ASTM E1398-91(2003)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation:E1398–91 (Reapproved 2003)
Standard Practice for
In Vivo Rat Hepatocyte DNA Repair Assay
This standard is issued under the fixed designation E1398; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope from the patterns of activation and detoxification that actually
occurinhepatocytes.Anextensiveliteratureisavailableonthe
1.1 This practice covers a typical procedure and guidelines
use of in vitro DNA repair assays (9-19).
forconductingtheratinvivohepatocyteDNArepairassay.The
2.2 Afurther advance was the development of an in vivo rat
procedures presented here are based on similar protocols that
hepatocyte DNA repair assay in which the test chemical is
have been shown to be reliable (1, 2, 3, 4, 5).
administered to the animal and the resulting DNA repair is
1.2 Mention of trade names or commercial products are
assessed in hepatocytes isolated from the treated animal (20).
meant only as examples and not as endorsements. Other
Numerous systems now exist to measure chemically induced
suppliers or manufacturers of equivalent products are accept-
DNA repair in specific tissues in the whole animal (4). The
able.
average of in vivo assays is that they reflect the complex
1.3 This standard does not purport to address all of the
patterns of uptake, distribution, metabolism, detoxification,
safety concerns associated with its use. It is the responsibility
and excretion that occur in the whole animal. Further, factors
of the user of this standard to establish appropriate safety and
such as chronic exposure, sex differences, and different routes
health practices and determine the applicability of regulatory
of exposure can be studied with these systems. This is
limitations prior to use.
illustrated by the potent hepatocarcinogen 2,6-dinitrotoluene
2. Significance and Use (DNT). Metabolic activation of 2,6-DNT involves uptake,
metabolism by the liver, excretion into the bile, reduction of
2.1 Measurement of chemically induced DNA repair is a
the nitro group by gut flora, readsorption, and further metabo-
means of assessing the ability of a chemical to reach and alter
lism by the liver once again to finally produce the ultimate
the DNA. DNA repair is an enzymatic process that involves
genotoxicant (21). Thus, 2,6-DNT is negative in the in vitro
recognition and excision of DNA-chemical adducts, followed
hepatocyte DNArepair assay (22) but is a very potent inducer
by DNA strand polymerization and ligation to restore the
of DNA repair in the in vivo DNA repair assay (23, 24).A
original primary structure of the DNA(6).This process can be
problem with tissue-specific assays is that they may fail to
quantitated by measuring the amount of labeled thymidine
detect activity of compounds that produce tumors in other
incorporated into the nuclear DNA of cells that are not in
target tissues. For example, no activity is seen in the in vivo
S-phaseandisoftencalledunscheduledDNAsynthesis(UDS)
DNA repair assay with the potent mutagen benzo(a)pyrene
(7). Numerous assays have been developed for the measure-
(BP), probably because limited tissue distribution and greater
ment of chemically induced DNA repair in various cell lines
detoxification in the liver yields too few DNA adducts to
and primary cell cultures from both rodent and human origin
produce a measurable response (3). In contrast, BP is readily
(4). The primary culture rat hepatocyte DNA repair assay has
detected in the less tissue-specific in vitro hepatocyte DNA
proven to be particularly valuable in assessing the genotoxic
repair assay (11). An extensive literature exists on the use of
activity of chemicals (8). Genotoxic activity often results from
the in vivo hepatocyte DNA repair assay (1-3, 5, 9, 25-33).
metabolites of a chemical. The in vitro rat hepatocyte assay
provides a system in which a metabolically competent cell is
3. Procedure
also the target cell. Most other in vitro short-term tests for
3.1 Treatment:
genotoxicityemployaratliverhomogenate(S-9)formetabolic
3.1.1 All personnel must be knowledgeable in the proce-
activation, which differs markedly in many important ways
dures for safe handling and proper disposal of carcinogens,
potential carcinogens, and radiochemicals. Disposable gloves
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
and lab coats must be worn.
