**Acrylamide Contamination of Lab Animal Diet**

Acrylamide (AA) is an industrial chemical produced for 50 years in
Europe, Japan, and the USA. The polymeric forms of acrylamide are widely
used as flocculating or flow control agents in the processing of water,
minerals, crude oil, pulp and paper. There is lab animal bedding made
from paper pulp that is flocculated during production. Is this a
potential source of AA contamination in lab animals? Monomeric
acrylamide is also used in biochemical applications for the
polyacrylamide gel electrophoresis analysis of proteins and is a
component of cigarette smoke.

Until 2002, AA was considered to be an industrial or occupational
toxicant, and the predominant routes of exposure were dermal absorption
and inhalation of aerosols. However, new data shows that AA is formed
from cooking many common types of food raising interest in its potential
for human toxicity.

Acrylamide was recently shown to be generated during the heating of
carbohydrate rich food. It has been shown that Maillard browning
reaction products derived from glucose and asparagine, a major amino
acid in grain and potatoes, is responsible for the formation of AA in
foods (Stadler et al., 2002) Acrylamide levels as high as 3 ppm have
been measured in coffee, french fries, potato chips, and bread (Dybing
and Sanner, 2003). Daily human consumption of AA from food has been
estimated by the World Health Organization to be in the range of 0.3-0.8
ug/kg body weight (WHO, 2002). These findings have piqued the interest
in this known genotoxicant and more research is being done to determine
the toxic mechanisms and internal exposures from experimental animal
models and humans in order to develop a relevant risk assessment.

The toxicological hazards associated with AA include, neurotoxicity
(LoPachin et al., 2003), germ cell mutagenicity (Dearfield et al.,
1995), and cancer in rodents (Friedman et al., 1995). Acrylamide has
been classified as a probable human carcinogen by IARC (IARC, 1995).
Oxidative metabolism of AA to glycidamide (GA) in experimental animals
has been shown to be critical for the genotoxicity of AA.

Hemoglobin (Hb) provides information about exposure to reactive
chemicals or electrophilic metabolites, such as AA and GA, through the
formation of covalent adducts. It has been shown that there is a
presence of adducts between AA and GA and the N-terminal valine residues
of Hb which allows AA-Hb and GA-Hb adduct concentrations to be used as
indicators of AA exposure (Tornqvist, 1994).

Early research showed measurable AA-Hb adduct levels in unexposed humans
(Bergmark, 1997). This finding was replicated in experimental animal
studies in which untreated rats, consuming only commercial rodent diets
had measurable AA-Hb adducts (Tareke et al., 2000). Tareke et al. showed
that feeding fried rodent diet resulted in a 8-14 fold increase in AA-Hb
adduct levels, indicating a link between diet and AA-Hb adduct
formation. Analysis of the diet before frying showed undetectable AA
levels (\< 10 ppb) but after frying the AA concentration ranged from
110-200 ppb.

In a study out of the National Center for Toxicology Research, Twaddle
et al., 2004, found AA-Hb and GA-Hb adducts in mice fed the basal NIH31
diet. These findings supported the idea that the basal diets were
contaminated with AA. Contamination of the basal diets by AA introduced
potential problems for plans to examine rodent carcinogencity and
neurotoxicity testing of AA. Dietary contamination of diets by AA could
have implications for toxicological testing in general. Therefore,
Twaddle et al. (2004) did a study with the following goals: Quantify
endogenous AA content in commercial rodent diets; determine the effects
of autoclaving and irradiation on AA formation in laboratory rodent
diets, and; examine the effects AA contaminated diet consumption on
B6C3F~1~ mice. Irradiated and autoclavable diets purchased from Harlan
Teklad, Zeigler Brothers, Purina Mills, and BioServ were analyzed for
AA. For the NIH31 diet produced by Purina Mills, the AA was determined
before and after autoclaving. The results show that the diets have AA
levels at 20 ppb or less except for Purina 5002 which had 130 ppb AA.
This indicates that the temperatures at which these diets are pelleted
(180^0^ F) do not typically increase AA concentrations in the feed.
However, autoclaving the NIH31 diet at 275^0^ F for 15 minutes and
drying for 115 minutes at temperatures ranging from 275^0^ F to the
ambient temperature resulted in a 14 fold increase in AA (from 17 to 240
ppb).

Examination of the stability of AA in lab animal diet over the course of
28 days storage at ambient temperature (21C) or refrigerator storage
(4C) showed a slow decline (t1/2= 41d) and no change in the AA
concentration, respectively.

A toxicokinetic study evaluated male B6C3F1 mice fed a basal diet,
irradiated NIH31 diet (NIH31IR), from weaning to 50 days of age (28
days). After feeding the basal diet the mice were fed autoclaved diet
containing 240 ppb AA. Analysis of AA and GA in serum showed that both
were present in mice fed the autoclaved NIH31 diet. The AA consumed was
equivalent to an average total administered dose of 4.5 ug/kg body wt.
The peak serum concentrations occurred 30 minutes after eating and were
0.035 and 0.027uM for AA and GA, respectively. In contrast, mice fed the
NIH31IR diet showed no measurable AA or GA.

