at the annual AAVLD meeting in Reno,
Nevada. The survey period was April 5 through October
31, 2007. The 17-item questionnaire is reproduced in
Responses were received from 474 veterinarians report-
ing 586 individual pets. In all, 135 cases (23%) were
excluded for the following reasons: 13 responses failed
to report species, five did not report health status of the
J. Med. Toxicol. (2010) 6:172184 173
animals, and 117 included no contact information or did
not want to be contacted. Because this study targeted
affected animals, another 27 cases were removed since
they report the animal as healthy, never ill.The
remaining 424 reported cases were evaluated.
The measurements discussed here are used in the Results
section. The question number on the survey is also
referenced when applicable. The following animal charac-
teristics were asked directly on the survey (see Appendix):
Q1, species (canine, feline, or other); Q2, breed (enter
name); Q3, age (reported in years/months); and Q4, sex
(male or female).
The survey asked (Q5), What is the current health
status of the affected animal?The possible responses
were healthy never ill, currently ill,”“previously ill,
now recovered,and deceased.For this study, we
definedananimalasaffectedif the response to the
current health status was currently ill, previously ill now
recovered, or deceased. Only the 424 affected animals
are used in any analysis, which compares recovered
cases to deceased cases, as recovery requires onset of an
illness from which recovery was made. Furthermore, a
dichotomous variable, deceased, is defined as either not
deceased if currently ill, previously ill now recovered, or
otherwise deceased. This variable is used in one of the
prediction models.
The survey asked (Q7), Did this animal have any
conditions that would predispose it to renal disease/failure?
The response was yes or no. This is a veterinarians
professional assessment of whether or not the pet had
preexisting diseases that might render it more vulnerable to
pet food-induced renal failure. For those who responded yes,
selections from a list of predisposing conditions could be
made (Q8). Multiple selections were allowed and respondents
also given a choice of otherwhere they freely listed those
predisposing conditions, which were not provided as a choice.
The survey asked (Q9) Pleaseindicatediagnostic
criteria used to arrive at your diagnosis (select all that
apply).The respondents could select from a list of eight
criteria plus other. Respondents who checked other were
asked to specify the other diagnostic criteria they used
(see Q9, Appendix). Veterinarians were asked (Q10) to
specify other chemical markers present in their cases,
which was another way of asking about other diagnostic
criteria they used to determine that the case they were
entering was indeed pet food related.
The survey asked (Q11) How soon after ingesting recalled
pet food did the animals become ill?Respondents chose from
a list of five choices plus other. Those who chose other were
asked to specify their answers.
The survey asked (Q12), Which dog/cat food(s) was
the affected animal eating at the time of (or immediately
preceding) the diagnosis? (Select all that apply).The
respondent could select from a list of food brands or
specify particular brands not on the selection list under
other. When other was entered, the brand was added to
the final list of reported food brands. See Q12,
Appendix, for the final list. Participants were able to
check multiple food brands that were fed to the affected
pet, if this were the case, so each case may have values in
more than one category. Each category is treated as yes/
no response. The rest of the questions (Q1317) were
designed to collect pet identification information, specific
food lot numbers, and for identification of veterinarians
completing the survey, their practice addresses, and
contact information in case a follow-up was necessary.
Statistical Analysis
Diagnostic criteria (Q9) were analyzed for all pets.
Statistical analyses focused on affected pets (defined by
responses currently ill, previously ill now recovered, or
deceased on the health status question, Q5). Univariate
descriptive statistics for each species are presented for the
animal characteristics, health status, and preexisting con-
ditions likely predisposing to renal failure. Then, also for
affected pets, bivariate analyses using cross-tabulation or
comparison of means are performed to relate the animal
characteristics, possible predisposition to renal disease, and
health status.
Two predictive models are considered. A multinomial
logistic model that considers the currently ill and
previously ill now recovered categories separately and
compares each category with deceased. A logistic model
assesses risk factors related to renal failure for affected
animals. Both model analyses are performed within
Univariate Analysis for Affected Pets
The regional distribution of all reported, affected pets is
summarized in Fig. 1. In North America, reports were
submitted from four provinces, 35 states, and Puerto Rico,
with the majority coming from California, Texas, Michi-
gan, Florida, and Ontario. The USA reported 94.5% of the
cases submitted, and the remaining 5.5% came from
Of the 424 affected pets, 278 (65.6%) cases were cats
and 146 (34.4%) cases were dogs, with a cat/dog ratio of
174 J. Med. Toxicol. (2010) 6:172184
Characteristics of Affected Pets
Among the affected pets, 44 dog breeds and 14 cat
breeds were represented. Dog breeds most frequently
reported were crossbreeds > Labrador retrievers >
Dachshund > Pomeranian. Sixty-six percent of all cat
cases were domestic short hair followed by the Siamese
cat (6.4%).
There was no significant difference for either dogs or
cats in the number of cases reported for males and
females (p<0.35; canine, 50.8% male and 49.2% female;
feline, 47% male and 53% female).
The average age of affected cats was 92 months
(7.6 years) with a standard deviation of 60 months
(range, 2 to 252 months). The median age of affected
cats was 84 months (7 years), and the median age was
within 8 months of the average. The average age of
affected dogs was 94 months (7.8 years) with a standard
deviation of 54 (range, 4 to 228 months). The median
age of affected dogs was 96 months (8 years), and the
average and median ages were within 3 months of each
When sex is accounted for, the average age of affected
female cats was 100 months (n= 135), while that of affected
male cats was 82 months (n=119). Using the two-sample
ttest, the reported male cats were affected significantly
younger than reported females (p=0.015). For dogs, the
average ages of affected females (93 months, n= 63) and
males (96 months, n=64) were not significantly different by
Diagnostic Criteria
A summary of diagnostic criteria (Q9) used for diagnosis
of pet food-induced nephrotoxicity is given in Tables 1
(cats) and 2(dogs). This question was asked to determine
the criteria on which clinicians relied for diagnosis/
definition of those cases. The questions were not mutually
exclusive, and respondents were told to mark all that
applied and write in additional helpful parameters. Among
deceased cats and dogs, the majority of veterinarians
relied on a history of ingesting recalled pet food and on
histopathology findings. In recovered or currently ill
categories, the majority of respondents relied on the
Fig. 1 Regional distribution of cases reported as affected in North America. Note that the states/provinces with most affected pets were California >
Texas > Michigan > Florida > Ontario. Blank state = no report
J. Med. Toxicol. (2010) 6:172184 175
history of ingesting recalled pet food to make their
diagnoses. Serum chemistry profiles indicative of azote-
mia were relied on by 41.7% of respondents. Additional
diagnostic criteria listed included polydipsia, polyuria,
low urinary specific gravity, renal biopsy, and ultrasound
results consistent with hyperechoic corticomedullary
junction. No response was received in response to Q10,
What other chemical marker was present in this case?
