title: Autoclave sterilization produces acrylamide in rodent diets: implications for toxicity testing.
abstract: Acrylamide (AA) is a neurotoxic and carcinogenic contaminant that is formed during the cooking of starchy foods. Assessment of human risks from toxicants is routinely performed using laboratory rodents, and such testing requires careful control of unintended exposures, particularly through the diet. This study describes an analytical method based on liquid chromatography with electrospray tandem mass spectrometry that was used to measure endogenous AA in rodent diets and to survey a number of commercial products for contamination. Method sensitivity permitted accurate quantification of endogenous levels of AA in raw diets below 20 ppb. Autoclaving a standard rodent diet (NIH-31) increased the AA content 14-fold, from 17 to 240 ppb. A nutritionally equivalent diet that was sterilized by irradiation was found to contain approximately 10 ppb of AA (NIH-31IR). A toxicokinetic study of AA and its epoxide metabolite, glycidamide, was performed by switching mice from NIH-31IR to the autoclaved diet for a 30 min feeding period (average AA dose administered was 4.5 microg/kg of body weight). The concentrations of AA and glycidamide were measured in serum collected at various times. The elimination half-lives and the areas under the respective concentration-time curves were similar for AA and glycidamide. Mice maintained on autoclaved NIH-31 diet, but otherwise untreated, showed elevated steady state levels of a glycidamide-derived DNA adduct in liver relative to mice maintained on the irradiated diet. This study demonstrates that a heat sterilization procedure used in laboratory animal husbandry (i.e., autoclaving) can lead to the formation of significant levels of AA in basal diets used for toxicity testing. AA in rodent diets is bioavailable, is distributed to tissues, and is metabolically activated to a genotoxic metabolite, which produces quantifiable cumulative DNA damage. Although the contribution of endogenous AA to the incidence of tumors in multiple organs of rodents otherwise untreated in chronic carcinogenicity bioassays (i.e., control groups) is not known, the reduction of endogenous AA through the use of a suitable irradiated diet was deemed to be critical for ongoing studies of AA carcinogenicity and neurotoxicity.
authors.affiliation: National Center for Toxicological Research, U S Food and Drug Administration, Jefferson, Arkansas 72079, USA.
authors.forename: Nathan C
authors.initials: NC
authors.lastname: Twaddle
authors.forename: Mona I
authors.initials: MI
authors.lastname: Churchwell
authors.forename: L Patrice
authors.initials: LP
authors.lastname: McDaniel
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Doerge
mesh_terms: D020106:Acrylamide / Q000032:analysis* / Q000097:blood / Q000633:toxicity*; D000821:Animal Feed / Q000032:analysis*; D000818:Animals; D005511:Food Handling / Q000379:methods; D006358:Hot Temperature; D008297:Male; D051379:Mice; D011312:Pressure; D021241:Spectrometry, Mass, Electrospray Ionization; D013242:Sterilization / Q000379:methods*
chemical_list: D020106:Acrylamide