Livers from adult mice fed the autoclaved NIH31 or NIH31IR basal diets
were assayed for DNA GA- adducts. The results showed the mean GA adduct
liver concentration from mice fed the autoclaved NIH31 diet was 7 times
greater than for those fed the NIH31IR diet (1 vs N7- GA-adducts in
10^8^ nucleotides). The AA concentration for the autoclaved diet was not
monitored when it was fed to the mice and therefore, the amount of AA
consumed is not known. A random sample of unknown age had 49 ppb of AA
compared to 240 ppb AA for diet assayed immediately after autoclaving.
The difference could be due to batch to batch variation or degradation
of AA over time (half life= 1.4 months).

The importance of AA contamination in rodent diets is emphasized by the
finding that AA ingested from one limited feeding of an autoclaved diet,
at concentrations comparable to those consumed by humans, is effectively
absorbed and metabolically converted to GA in mice. This study
demonstrates the bioavailability of AA from autoclaved laboratory animal
diet, AA distribution to tissues, and AA metabolic activation to GA
resulting in quantifiable DNA damage. Twaddle et al, concluded that in
controlled environments of animal toxicity testing facilities, the basal
diet is a critical control point for introduction of uncertainty into
research and for toxicity testing of AA the diets must be analyzed to
minimize unintentional contamination.

There appears to be a difference between the autoclaving techniques used
at the Division of Veterinary Resources (DVR), Office of Research
Services, NIH and National Center for Toxicology Research (NCTR). DVR
sterilizes the diet for 15 minutes at 250^0^ F and dries it at 250^0^ F
for 15 minutes compared to 275^0^ F for 15 minutes and drying for 115
minutes at temperatures ranging from 275^0^ F to the ambient temperature
at NCTR. To evaluate differences in autoclaving techniques on AA
concentration of the diet, samples of both autoclaved and non-autoclaved
NIH31 diet from DVR were submitted to Dr. Twaddle's lab for analyses.
The AA concentrations before and after autoclaving were 10 and 125 ppb,
respectively. It appears the AA concentrations for the diet that was not
autoclaved agrees with the publication. However, the DVR autoclaved diet
levels were 125 versus 240 ppb reported by Twaddle et al. It is not
possible without further investigation to know if batch to batch
variation, degradation of AA over time (AA half life = 1.4 months) or
autoclaving technique caused the differences in AA concentrations
between the NIH31 diets autoclaved at DVR and NCTR. This data supports
the idea that diet is a potential experimental variable and in this case
that autoclaving increases AA concentrations.

Bergmark, E. (1997). Hemoglobin adducts of acrylamide and acrylonitrile
in laboratory workers, smokers, and nonsmokers. Chem. Res. Toxicol. 10,
78-84.

Dearfield, K. L, Douglas, G. R., Ehling, U. H., Moore, M. H., Saga, G.
A., Brusick, D. J. (1995) Acrylamide: a review of its genotoxicity and
an assessment of heritable genetic risk. Mutat. Res. 330, 71-99.

Dybing and Sanner, 2003

Friedman, M. A., Dulak, L. H., and Stedman, M. A. (1995). A lifetime
oncogenicity study in rats with acrylamide. Fundam. Appl. Toxicol. 27,
95-105.

International Agency for Research on Cancer (1995) Acrylamide. IARC,
Monogr. Ser. 60, 1-45.

LoPachin, R. MM., Balaban, C. D., and Ross, J. F. (2003). Acrylamide
axonopathy revisited. Toxicol. Appl. Pharmacol. 188, 135-153.

Stadler, R. H., Blank, I., Varga, N., Robert, F., Hau, J., Guy, P. A.,
Robert, M. C., and Reidiker, S. (2002). Acrylamide from Maillard
reaction products. Nature 419, 449-450.

Tareke, E, Rydberg, P., Karlsson, P., and Tornqvist, M. (2000). A
cooking carcinogen? Chem. Res. Toxicol. 13, 517-522.

Tornqvist, M. (1994). Epoxide adducts to N-terminal valine of
hemoglobin. Methods Enzymol. 231, 650-657.

Twaddle, N. C., Churchwell, M. I., McDaniel, L. P., and Doerge, D. R.
(2004). Autoclave sterilization produces acrylamide in rodent diets:
Implications for toxicity testing. J. Agric. Food Chem. 52, 4344-4349.

World Health Organization (2002). FAO/WHO Consultations on the health
implications of acrylamide in foods. Summary report of a meeting held in
Geneva, June 25-27, 2002; available at
http://www.who.int/foodsafety/publications/chem/en/acrylamide/\_full.pdf.