Predisposition to Renal Disease
The majority of affected pets (330 of 419, 78.8%) did not
have any reported preexisting conditions that predisposed
them to renal failure.
The conditions listed (gastrointestinal disease, renal
disease, cardiovascular disease, neurological disease,
cancer, and other) were not mutually exclusive, and
70% of those reporting preexisting conditions selected
more than one condition in a given animal. By species,
22.5% of affected dogs (n=142) and 20.6% of affected
cats (n=277) reported a predisposing conditions. Of the 19
positive canine responses, 15 (78.9%) reported preexisting
renal disease and four (21.1%) reported cardiovascular
disease. Of the 43 feline responses, 32 (74.4%) reported
preexisting renal disease, five (11.6%) reported cardiovas-
cular disease, and six (14.0%) reported something outside
of the list of categories. The most commonly reported
preexisting renal diseases were diabetes, membranous
glomerulonephritis, chronic interstitial nephritis and fibro-
sis, cancer (renal lymphoma and carcinoma), nephroscle-
rosis, unilateral kidney agenesis, and prior exposure to
toxins such as Easter lilies (Q8).
Table 1 Diagnostic criteria used for cats
Cats Deceased (171) Recovered (49) Currently ill (58)
Gross necropsy 28 (16.4%) 3 (6.1%) 2 (3.4%)
Histopathology 121 (70.8%) 0 2 (3.4%)
Urinalysis 24 (14%) 13 (26.5%) 11 (19%)
Melamine in food 5 (2.9%) 1 (2%) 0
Melamine in tissue 4 (2.3%) 0 0
Melamine in urine 7 (4.1%) 0 1 (1.7%)
Other marker in food, urine, or tissues 11 (6.4%) 1 (2%) 0
History of eating recalled pet food 123 (71.9%) 45 (91.8%) 50 (86.2%)
Note that multiple diagnostic criteria could be used. Note that for deceased cats, histopathology of the kidneys and a history of eating recalled
food were the most commonly used criteria. For recovered and currently ill, urinalysis and history of ingesting recalled pet food were the most
commonly used criteria. Not reflected in this table is Serum chemistry profileconfirming azotemia, which was self-reported as widely used as
one of the diagnostic criteria among recovered or currently ill
Table 2 Reports of criteria for diagnosis (dog)
Dogs Deceased (107) Recovered (15) Currently ill (24)
Gross necropsy 20 (18.7%) 1 (6.7%) 1 (4.2%)
Histopathology 69 (64.5%) 0 2 (8.3%)
Urinalysis 10 (9.3%) 5 (33.3%) 3 (12.5%)
Melamine in food 1 (0.9%) 0 0
Melamine in tissue 2 (1.9%) 0 0
Melamine in urine 2 (1.9%) 0 1 (4.2%)
Other marker in food, urine, or tissues 3 (2.8%) 0 1 (4.2%)
History of eating recalled pet food 78 (72.9%) 13 (86.7%) 17 (70.8%)
Note that multiple diagnostic criteria could be used. Note that for deceased dogs, histopathology of the kidneys and a history of eating recalled
food were the most commonly used criteria. For recovered and currently ill, urinalysis and history of ingesting recalled pet food were the most
commonly used criteria. Not reflected in this table is Serum chemistry profileconfirming azotemia, which was self-reported as widely used as
one of the diagnostic criteria among recovered or currently ill
176 J. Med. Toxicol. (2010) 6:172184
Foods Consumed Prior to Sickness
In the USA, 20 food brands were reported to have
poisoned dogs and 28 food brands for cats. In Canada,
there were three food brands reported to have poisoned
dogs and 10 food brands for cats. Figure 2lists those
food brands with a frequency of >10 cases reported, with
the remainder included as other. Iams (Proctor & Gamble)
and Special Kitty (Menu Foods, Inc.) were the top brands
most often consumed by affected cats, while Alpo (Nestle
Purina), Mighty Dog Pouch (Nestle Purina), and OlRoy
US (Menu Foods, Inc.) were those most often eaten by
affected dogs.
Only 67% of respondents addressed Q11, regarding
the time interval between consumption and onset of
illness. The majority of these (130 out of 394) indicated
that the food brands were routinely fed, and it was
unclear when a contaminated bag was opened. Of those
who knew, four respondents indicated feeding less than
3 to 7 days, 38 indicated 7 to 10 days, and 79 indicated
more than 10 days. Overall, these results suggest that
most of the cases occurred after multiple ingestions of
the contaminated pet food.
Bivariate Analyses Relating Health Status
and Predisposition to Other Variables
Health Status Related to Other Variables
The cross-tabulation of sex with health status showed
no significant relationship for dogs (p=0.663) or for cats
(p=0.164). To compare the mean ages of affected pets in
each health status category (currently ill, previously ill
now recovered, and deceased), a one-way analysis of
variance was run separately for cats and dogs (Table 3).
For the affected animals, the mean age of the currently ill
was higher for both dogs and cats, while the mean age for
the recovered group is the lowest for both dogs and cats.
However, the differences are not significant (p= 0.078).
The correlation of preexisting renal disease (Q7) with
health status (Q5) is reported in Table 4. The association of
preexisting renal disease with death is statistically signifi-
cant in both dogs (p=0.090) and cats (p= 0.023). For cats,
57.3% of those with no noted preexisting conditions died,
compared to 77.2% of cats with known preexisting renal
diseaserenal impairment increased the incidence of death
by 20%. In dogs, 19.3% more animals died when
preexisting renal conditions were reported (87.5% vs
The Relationship Between Preexisting Renal Disease (Q7)
and Sex and Age
The cross-tabulation of sex and whether the animal had
any preexisting renal disease is reported in Table 5.The
relationship is not significant for dogs (p=0.677), but is
highly significant for cats (p=0.007). These results imply
that male cats were more likely than female cats to have
preexisting renal diseases that rendered them more
vulnerable to the melamine-cyanurate pet food-induced
renal failure.