Surgical Materials and Devices and is the direct responsibility of Subcommittee
3.1.2 Any strain or sex of rat may be used. The largest
F04.16 on Biocompatibility Test Methods.
database is for male Fischer-344 rats.Young adult animals are
Current edition approved Sept. 10, 2003. Published September 2003. Originally
published in 1991. Last previous edition published in 1998 as E1398–91 (1998).
preferred. It is possible that factors such as sex, age, and strain
The boldface numbers in parentheses refer to the list of references found at the
of the rat could affect the outcome of the DNA repair
end of this practice.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1398–91 (2003)
experiments. Therefore, for any one series of experiments 3.2.2 DMN exhibits a maximum UDS response 1 h after
(including controls) these variables should be kept constant. treatment. However, the response remains elevated and mea-
surable for at least 16 h after treatment. More commonly,
3.1.3 Administration is usually by gavage with chemicals
however, chemicals (for example, 2,6-DNT and
dissolved or suspended in an appropriate vehicle such as water
2-acetylaminofluorene (2-AAF)) show a peak response 12 to
or corn oil, depending on solubility.An advantage of the assay
16 h post-treatment.The time from treatment to perfusion may
is that various routes of administration may be chosen. Thus,
bevariedtoobtainatimecourseofinducedrepair.Theroutine
chemicals may also be administered by intraperitoneal injec-
protocol for primary screening involves a time point between
tion or inhalation or in the diet. For gavage administration, 0.2
12 and 16 h with an optional time point between 1 and 4 h.
to 1.0 mL of test chemical solution is administered per 100 g
3.2.3 Anesthetize the rat by intraperitoneal injection with a
bodyweight.Controlsreceivetheappropriatevehiclesolution.
Stock corn oil should be replaced with fresh monthly. 50-mg/mLsolution of sodium pentobarbital (0.2 mLper 100 g
body weight) 10 min prior to the perfusion procedure. Other
3.1.4 ForDNArepairstudies,animalsmaybetakenofffeed
proven anesthetics are also acceptable. Make sure that the
for a few hours prior to sacrifice to make the process of
animal is completely anesthetized, so that it feels no pain.
perfusion a little easier with less food in the stomach. The
3.2.4 Secure the animal with the ventral surface up on
period without food should never exceed 12 h because of the
absorbent paper attached to a cork board. Fold the paper in on
possibility of altered metabolism or uptake. Water should be
each edge to contain perfusate overflow. Thoroughly wet the
continuously available.
abdomen with 70% ethanol and wipe with gauze for cleanli-
3.1.5 Dose selection will depend on the characteristics of
nessandtodiscourageloosefurfromgettingontheliverwhen
each chemical and the purpose of the experiment. If one is
the animal is opened.
investigating whether a chemical can produce a genotoxic
3.2.5 Make a V-shaped incision through both skin and
effect in the animal, even at massive doses and by routes of
muscle from the center of the lower abdomen to the lateral
administration that may overwhelm natural defense mecha-
aspects of the rib cage. Do not puncture the diaphragm or cut
nisms, then high doses (such as the LD50, or higher) that do
theliver.Foldbacktheskinandattachedmuscleoverthechest
not kill the animal before the 16-h sacrifice point may be
to reveal the abdominal cavity.
employed. Even in such a case, doses above 1000-mg/kg body
3.2.6 Placeatubeapproximately1cmindiameterunderthe
weightarenotrecommended.Insomeinstanceshepatotoxicity
back to make the portal vein more accessible.
at high doses may result in inhibition of cell attachment or
DNA repair. More commonly, the purpose of employing the 3.2.7 Movetheintestinesgentlyouttotherighttorevealthe
whole animal is to evaluate the genotoxic effects of realistic portal vein.Adjust the tube under the animal so that the portal
exposures and routes of administration in the target tissue. In vein is horizontal.
this case, doses above 500 mg/kg and the intraperitoneal route 3.2.8 Put a suture in place (but not tightened) in the center
of administration are not recommended. The usual range of
of the portal vein and another around the vena cava just above
doses is from 10 to 500 mg compound per kilogram body the right renal branch.
weight. Target doses with a new compound are usually the
3.2.9 Perform perfusions with a peristaltic pump, the tubing
LD50 and 0.2 3the LD50, with 500 mg/kg as an upper limit.
of which is sterilized by circulation of 70% ethanol followed
The potent mutagen dimethylnitrosamine (DMN) (often used
by sterile water. Place a valve in the line so that the operator
as a positive control) can be detected with doses as low as 1
mayswitchfromtheEGTAsolutiontothecollagenasesolution
mg/kg. Normally, an initial range finding experiment is con-
without disrupting the flow. Solutions should be kept at a
ducted using single animals to cover a range of times and
temperature that results in a 37°C temperature at the hepatic
doses.Ifapositiveresponseisseen,additionalexperimentsare
portal vein.
conducted to establish the dose-response relationship. If no
3.2.9.1 Aperistalticpumpwithachargeablepumpheadand
response is seen, the highest dose(s) is repeated. The final
silicone tubing is suitable for performing the perfusion.
report should contain results from at least three animals per
3.2.9.2 Begin the flow of the 37°C EGTA solution at 8
datapoint.
mL/min and run to waste.
