ASTM_E_1397_-_91_2003.pdf

Designation:E 1397 91(Reapproved 2003)Standard Practice forIn VitroRat Hepatocyte DNA Repair Assay1This standard is issued under the fixed designation E 1397;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon(e)indicates an editorial change since the last revision or reapproval.1.Scope1.1 This practice covers a typical procedure and guidelinesfor conducting the rat in vitro hepatocyte DNA repair assay.The procedures presented here are based on similar protocolsthat have been shown to be reliable(1-6)2.1.2 Mention of trade names or commercial products aremeant only as examples and not as endorsements.Othersuppliers or manufacturers of equivalent products are accept-able.1.3 This standard does not purport to address all of thesafety concerns associated with its use.It is the responsibilityof the user of this standard to establish appropriate safety andhealth practices and determine the applicability of regulatorylimitations prior to use.2.Significance and Use2.1 Measurement of chemically induced DNA repair is ameans of assessing the ability of a chemical to reach and alterthe DNA.DNArepair is an enzymatic process that involves therecognition and excision of DNA-chemical adduct followed byDNA strand polymerization and ligation to restore the originalprimary structure of the DNA(7).This process can bequantitated by measuring the amount of labeled thymidineincorporated into the nuclear DNA of cells that are not inS-phase and is often called unscheduled DNA synthesis(UDS)(8).Numerous assays have been developed for the measure-ment of chemically induced DNA repair in various cell linesand primary cell cultures from both rodent and human origin(9).The primary rat hepatocyte DNArepair assay developed byWilliams(10)has proven to be particularly valuable inassessing the genotoxic activity and potential carcinogenicityof chemicals(11),(12).Genotoxic activity is often produced byreactive metabolites of a chemical.The in vitro rat hepatocyteassay provides a system in which a metabolically competentcell is itself the target cell for measured genotoxicity.Mostother short-term tests for genotoxicity employ a rat liverhomogenate(S-9)for metabolic activation,which differsmarkedly in many important ways from the patterns ofactivation and detoxification that actually occur in hepatocytes.An extensive literature is available on the use of in vitrohepatocyte DNA repair assays(2,3,6,13-28).3.Procedure3.1 Liver Perfusion:3.1.1 All personnel must be knowledgeable in the proce-dures for safe handling and proper disposal of carcinogens,potential carcinogens,and radiochemicals.Disposable glovesand lab coats must be worn.3.1.2 Any proven technique which allows the successfulisolation and culture of rat hepatocytes can be used.Anexample of one such procedure is given in 3.1.3-3.1.20.3.1.3 Any strain or sex of rat may be used.The largestdatabase is for male Fischer-344 rats.Young adult animals arepreferred.It is possible that factors such as sex,age,and strainof the rat could affect the outcome of the DNA repairexperiments.Therefore,for any one series of experiments thesevariables(including controls)should be kept constant.3.1.4 Anesthetize the rat by intraperitoneal injection with a50-g/mL solution of sodium phenabarbitol(0.2 mL per 100 gbody weight)10 min prior to the perfusion procedure.Otherproven anesthetics are also acceptable.Make sure that theanimal is completely anesthetized,so that it feels no pain.3.1.5 Wet the abdomen thoroughly with 70%ethanol andwipe with gauze for cleanliness to discourage loose fur fromgetting on the liver when the animal is opened.3.1.6 Make a V-shaped incision through both skin andmuscle from the center of the lower abdomen to the lateralaspects of the rib cage.Do not puncture the diaphragm or cutthe liver.Fold the skin and attached muscle back over the chestto reveal the abdominal cavity.3.1.7 Place a tube approximately 1 cm in diameter under theback to make the portal vein more accessible.3.1.8 Move the intestines gently out to the right to reveal theportal vein.Adjust the tube under the animal so that the portalvein is horizontal.3.1.9 Put a suture in place(but not tightened)in the centerof the portal vein and another around the vena cava just abovethe right renal branch.1This practice is under the jurisdiction ofASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Sept.10,2003.Published September 2003.Originallyapproved in 1991.Last previous edition approved in 1998 as E 1397 91(1998).2The boldface numbers in parentheses refer to the list of references at the end ofthis practice.1Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959,United States.3.1.10 Perform perfusi