The two-sample ttest was used to analyze for differences
in mean age between animals with any reported preexisting
disease and those without preexisting conditions (Q7). For
dogs, the mean age of the group with preexisting disease
conditions was 106 months (8.9 years) with standard
Fig. 2 Food brands consumed by affected pets. Note that for cats,
Iams and Special Kitty were the two top brands that were consumed
by affected cats. For dogs, the two top food brands were OlRoy US
and Mighty Dog Pouch
Species Health status Fpvalue
Currently ill Recovered Deceased
Dogs (N=133)
Mean age (standard deviation) 104.7 (48.3) 81.1 (54.1) 93.5 (54.8) 0.777 0.462
Cats (N=268)
Mean age (standard deviation) 107.3 (53.0) 82.9 (54.6) 89.1 (64.0) 2.570 0.078
Table 3 Mean age in months
for each health status group
J. Med. Toxicol. (2010) 6:172184 177
deviation of 59 months, and for the non-pre-existing
disease group, the mean age was 89 months (7.4 years)
with standard deviation of 52 months. The difference in
mean age was not significant (p= 0.150). On the other hand,
the mean age of the group of cats with preexisting disease
conditions was 125 months (10.5 years) with standard
deviation of 60 months, and for those without preexisting
conditions, the mean age was 82 months (6.8 years) with
standard deviation of 57 months. The difference in mean
age is highly significant for cats, p< 0.001. This means that
older cats were more likely to have preexisting disease
conditions predisposing them to melamine-contaminated
pet food than younger cats or dogs. By far, the preexisting
disease conditions were kidney-related (74.4%).
Predictive Models
Multinomial Logistic Model to Predict Death
This model considers the currently ill and previously ill
now recovered categories separately, comparing each
category with deceased. The same predictor variables as
in the logistic model are used. Results are reported in
Table 6. For dogs, none of the variables are significant
predictors of either currently ill vs deceased, or recovered
vs deceased. However, for cats, age and predisposition to
renal disease significantly distinguish currently ill from
deceased, and predisposition is significant for recovered vs
deceased. A 1-year increase in age increases the odds of
being currently ill rather than deceased by about 11%. This
is likely the result of the cross-sectional nature of the data
since the final outcome for the ill animals is not known.
However, the presence of preexisting predisposing diseases
is a strong risk factor in death. Those cats with preexisting
disease conditions have more than triple the odds of being
deceased rather than currently ill, and more than 2.5 times
the odds of being deceased rather than recovered. Although
the average age of the recovered cats was slightly below the
average age of deceased cats, the difference was not enough
to conclude that age is a significant risk factor (comparing
recovered and deceased) using this multinomial logistic
model to predict death.
Logistic Model to Predict Predisposition to Renal Disease
Since preexisting disease plays an important role in
survival, the question of whether it is a mediating factor
is important. That is, can preexisting conditions predispos-
ing to renal disease be predicted by sex and age? Results
are reported in Table 7. For dogs, age and sex there are no
significant findings. However, for cats, age and sex are
significant risk factors in predisposition to renal disease.
The odds ratio for sex is interpreted as the increase in odds
of predisposition to renal disease when comparing males to
females. Thus, for cats, the odds of predisposition to renal
disease decrease by about 34% when the cat is female. In
addition, for cats, the odds of predisposition to renal disease
increase by about 14% for each year of age. This
predisposition to renal disease, in turn, increases the chance
of death or euthanasia. This suggests that following
ingestion of melamine-cyanurate-contaminated pet food,
older male cats were most at risk to die or be euthanized
than any other segment of the pet population examined in
this survey.
This study was voluntary, and therefore, it was not
possible to collect data on all pets that were affected by
Currently ill Previously ill, recovered Deceased χ
Preexisting conditions 7 (12.3%) 6 (10.5%) 44 (77.2%) 0.023
No preexisting conditions 51 (23.2%) 43 (19.5%) 126 (57.3%)
Preexisting conditions 3 (9.4%) 1 (3.1%) 28 (87.5%) 0.090
No preexisting conditions 21 (19.1%) 14 (12.7%) 75 (68.2%)
Table 4 Health status by
species and preexisting
Table 5 Percentage of each sex with preexisting conditions
Species/sex Preexisting conditions pvalue
Yes No
Male 16 (24.6%) 49 (75.4%) 0.677
Female 14 (21.5%) 51 (78.5%)
Male 17 (13.9%) 105 (86.1%) 0.007
Female 38 (27.7%) 99 (72.3%)
178 J. Med. Toxicol. (2010) 6:172184
the melamine-cyanuric acid pet food contamination. The
focus of the study was to determine the minimum
number of confirmed cases of pet food-induced nephro-
toxicity, to determine how geographically widespread the
confirmed cases were, and, using this database of
confirmed cases, to identify common factors, signalment,
and disease outcome among clinically affected pets. In
this web-based survey, 474 veterinarians responded
reporting a total of 586 individual pets. As no control
group was surveyed, a strong bias is undoubtedly present
since only animals whose illness affected them enough to
be noticed by the owners were likely presented to
veterinarians. Many questions could only be answered
descriptively in terms of the survey results. Inferential
statistics were used where possible to extend predictions
beyond the sample group.
Reported affected pets were fairly widely distributed
throughout the USA, although no cases were reported in
some states. This may be due to the uneven distribution of
participating clinics or diagnostic laboratories. Respondents
had an option to list species affected other than dogs and
cats, but none were reported (Q1). Reports from experi-
mental studies indicate that pigs and fish are also affected
by melamine-cyanurate-contaminated food [14].
Results show that 65.5% of the affected pets were cats
and 34.4% were dogs. According to the US Pet Ownership
and Demographics Sourcebook information posted on the
AVMA website, in 2007, there were 72.1 million dogs (in
43.0 million homes) and 81.7 million cats (in 37.5 million
homes) in the USA [15]. Whereas there were proportionally
more cats than dogs (53.1% vs 46.9%), there were fewer
homes with cats than with dogs (46.5% vs 53.5%) in the
USA in 2007, suggesting that more cats were kept per
household. Comparing pet populations, more cats were
affected than were represented in the general pet population
(65.5% affected vs 53.5% in pet population) in 2007. It is
possible that more cats were affected than dogs because
multiple cats in the same household may have been exposed
to contaminated pet food. Alternatively, since published data
show that daily dry matter intake in cats of 0.05
0.8 kg animal
is similar to that of dogs (0.07
0.8 kg animal
), it is possible that cats are more
sensitive than dogs to melamine-cyanurate-contaminated pet
food [16].
