ASTM_E_1398_-_91_2003.pdf

Designation:E 1398 91(Reapproved 2003)Standard Practice forIn VivoRat Hepatocyte DNA Repair Assay1This standard is issued under the fixed designation E 1398;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 vivo hepatocyte DNArepair assay.Theprocedures presented here are based on similar protocols thathave been shown to be reliable(1,2,3,4,5).21.2 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.DNA repair is an enzymatic process that involvesrecognition and excision of DNA-chemical adducts,followedby DNA strand polymerization and ligation to restore theoriginal primary structure of the DNA(6).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)(7).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(4).The primary culture rat hepatocyte DNA repair assay hasproven to be particularly valuable in assessing the genotoxicactivity of chemicals(8).Genotoxic activity often results frommetabolites of a chemical.The in vitro rat hepatocyte assayprovides a system in which a metabolically competent cell isalso the target cell.Most other in vitro short-term tests forgenotoxicity employ a rat liver homogenate(S-9)for metabolicactivation,which differs markedly in many important waysfrom the patterns of activation and detoxification that actuallyoccur in hepatocytes.An extensive literature is available on theuse of in vitro DNA repair assays(9-19).2.2 A further advance was the development of an in vivo rathepatocyte DNA repair assay in which the test chemical isadministered to the animal and the resulting DNA repair isassessed in hepatocytes isolated from the treated animal(20).Numerous systems now exist to measure chemically inducedDNA repair in specific tissues in the whole animal(4).Theaverage of in vivo assays is that they reflect the complexpatterns of uptake,distribution,metabolism,detoxification,and excretion that occur in the whole animal.Further,factorssuch as chronic exposure,sex differences,and different routesof exposure can be studied with these systems.This isillustrated by the potent hepatocarcinogen 2,6-dinitrotoluene(DNT).Metabolic activation of 2,6-DNT involves uptake,metabolism by the liver,excretion into the bile,reduction ofthe nitro group by gut flora,readsorption,and further metabo-lism by the liver once again to finally produce the ultimategenotoxicant(21).Thus,2,6-DNT is negative in the in vitrohepatocyte DNA repair assay(22)but is a very potent inducerof DNA repair in the in vivo DNA repair assay(23,24).Aproblem with tissue-specific assays is that they may fail todetect activity of compounds that produce tumors in othertarget tissues.For example,no activity is seen in the in vivoDNA repair assay with the potent mutagen benzo(a)pyrene(BP),probably because limited tissue distribution and greaterdetoxification in the liver yields too few DNA adducts toproduce a measurable response(3).In contrast,BP is readilydetected in the less tissue-specific in vitro hepatocyte DNArepair assay(11).An extensive literature exists on the use ofthe in vivo hepatocyte DNA repair assay(1-3,5,9,25-33).3.Procedure3.1 Treatment: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 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 repair1This 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.Originallypublished in 1991.Last previous edition published in 1998 as E 1398 91(1998).2The boldface numbers in pa