3.1.6 Thus far, no examples have been seen of a compound 1
3.2.10 Cannulate the portal vein with a 20 GA 1 ⁄4-in.
thatproducesaUDSresponseinfemaleratsbutnotmales.For
catheter about 3 mm below the suture. Remove the inner
thosecaseswhereacomparisonhasbeenmade,malesrespond
needle and insert the plastic catheter further to about ⁄3 the
more strongly than females in this assay.Thus, for the purpose
length of vein and tie in place by the suture. Blood should
of routine screening only male rats need to be used.
emerge from the catheter. Insert the tube with the flowing
3.1.7 Treated animals should be maintained in a ventilated
EGTA in the catheter (avoid bubbles) and tape in place.
area or other suitable location to prevent possible human
3.2.11 Immediately cut the vena cava below the right renal
exposure to expired chemicals. Contaminated cages, bedding,
branch and allow the liver to drain of blood for 1.5 min. The
excreta, and carcasses should be disposed of safely according
liver should rapidly clear of blood and turn a tan color. If all
to standard published procedures.
lobes do not clear uniformly, the catheter has probably been
3.2 Liver Perfusion:
inserted too far into the portal vein.
3.2.1 Any proven technique which allows the successful 3.2.12 Tightenthesuturearoundthevenacavaandincrease
isolation and culture of rat hepatocytes can be used. An the flow to 20 mL/min for 2 min.The liver should swell at this
example of one such procedure is given in 3.2.2-3.2.17. point. In some cases gentle massaging of the liver or adjusting
E1398–91 (2003)
the orientation of the catheter may be necessary for complete for control cultures. With practice and the proper technique,
clearing. At this point the vena cava above the suture may be viabilitiesofabout90%canroutinelybeobtained.Attachment
clipped to release some of the pressure in the liver. of the cells to the substrate is an active process. Thus, if a
3.2.13 Switch the flow to the 37°C collagenase solution for sufficientnumberofcellsattachtoconducttheexperiment,this
is a further indication of the viability of the culture.
12 min. During this period, cover the liver with sterile gauze
wetted with sterile saline or WEI (see Annex A1) and place a 3.3.10 Placea25-mmroundplasticcoverslipintoeachwell
40-W lamp 2 in. above the liver for warming. It is valuable to of 6-well culture dishes. 10.5 by 22-mm plastic coverslips and
screen each new batch of collagenase to be ensured that it will 26 by 33-mm eight-chamber culture dishes can also be used.
function properly. Be sure to keep the proper side up as marked on the package.
FourmillilitresofWEC(seeAnnexA1)areaddedtoeachwell.
3.2.14 Allowtheperfusatetoflowontothepaperandcollect
by suction into a vessel connected by means of a trap to the Hepatocyteswillnotattachtoglassunlesstheslideshavebeen
boiled. The use of collagen-coated thermanox coverslips im-
vacuumline.Theperfusateshouldbedisposedofashazardous
waste. proves cell attachment and morphology.
3.2.15 Aftertheperfusioniscompleted,removethecatheter 3.3.10.1 Theseproceduresyieldpreparationsconsistingpri-
marily of hepatocytes.Approximately 400000 viable cells are
and gauze. Remove the liver carefully by cutting away the
membranes connecting it to the stomach and lower esophagus, seeded into each well and distributed over the coverslip by
shaking or stirring gently with a plastic 1-mL pipet. Glass
cutting away the diaphragm, and cutting any remaining attach-
ments to veins or tissues in the abdomen. pipettes can scratch the coverslips.
3.2.16 Hold the liver by the small piece of attached dia- 3.3.11 Incubate the cultures for 90 to 120 min in a 37°C
incubator with 5% CO and 95% relative humidity to allow
phragm and rinse with sterile saline or WEI (see Annex A1).
the cells to attach.
3.2.17 Place the liver in a sterile petri dish and take to a
sterile hood to prepare the cells. 3.4 Labeling the Cultures:
3.3 Preparation of Hepatocyte Cultures: 3.4.1 After the 90-min attachment, wash cultures once with
3.3.1 Place the perfused liver in a 60-mm petri dish and 4mLWEI(seeAnnexA1)perwelltoremoveunattachedcells
and debris. This is done by tilting the culture slightly, aspirat-
rinse with 37°C WEI (see Annex A1). Remove extraneous
tissues (fat, muscle, and so forth). ing the media, and adding the fresh media at 37°C. Be careful
not to direct the stream from the pipet directly onto the cells.
3.3.2 Place the liver in a clean petri dish and add 30 mL of
3.4.2 Remove the WEI (see Annex A1) and replace with 2
fresh collagenase solution at 37°C.
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