Among dogs, crossbreeds were the most frequently
reported to have been affected, likely reflecting the
overall popularity of mixed-breed dogs as pets. Unfortu-
nately, animal weight was not included in the question-
naire; therefore, the size of these mixed dog breeds is
Species Variable Odds ratio pvalue
Currently ill Age in years 1.10 0.143
Sex 0.730 0.569
Preexisting predisposing diseases 2.915 0.186
Recovered Age in years 0.963 0.571
Sex 0.617 0.400
Preexisting predisposing diseases 4.80 0.141
Currently ill Age in years 1.11 0.004*
Sex 1.832 0.074
Preexisting predisposing diseases 3.21 0.015*
Recovered Age in years 1.01 0.806
Sex 1.27 0.481
Preexisting predisposing diseases 2.63 0.051*
Table 6 Multinomial logistic
regression with reference
category deceased
*Significantly different from
deceased cats
Table 7 Logistic regression for predicting disposition to renal failure
Species/variable Odds ratio pvalue
Age in years 1.077 0.131
Sex 1.208 0.664
Age in years 1.142 <0.001**
Sex 0.462 0.024*
J. Med. Toxicol. (2010) 6:172184 179
unknown. Among the purebreds, the Labrador retriever,
Dachshund, and Pomeranian were most represented. The
Labrador retriever is a large breed, while the Pomeranian
is a small dog. Dog size does not appear to be a factor as
both large and small dogs were affected. However, the
Labrador retriever is the most popular dog in the USA,
and the results may simply reflect that fact. With respect
to affected cat breeds, the domestic short hair cat was the
most frequently affected. This is probably because it is
the most common household breed of cat.
News reports indicated that thousandsof pets died
as a result of pet food-induced nephrotoxicity [13].
Results of this study show that the numbers of confirmed
affected pets were at least 586 cases, but the total will
probably never be known. Banfield, a large small animal
practice in North America, indicated that only a small
population of pets was actually affected by the contami-
nated pet food [17]. It is likely, however, that some pets
had died before this outbreak was recognized and
therefore were not reported. Furthermore, many affected
pets may not have been presented for treatment, or for
necropsy if deceased. It is also likely that despite the effort
to publicize the survey as widely as was done, some
veterinarians were not aware of it or chose not to
Despite its shortcomings, this study yielded interesting
data. Results show that overall within species, male and
female pets were affected at about the same frequency. In
addition, animals of all ages were affected, although the
average and median age of affected pets was about
8 years old. A higher proportion of affected dogs died
than affected cats, despite the observation that more cats
than dogs were affected. The reasons for this are not
clear. It is also noteworthy that the older male cat was
most at risk to die or to be euthanized after ingesting
contaminated pet food. It is not clear why older male
cats experienced a more severe outcome than older
female cats, this in spite of the finding that significantly
more older female cats were affected than males.
Another interesting finding was that the majority of
affected pets (>78%) did not have preexisting diseases.
This suggests that melamine cyanurate is a potent toxin
that will affect even healthy pets. However, among pets
reported to have preexisting conditions, 74.4% of cats
and 78.9% of dogs had kidney-related diseases of which
diabetes, glomerulonephritis, chronic interstitial nephritis,
and kidney cancer were the most common. The results
show that these preexisting diseases increased the
incidence of death.
Many food brands were reported to have affected pets.
However, some food brands were more frequently
reported than others. For example, for cats, Iams and
Special Kitty U.S. were two top foods eaten by affected
pets. It is not clear if these food brands contained
proportionally more contaminants than others, or if
simply they were the most popular cat food brands.
Also, the majority of veterinarians indicated that illness
was observed in pets following several days of feeding
contaminated pet food. This suggests cumulative effect(s)
of the toxicants.
In conclusion, this study has shown that more cats
than dogs were reported poisoned by the melamine-
cyanuric acid pet food contaminants. The proportion of
affected cats to dogs was 1.9:1 compared to a ratio of
1.1:1 in the general population in 2007. Of those cases
submitted in this survey, 278 pets were reported to have
died. The cat to dog ratio of pets that died was 1.6:1.
Within species however, a higher percentage of affected
dogs (73.3%) were reported to have died or euthanized
compared to only 61.5% of cats. The vast majority of
affected pets were healthy, without preexisting disease
conditions. Of the pets reported to have preexisting
conditions, the vast majority had preexisting renal
diseases. Preexisting renal diseases and old age pre-
dicted the most severe outcome (death or euthanasia)
than any other factors. Older male cats with preexisting
diseases were the most vulnerable and more likely to
die or be euthanized among pets that ate contaminated
pet food.
Acknowledgments This study would not have been successful if
there was no participation from veterinary clinicians and diagnos-
ticians across North America. Special gratitude goes to diagnosticians
who entered multiple cases including Drs. Nikos Gurfield (San Diego
County Animal Disease Diagnostic Laboratory), Catherine Barr (TX),
Jennifer Cobin (St Francis Veterinary Center), Tracy Darling (Cat
Clinic Orange County), Pat Halbur (Iowa State University), Rebecca
Harrod (Hazel Ridge Vet Clinic, CA), and Nikos Gurfield (San
Diego County Diagnostic Lab). A special thank-you also goes to Liz
Connelly who helped with the logistics of the survey instrument in
April of 2007. Finally, we acknowledge all professional organizations
such as the AAVLD, the AVMA, the ACVIM, and the ACVP for
publicizing the survey.
Funding No funds were used on this project. The Diagnostic Center
for Population and Animal Health, College of Veterinary Medicine,
Michigan State University, allowed use of the software.
Previous presentation of data Preliminary results were presented at
the annual AAVLD meeting in Reno Nevada in October 2008. These
data have not been published or presented at another venue.
180 J. Med. Toxicol. (2010) 6:172184
A Complete list of Questions on the Survey Questionnaire
1. What is the species of the affected animal?
Other (please specify)
2. What is the breed of the affected animal?
3. What is the age of the affected animal? Years Months
4. What is the sex of the affected animal? Male Female
5. What is the current health status of the affected animal?
Healthy never ill
Currently ill
Previously ill now recovered
6. What geographic location was the affected animal living in at the time of illness onset?
State or Province
7. In your opinion, did this animal have any conditions that would predispose it to renal disease/failure?
8. Define the predisposing condition:
Gastrointestinal disease
Renal disease
Cardiovascular disease
Neurologic disease
Other (please specify)
9. Please indicate diagnostic criteria used to arrive at your diagnosis: (select all that apply)
Gross findin
stal-induced renal failure (with distinct ID of characteristic cr
J. Med. Toxicol. (2010) 6:172184 181
Histopathologic findings consistent with crystal-induced renal failure (with distinct ID of characteristic crystals)
Urinalysis consistent with characteristic crystals
Analytical confirmation of melamine marker in food
Analytical confirmation of melamine marker in animal tissues
Analytical confirmation of melamine marker in urine
Analytical confirmation of any chemical marker in tissue, urine, or food.
History of having eaten recalled pet food
Other (please specify)
10. What other chemical marker was present in this case?
11. How soon after ingesting the recalled pet food did the animal become ill?
Less than 1 day
More than 10 days
Other (please specify)
12. Which dog/cat food(s) was the affected animal eating at the time of (or immediately preceding) the diagnosis?
(Select all that apply)
182 J. Med. Toxicol. (2010) 6:172184
13. Dog/cat food lot number (if known)
14. Dog/cat food UPC (Universal product code) number (if known)
15. Please
rovide the uni
ue animal-identification number for this case.
