Current Protocols in Toxicology || Determination of ADME and Bioavailability Following Intravenous, Oral, and Dermal Routes of Exposure

UNIT 5.8Determination of ADME andBioavailability Following Intravenous,Oral, and Dermal Routes of Exposure

Shakil A. Saghir1, 2

1The Dow Chemical Company, Midland, Michigan2Department of Biological and Biomedical Sciences, The Aga Khan University,Karachi, Pakistan

ABSTRACT

Humans are exposed to chemicals either voluntarily or involuntarily through severalroutes. Therapeutic drugs are introduced into the human system via a number of routesincluding, but not limited to, oral, inhalation, intravenous (i.v.), topical, and subcutaneous.For occupational and environmental chemicals, the major routes of human exposureare inhalation, dermal, and oral. To determine the extent of exposure to chemicals, theconcentration of the active molecules is measured in a biological medium. Determinationof absolute and/or relative bioavailability of occupational and environmental chemicalexposure through different routes is critical in understanding the risk to the generalpopulation of a low-level exposure to these chemicals. This unit describes typical protocoldesigns to generate data for the calculation of absorption, distribution, metabolism, andelimination (ADME) and absolute and relative bioavailability of chemicals when exposedthrough i.v., oral, and dermal routes. Curr. Protoc. Toxicol. 41:5.8.1-5.8.19. C© 2009 byJohn Wiley & Sons, Inc.

Keywords: oral ADME � intravenous (IV) ADME � dermal ADME � bioavailability

INTRODUCTION

Humans are exposed to chemicals either voluntarily (i.e., therapeutic drugs or substancesof abuse) or involuntarily (i.e., occupational or environmental chemicals) through severalroutes. The routes by which therapeutic drugs are introduced into the human system in-clude, but are not limited to, oral, inhalation, intravenous (i.v.), topical, and subcutaneous.For occupational and environmental chemicals, the major routes of human exposure areinhalation, dermal, and oral.

For pharmaceuticals, determination of bioavailability is essential in calculating dosagesfor non-i.v. routes of administration. Determination of absolute bioavailability (com-parison between vascular and non-vascular routes of administration) and/or relativebioavailability (comparison of different non-vascular routes or different formulations bythe same route of administration) of occupational and environmental chemical exposurethrough different routes is critical for understanding the risk to the general populationof low-level exposure to these chemicals. Bioavailability of chemicals is determined aspart of the preclinical (pharmaceuticals)/registration (occupational and environmentalchemicals) pharmacokinetic/toxicokinetic studies conducted in early stages of pharma-cology/toxicology program studies in animals.

Typical protocol designs to generate data for the calculation of absolute and relativebioavailability of chemicals when exposed through i.v. (see Basic Protocol 1), oral (seeAlternate Protocol 1), and dermal (see Alternate Protocol 2) routes are described in thisunit. A modification of these protocols can also be used for determining absorption, dis-tribution, metabolism, and elimination (ADME) and bioavailability of a test compound

Current Protocols in Toxicology 5.8.1-5.8.19, August 2009Published online August 2009 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/0471140856.tx0508s41Copyright C© 2009 John Wiley & Sons, Inc.

Toxicokinetics

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Supplement 41

Determination ofADME and

Bioavailability

5.8.2

Supplement 41 Current Protocols in Toxicology

from other routes of exposure. These ADME studies provide information on distribution,elimination, and metabolism of a compound. An assortment of methods describe thecollection and analysis of samples—plasma and red blood cells (see Support Protocol 1),urine (see Support Protocol 2), feces (see Support Protocol 3), expired volatiles (seeSupport Protocol 4), expired CO2 (see Support Protocol 5)—determination of oral ab-sorption (see Support Protocol 6), collection of dermal samples—by dermal wash (seeSupport Protocol 7), sample from application devices (see Support Protocol 8), by tapestripping (see Support Protocol 9), and from the dermal application site (see SupportProtocol 10)—determination of dermal absorption (see Support Protocol 11), 14C analysisof samples (see Support Protocol 12), tissue collection (see Support Protocol 13), collec-tion of the final cage wash (see Support Protocol 14), determining metabolic profile andidentification (see Support Protocol 15), and collection of control samples (see SupportProtocol 16). Data analysis is described in Basic Protocol 2.

NOTE: All protocols that use live animals must be reviewed and approved by an Institu-tional Animal Care and Use Committee (IACUC) prior to initiation of the study.

NOTE: Appropriate precautions must be taken when working with radioactive chemi-cals to avoid contamination of personnel conducting the study and surroundings. Theradioactive waste should be collected and disposed of appropriately following the Nu-clear Regulatory Commission (NRC) and institutional guidelines that are provided bythe institution radiation safety officer.