J. Med. Toxicol. (2010) 6:172184 183
1. Burns K, Nollen S, Kahler S, Rezendies A (2007) Recall of pet
food leaves veterinarians seeking solutions. JAVMA 230
(12):1795, April 15, 2007 News
2. OECD (2002) SIDS analysis, melamine. UNEP, Nairobi, pp 111
3. Hau KA, Kwan TH, Li PK (2009) Melamine toxicity and the
kidney. J Am Soc Nephrol 20:245250
4. Clark R (1966) Melamine crystalluria in sheep. J S Afr Vet Med
Assoc 37:349351
5. Lipschitz WI, Stokey E (1945) The mode of action of three new
diuretics: melamine, adenine and formoguanamine. J Pharmacol
Exp Ther 83:235249
6. Melnick RL, Boorman GA, Haseman JK, Montali RJ, Huff J
(1984) Urolithiasis and bladder carcinogenicity of melamine in
rodents. Toxicol Appl Pharmacol 72:292303
7. Ogasawara H, Imaida K, Ishiwata H, Toyoda K, Kawanishi T,
Uneyama C et al (1995) Urinary bladder carcinogenesis induced by
melamine in F344 male rats: correlation between carcinogenicity and
urolith formation. Carcinogenesis 16:277327777
8. Puschner B, Poppenga RH, Lowenstine LJ, Filigenzi MS,
Pesavento PA (2007) Assessment of melamine and cyanuric acid
toxicity in cats. J Vet Diagn Invest 19:616624
9. Dobson RLM, Motlagh S, Quijano M, Cambron T, Baker RT,
Pullen MA et al (2008) Identification and characterization of
toxicity of contaminants in pet food leading to an outbreak of
renal toxicity in cats and dogs. Tox Sci 106:251262
10. Hodge HC, Panner BJ, Downs WL, Maynard EA (1965) Toxicity
of cyanurate. Toxicol Appl Pharmacol 17:667674
11. Brown CA, Jeong K, Poppenga RH, Puschner B, Miller DM, Ellis
AE et al (2007) Outbreaks of renal failure associated with
melamine and cyanuric acid in dogs and cats in 2004 and 2007.
J Vet Diagn Invest 19:525531
12. Thompson ME, Lewen-Smith MR, Kalasinsky VF, Pizzolato M,
Fleetwood ML, McElhaney MR et al (2008) Characterization of
melamine-containing and calcium oxalate crystals in three dogs
with suspect pet food-induced nephrotoxicosis. Vet Pathol
13. New York Times (2007) Editorial. After the pet food contamination,
May 7, 2007
14. Reimschuessel R, Gieseker CM, Miller RA, Ward J, Boehmer J,
Rummel N et al (2008) Evaluation of the renal effects of
experimental feeding of melamine and cyanuric acid to fish and
pigs. Am J Vet Res 69:12171228
15. US pet ownership and demographics sourcebook 2007 edition. Cited
on 09/09/09
16. Puls R (1994) Water consumption and dry matter intake guide-
lines. In: Puls R (ed) Mineral levels in animal health. Sherpa
International, Clearbrook, pp 307307
17. Lundeen T (2007) Fewer pets ill from recall. Feedstuffs 79, April
16, pp 1 and 5
16. Can we contact you regarding this case if further review is deemed beneficial?
17. Please provide contact information:
Contact name
Address 1:
Address 2:
Contact Phone number
Contact e-mail
184 J. Med. Toxicol. (2010) 6:172184
... According to a US survey, this resulted in health effects and death in pets in 35 states, Puerto Rico, and in four Canadian provinces. (Rumbeiha et al., 2010) Melamine has also unintentionally become an agricultural food contaminant due to the use of the insecticide cyromazine. (Hilts and Pelletier, 2008) Cyromazine inhibits chitin synthesis and is commonly added to animal feeds to control flies in manure (Keiding, 1999;EPA, 2013). ...
Melamine is commonly used in a variety of consumer products such as furniture, dining ware, and food utensils. The chemical infamously gained worldwide attention by its illegal addition to a variety of foodstuffs in order to falsify protein content, which led to serious, sometimes fatal, health impacts in children and pets. This resulted in a large amount of published primary studies and reviews of the impacts of melamine exposure on kidney function. However, a growing body of literature suggests that melamine may have impacts beyond renal dysfunction. We conducted a scoping review of this literature which yielded more than 40 studies with human, animal, and in vitro findings. Neurological impacts, reproductive function, and anthropometric outcomes were identified as possible candidates for systematic review based on evidence stream and replication of endpoints. The results of this analysis provide a basis for prioritizing future research on health impacts associated with melamine exposure.
... In 2008, WHO arose a serious food safety alert due to the elevated occurrence of kidney stones and renal failure in infants due to ingestion of MEL-adulterated infant formula in many countries. Also, a huge number of accidental pet deaths following ingestion of MEL-tainted foods have been lately reported (Dorne et al., 2013;Rumbeiha et al., 2010). Additionally, various hazards have been accompanied with MEL consumption including bladder cancer (Ogasawara et al., 1995), kidney injury (Dobson et al., 2008), nerve dysfunction (Wang et al., 2011), and reproductive toxicity (Khalil et al., 2017;Yin et al., 2013). ...
... According to a US survey, this resulted in health effects and death in pets in 35 states, Puerto Rico, and in four Canadian provinces. (Rumbeiha et al., 2010) Melamine has also unintentionally become an agricultural food contaminant due to the use of the insecticide cyromazine. (Hilts and Pelletier, 2008) Cyromazine inhibits chitin synthesis and is commonly added to animal feeds to control flies in manure (Keiding, 1999;EPA, 2013). ...
Polycyclic aromatic hydrocarbons (PAHs) are a class of common persistent environmental pollutants found in water, air, soil, and plants and can be released by natural sources. However, the majority of atmospheric PAHs are from vehicular emissions, coal-burning plants, and the production and use of petroleum-derived substances. Exposure to PAHs has been implicated in cancer and other diseases, including reproductive disorders. This scoping review is a preliminary step that explores the utility and feasibility of completing a systematic review evaluating the effect of PAHs on female reproduction. We performed literature searches in PubMed, Web of Science, and Scopus, then screened, identified, and categorized relevant studies. Our results identified fertility and pregnancy/fetal viability as outcomes with sufficient research for systematic review. In addition to presenting the relevant studies, the review identifies data gaps, and provides the groundwork to develop the most appropriate research questions for systematic review.
... In recent years, international food safety agencies have examined toxicities of MA due to a large number of accidental pet deaths following ingestion of MA-tainted foods (Rumbeiha et al. 2010;Dorne et al. 2013). Moreover, a serious food safety alert regarding MA poisoning arose due to contaminated infant formula found in various countries (WHO 2008). ...
Full-text available
The aim of the present study was to explore the potential hematotoxic and immunotoxic effects of melamine (MA) in the absence and presence of formaldehyde (FA) in mice. Forty adult Swiss mice were equally allocated into four groups and daily treated with water, MA (50 mg/kg), FA (25 mg/kg), and MA þ FA respectively via feeding needle for 60 consecutive days. Hematological status was evaluated using erythrogram and leukogram profiling. Innate immune functions were assessed by measuring white blood cells lysozyme and phagocytic activities. Serum immunoglobulin levels were evaluated as indicators of humoral immunity. In addition, histologic and immunohistochemical evaluations of splenic tissues were performed.The results indicated that either MA or FA treatment resulted in significant decreases in RBCs, Hb, MCHC, total WBC, lymphocyte, and basophile levels as well as in WBCs phagocytosis and lysozyme activity. In contrast, MCV, PCV%, and reticulocyte levels were significantly increased in these hosts. The total IgM level was significantly reduced in the MA-only-exposed mice but markedly increased in the FA-only-treated ones. A significant decrease in serum IgG levels was detected following either MA or FA treatment. The combined exposure to MA and FA, compared to levels of either toxicant alone, was revealed to evoke a significant improvement in Hb, PCV%, MCV, MCHC, neutrophil, eosinophil, total IgM level, and lysozyme activity; however these values did not reach that of the controls. Furthermore, compared to control mice, both MA-only- and FA-only-treated mice showed a strong distribution of CD4þ and CD8þ cells in their spleens, while a moderate presence of the former cells was obvious at their co-exposure. Taken together, these findings revealed that exposure to MA or FA resulted in significant alterations in hemato-immune parameters at variable degrees while a co-exposure resulted in the mitigation of most effects of either toxicant alone.
Melamine is an industrial chemical with a high LD50 in laboratory animals. Because it is nitrogen rich, melamine has found illicit use as a substitute for protein in the production of foods and feeds with the unexpected consequences of high morbidity and mortality. There was an incident of pet food–contamination incident in 2007 in which melamine, combined with co-contaminant cyanuric acid, crystalized in the distal convoluted tubules of cats and dogs, resulting in renal failure. There was little or no cyanuric acid present in Chinese baby formula contaminated with melamine in 2008; however, neonates excrete uric acid, which combines with melamine in the urinary tract, forming uroliths in affected infants. Melamine is not currently considered to be mutagenic.
Melamine is a chemical substance used as a food adulterant because of its high nitrogen content; it is known to induce neurotoxicity, thereby adversely affecting the central nervous system. The biocompatibility, bioavailability, lower toxicity, and the large surface area of nanosized selenium relative to its other forms indicate that selenium nanoparticles (SeNPs) have a potential ameliorative effect against melamine-induced neurotoxicity. In this study, we tested this hypothesis using 40 adult male albino rats that were randomly assigned into four groups (n = 10 per group): group I rats served as the untreated negative controls and were fed with standard diet and distilled water; group II rats were orally treated with melamine (300 mg/kg body weight/d); group III rats orally received melamine (300 mg/kg body weight/d) and SeNPs (2 mg/kg body weight/d); and group IV rats received SeNPs only (2 mg/kg body weight/d) for 28 days. Blood and brain samples were collected from all rats and processed for biochemical, histopathological, and immunohistochemical investigations. SeNPs were encapsulated in starch as a natural stabilizer and a size-controlling agent (SeNP@starch). The prepared SeNPs were characterized using different techniques. Inductively coupled plasma-optical emission spectrometry (ICP-OES) indicated that the percentage of selenium loaded in starch was 1.888%. Powder x-ray diffractometer (XRD) was used to investigate the crystalline structure of the Se-NP@starch, to be tubular and composed of amorphous starch as well as metallic selenium. Thermogravimetric analysis confirmed the thermal stability of the product and determined the interactions among the different components. Transmission electron microscope demonstrated the spherical shape of SeNPs and their dispersion into starch surface as well as evaluating their size in nanoscale (range 20-140 nm). Our results revealed that the melamine- exposed rats had significantly elevated in malondialdehyde levels, significantly reduced in total antioxidant capacity, down-regulated expression of the antioxidant related genes Nrf2 (nuclear factor erythroid 2–related factor 2) and GPx (glutathione peroxidase), as well as up-regulated expression of the apoptosis-related gene Bax (B-cell lymphoma 2–associated X protein), with down regulation of Bcl-2 (B-cell lymphoma 2). Histopathological examination exhibited several alterations in the cerebrum, cerebellum, and hippocampus of the treated rats compared with the controls. Neuronal degeneration, vacuolation of the neuropils, and pericellular and perivascular spaces were observed. In addition, the pyramidal and granular cell layers of the hippocampus and cerebellum, respectively, were found to have significantly reduced thickness. Furthermore, a significant decrease in the percentage area of the glial fibrillary acidic protein and a significant increase in the percentage area of caspase-3 were noted. On the other hand, co-treatment with SeNPs partially ameliorated these alterations. A significant reduction in malondialdehyde levels; a non- significant elevation in total antioxidant capacity; up-regulation, upregulation of Nrf2, GPx, and Bcl-2 and downregulation of Bax were recorded. Neuronal degeneration, vacuolation of neuropils, and pericellular spaces were reduced. The pyramidal and granular cell layers restored their normal thickness. The percentage area of the glial fibrillary acidic protein significantly increased, whereas that of caspase-3 significantly decreased. In conclusion, SeNPs have an ameliorative effect against melamine-induced neurotoxicity in albino rats.
Veterinarians are faced with the challenge of understanding and discussing the wide array of commercial pet foods with their clients. To promote these discussions, a pet food processing categorization system is applied to define familiar heat processed dry and can pet foods as ultra-processed commercial diets (UPCD) and other less processed diets as minimally processed commercial diets. A review of the FDA pet food recalls on commercial diets are used to discuss well-known health risks, such as nutritional imbalances, bacterial pathogens, aflatoxin, and toxic contaminations. A less-known concern of advanced glycation end products found in UPCDs is presented.
Background /aims Melamine (ML) is a common food adulterant and contaminant. Moringa oleifera is a well-known medicinal plant with many beneficial biological properties. This study investigated possible prophylactic and therapeutic activity of an ethanolic extract of M. oleifera (MEE) against ML-induced hepatorenal damage. Method Fifty male Sprague Dawley rats were orally administered distilled water, MEE (800 mg/kg bw), ML (700 mg/kg bw), MEE/ML (prophylactically) or MEE+ML (therapeutically). Hepatic aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphate (ALP) in plasma were measured. Serum total bilirubin, direct bilirubin, indirect bilirubin, protein, albumin, and globulin contents were also assayed, and urea and creatinine levels determined. Moreover, antioxidant enzyme activity of glutathione peroxidase (GPx) and catalase (CAT) in serum levels were quantified. Complementary histological and histochemical evaluation of renal and hepatic tissues was conducted, and expression of oxidative stress (GPx and CAT) and apoptosis related genes, p53 and Bcl-2, in hepatic tissue were assessed. In parallel, transcriptional expression of inflammation and renal injury-related genes, including kidney injury molecule 1 (KIM-1), metallopeptidase inhibitor 1 (TIMP1), and tumor necrosis factor alpha (TNF-α (in the kidney tissue were determined. Results ML caused significant increases in serum levels of ALT, AST, ALP, total bilirubin, direct bilirubin, indirect bilirubin, urea, and creatinine. Further, ML treated rats showed significant reductions in serum levels of protein, albumin, globulin, GPx, and CAT. Distinct histopathological damage and disturbances in glycogen and DNA content in hepatic and renal tissues of ML treated rats were observed. KIM-1, TIMP-1, and TNF-α gene expression was significantly upregulated in kidney tissue. Also, GPx, CAT, and Bcl-2 genes were significantly downregulated, and p53 was significantly upregulated in liver tissue after ML treatment. MEE significantly counteracted the ML-induced hepatorenal damage primarily for co-exposed rats. Conclusion MEE could be an effective therapeutic supplement for treatment of ML-induced hepato-renal damage, probably via modulating oxidative stress, apoptosis, and inflammation.
Melamine is a nitrogen-containing heterocyclic organic compound with a triazine skeleton, which has been widely applied in industrial and chemical fields. Previous toxicity studies of melamine mainly focused on renal toxicity and hepatic pathological changes, but its toxicity against the reproductive system has seldom been assessed. We investigated the effects of melamine on the reproductive system of male mice. Forty healthy male Kunming mice were randomly divided into a normal saline negative control group, a low-dose melamine group, a medium-dose melamine group and a high-dose melamine group (n = 10). The mice were administered for 5 consecutive days, and killed on the 35th day after first administration. In melamine administration groups, seminiferous tubules had disordered, loose arrangement, and spermatogenic cells at all levels obviously decreased. The sperm count and motility decreased significantly, and the sperm deformity rate increased significantly. Melamine induced apoptosis of testicular spermatogenic cells. To further explore the mechanism, we detected metabolism-related enzymes sorbitol dehydrogenase (SDH) and lactate dehydrogenase (LDH) as well as oxidative stress indices superoxide dismutase (SOD)and malondialdehyde (MDA). The activities of SDH, LDH and SOD in melamine treatment groups decreased significantly, and the MDA level increased obviously. The expressions of apoptosis-related proteins Bcl-2, Bax and caspase-3 were detected by immunohistochemistry. The expression of Bcl-2 significantly increased, but those of Bax and caspase-3 significantly reduced (P < 0.05). In conclusion, melamine damaged the reproductive system of mice via the oxidative stress pathway and by inducing cell apoptosis.
Poor quality melamine containing cyanuric acid was fraudulently added to pet-food ingredients by Chinese distributors, producing morbidity and death in dogs and cats in 2007. The following year, a more pure grade of melamine was added to milk products, including baby formula, causing uroliths in infants and young children. Melamine and cyanuric acid interact in the renal tubules forming crystals, producing renal failure in cats and dogs. Melamine also interacts with uric acid, which caused urolithiasis in young children in 2008. Biomarker studies suggest that the physiological disruptions caused by melamine and melamine and cyanuric acid affect amino acid and energy metabolism and induce apoptosis in renal tubular epithelium in vitro. N-acetyl-ß-D glycosidase is a potential indicator of persistent urolithiasis due to melamine. Upregulation of mRNA for TGF-ß1, VCAM-1, MCP-1, and IL-6 was found to be an early indicator of melamine exposure in vitro.
Full-text available
To determine whether renal crystals can be experimentally induced in animals fed melamine or the related triazine compound cyanuric acid, separately or in combination, and to compare experimentally induced crystals with those from a cat with triazine-related renal failure. 75 fish (21 tilapia, 24 rainbow trout, 15 channel catfish, and 15 Atlantic salmon), 4 pigs, and 1 cat that was euthanatized because of renal failure. Fish and pigs were fed a target dosage of melamine (400 mg/kg), cyanuric acid (400 mg/kg), or melamine and cyanuric acid (400 mg of each compound/kg) daily for 3 days and were euthanatized 1, 3, 6, 10, or 14 days after administration ceased. Fresh, frozen, and formalin-fixed kidneys were examined for crystals. Edible tissues were collected for residue analysis. Crystals were examined for composition via Raman spectroscopy and hydrophilic-interaction liquid chromatography-tandem mass spectrometry. All animals fed the combination of melamine and cyanuric acid developed goldbrown renal crystals arranged in radial spheres (spherulites), similar to those detected in the cat. Spectral analyses of crystals from the cat, pigs, and fish were consistent with melamine-cyanurate complex crystals. Melamine and cyanuric acid residues were identified in edible tissues of fish. Although melamine and cyanuric acid appeared to have low toxicity when administered separately, they induced extensive renal crystal formation when administered together. The subsequent renal failure may be similar to acute uric acid nephropathy in humans, in which crystal spherulites obstruct renal tubules.
Full-text available
This paper describes research relating to the major recall of pet food that occurred in Spring 2007 in North America. Clinical observations of acute renal failure in cats and dogs were associated with consumption of wet pet food produced by a contract manufacturer producing for a large number of companies. The affected lots of food had been formulated with wheat gluten originating from China. Pet food and gluten were analyzed for contaminants using several configurations of high-performance liquid chromatography (HPLC) and mass spectrometry (MS), which revealed a number of simple triazine compounds, principally melamine and cyanuric acid, with lower concentrations of ammeline, ammelide, ureidomelamine, and N-methylmelamine. Melamine and cyanuric acid, have been tested and do not produce acute renal toxicity. Some of the triazines have poor solubility, as does the compound melamine cyanurate. Pathological evaluation of cats and dogs that had died from the acute renal failure indicated the presence of crystals in kidney tubules. We hypothesized that these crystals were composed of the poorly soluble triazines, a melamine-cyanuric acid complex, or a combination. Sprague dawley rats were given up to 100 mg/kg ammeline or ammelide alone, a mixture of melamine and cyanuric acid (400/400 mg/kg/day), or a mixture of all four compounds (400 mg/kg/day melamine, 40 mg/kg/day of the others). Neither ammeline nor ammelide alone produced any renal effects, but the mixtures produced significant renal damage and crystals in nephrons. HPLC-MS/MS confirmed the presence of melamine and cyanuric acid in the kidney. Infrared microspectroscopy on individual crystals from rat or cat (donated material from a veterinary clinic) kidneys confirmed that they were melamine-cyanuric acid cocrystals. Crystals from contaminated gluten produced comparable spectra. These results establish the causal link between the contaminated gluten and the adverse effects and provide a mechanistic explanation for how two apparently innocuous compounds could have adverse effects in combination, that is, by forming an insoluble precipitate in renal tubules leading to progressive tubular blockage and degeneration.
Full-text available
Sixteen animals affected in 2 outbreaks of pet food-associated renal failure (2 dogs in 2004; 10 cats and 4 dogs in 2007) were evaluated for histopathologic, toxicologic, and clinicopathologic changes. All 16 animals had clinical and laboratory evidence of uremia, including anorexia, vomiting, lethargy, polyuria, azotemia, and hyperphosphatemia. Where measured, serum hepatic enzyme concentrations were normal in animals from both outbreaks. All animals died or were euthanized because of severe uremia. Distal tubular lesions were present in all 16 animals, and unique polarizable crystals with striations were present in distal tubules or collecting ducts in all animals. The proximal tubules were largely unaffected. Crystals and histologic appearance were identical in both outbreaks. A chronic pattern of histologic change, characterized by interstitial fibrosis and inflammation, was observed in some affected animals. Melamine and cyanuric acid were present in renal tissue from both outbreaks. These results indicate that the pet food-associated renal failure outbreaks in 2004 and 2007 share identical clinical, histologic, and toxicologic findings, providing compelling evidence that they share the same causation.
The toxicity of melamine caught the attention of physicians as a result of a recent spate of renal injury after exposure to melamine-tainted milk in China. Melamine is an organic nitrogenous compound used in the production of plastics, dyes, fertilizers, and fabrics. In the current incident, melamine was added to milk to elevate falsely assay results for protein content. A variety of toxic effects from melamine, including nephrolithiasis, chronic kidney inflammation, and bladder carcinoma, all have been studied in animals. We review here the epidemiology, clinical features, and investigative findings concerning the only outbreak of melamine poisoning in humans. We also examine the renal toxicities of melamine and cyanuric acid--a by-product of its synthesis--and the associated risk factors on exposure and provide guidance on levels in foods.
Sodium cyanurate fed to rats for 20 weeks and to dogs for 6 months was without evident adverse effects at a dietary concentration of 0.8%. Rats fed 8% of sodium cyanurate in the diet for 20 weeks showed consistent and characteristic histologic changes in the kidneys, consisting of dilatation of the distal collecting tubules and ducts of Bellini with focal areas of epithelial proliferation. Similar renal changes were observed in dogs fed 8% of sodium cyanurate in the diet for 16–24 months.Five milliliters of an 0.8% or 8% aqueous suspension of sodium cyanurate applied daily, 5 days/week, to the skin of rabbits for 3 months caused no adverse effects at the lower concentration. At the higher concentration, kidney changes similar to, but less marked than, those described in the rats and dogs were noted. No skin irritation was caused by either concentration.No eye irritation was observed in rabbits receiving 0.1 ml daily, 5 days/week, of an 8% aqueous suspension of sodium cyanurate instilled into one eye over a period of 3 months.
Melamine (2,4,6-triamino-s-triazine) was administered in the diet to F344 rats or B6C3F1 mice for 13 weeks (subchronic) or for 103 weeks (chronic) to determine its toxicologic profile, including carcinogenic potential in the chronic study. The dose levels of melamine in the subchronic studies ranged from 750 to 18,000 ppm for rats, and 6000 to 18,000 ppm for mice. In the chronic studies the dose levels of melamine were 2250 or 4500 ppm for male rats and mice of each sex, and 4500 or 9000 ppm for female rats. In these studies, compound-related lesions were observed in the urinary tract. Most noticeable was the development of uroliths (urinary bladder stones), which occurred at a greater frequency in males than females of either species. Increased incidences of urinary bladder stones and hyperplasia of the bladder epithelium were observed at 13 weeks in male rats fed diets containing melamine. In the chronic study, transitional-cell carcinomas in the urinary bladder of male rats occurred at a significantly (p less than or equal to 0.016) higher incidence in the 4500 ppm (high dose) group (8/49) than in the controls (0/45). Seven of the eight male rats with transitional-cell carcinomas of the urinary bladder also had bladder stones. There was a statistically significant association (p less than or equal to 0.001) between bladder stones and bladder tumors in male rats fed melamine (4500 ppm). Urinary bladder tumors were not observed in the low-dose (2250 ppm) male rat group, while bladder stones were observed in one rat in this group. In the female rat chronic study, chronic inflammation of the kidney was observed at an increased incidence (relative to controls) in both the low (4500 ppm) and high (9000 ppm) dose groups. Ulceration of the bladder epithelium was observed in male and female mice in the 13-week study. The distribution of these toxic lesions was not correlated statistically with the distribution of urinary bladder stones. Acute and chronic inflammation and epithelial hyperplasia of the urinary bladder were found in increased incidence in dosed male mice (2250 and 4500 ppm) in the chronic study. In addition, a high incidence of urinary bladder stones was observed in dosed male mice relative to controls. However, there was no evidence of bladder tumor development in this species.
Urinary bladder carcinogenesis associated with melamine treatment was examined with concomitant use of NaCl to allow assessment of the relationship between uroliths and lesion development. Analysis of the chemical composition of calculi was also performed. F344/DuCrj male rats received diets containing 3 or 1% melamine alone or in combination with either 10 or 5 % NaCl, or 10% NaCl alone for 36 weeks, and then diet without NaCl supplement for a further 4 weeks. The water intake, used as an index of urinary output, was increased by NaCl treatment. The incidences of bladder transitional cell carcinomas and papillomas were 90 and 55% in the group treated with 3% melamine alone; 0 and 15% in the group treated with 3% melamine and 10% NaCl; and 21 and 42% in group treated with 1% melamine alone; and zero in the other groups. Calculus formation resulting from melamine administration was suppressed dose-dependently by the simultaneous NaCl treatment, along with the occurrence of hyperplasia of the papilla in the kidneys. The main constituent of calculi were melamine itself and uric acid (total contents 61.1-81.2%), contained in equal molar ratio. The results indicate that melamine-induced proliferative lesions of the urinary tract of rats were directly due to the irritative stimulation of calculi, and not molecular interactions between melamine itself or its metabolites with the bladder epithelium.