BASICPROTOCOL 1

CONDUCTING AN INTRAVENOUS ADME STUDY

The bolus i.v. dose is the most important technique for obtaining kinetic (distributionand elimination) information of a chemical without the process of absorption, as the doseis directly delivered into the general circulation. The kinetic information obtained fromi.v. administration serves as the basis for comparison with other routes of exposure todetermine absolute bioavailability of a chemical from the respective routes of exposure.

NOTE: Use sterile technique and reagents to prepare i.v. solutions.

Materials

Saline or a balanced salt solution (BSS)10% Intralipid (lipid emulsion; Fresenius Kabi or prepared as described by Saghir

et al., 2005) or other suitable aqueous miscible solvent/emulsifier1 to 5 N HCl or NaOH14C- or 3H-radiolabeled test compound (ideally ≥97% pure)Non-radiolabeled test compound (≥97% pure), if necessaryScintillation cocktailJugular vein–cannulated (JVC) rats (Fig. 5.8.1; at least 8 weeks old; Taconic)Rodent chow in pellet formCannula flush solution (see recipe)Cannula lock solution (see recipe)Isopropyl alcoholSilicone greaseActivated charcoal3:7 (v/v) monoethanolamine/1-methoxy-2-propanol solution

Scintillation counterGlass rat metabolism cages that separate urine and feces and collect exhaled air

(see Fig. 5.8.2 for detailed setup)1- to 3-ml Luer-lok glass syringeLuer-stub adapter (23-G) with blunt endForceps and hemostats

Toxicokinetics

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Current Protocols in Toxicology Supplement 41

cannula plug

exteriorized JVC

Figure 5.8.1 A jugular vein cannulated rat showing the exteriorized cannula with plug.

Warming lamp with 40- to 100-W bulb or ∼45◦C incubator equipped withmechanical convection

Hard Plexiglas rat holder or preferably cone-shaped flexible plastic bag(e.g., Decapicone, Braintree Scientific)

Board and tape0.5-in. long, precision glide 23-G needle with short beveled tip0.5-in. long blunt-end Luer-stub adapter (23-G)1- or 2-ml Luer-lok glass syringeCotton pledget or gauze

Additional reagents and equipment for sample collection (see Support Protocols 1through 5)

Prepare doses1. Prepare doses as a solution in saline or a balanced salt solution (BSS). For lipophilic

chemicals, make emulsions for injection either in 10% intralipid (lipid emulsion) orother suitable aqueous miscible solvents and emulsifiers.

Test compounds can also be administered as aqueous suspensions in a mixture of BSS and∼20% suspending agent (e.g., Alkamuls from Rhodia; http://www.rhodia-novecare.com).

2. Prepare the dose solution/suspension/emulsion under sterile conditions; sterilize alldosing media prior to addition of the test compound.

3. Adjust pH of the dosing solutions/suspensions/emulsions to ∼7.5 with 1 to 5 N HClor NaOH.

4. Confirm the homogeneity and concentration of test compound in dose solu-tions/suspensions/emulsions by removing aliquots from at least three locations withinthe container and measuring radioactivity by liquid scintillation counting (LSC) andchemical analysis.

Use a non-radiolabeled test compound (≥97% pure), if necessary, to adjust the radioac-tivity of the dosing solutions/suspensions/emulsions or to deliver the desired mass ofchemical.


Bioavailability

5.8.4


air flow meter

drinking water sipper

rat housing unit

food supply area

vacuum line to draw air

charcoal trap

feces collection unit

CO2 trap

urine collection unit

Figure 5.8.2 A metabolism cage setup for ADME study.

5. Confirm stability of the test compound in the vehicle over a period that is at least aslong as the intended duration of its use.

Care for animals and dose6. House JVC rats individually and acclimate them to metabolism cages for a minimum

of 3 days under controlled conditions (22 ± 3◦C, 40% to 70% relative humidity, 12 hrlight/dark photocycle, air exchange of 12 to 15 times/hr) prior to dosing.

Use at least four JVC rats per dose group. The gender and strain of the rat depends onthe objectives of the study. The JVC rats are commercially available or can be fitted witha JVC by the surgical procedure described by Waynforth and Flecknell (1992).

A shorter acclimation period, when needed, should be evaluated and approved by theIACUC.

7. Provide animals ad libitum with certified rodent chow in pellet form and municipalwater.

8. After acclimation, slowly dose animals intravenously with a sterile 1- to 3-ml Luer-lok glass syringe and needle and a small volume of sterile test compound eitherthrough the JVC (if the compound is not absorbed by the tubing; see step 11a) or tailvein (see step 11b). Use a moderate administration rate (0.1 to 0.2 ml/min) to avoidany side-effects.

Use of a glass syringe avoids binding/absorption of the test compound to the syringe. Useof a plastic syringe is possible for test compounds that do not bind/absorb to plastic. Itis recommended to ensure compatibility of JVC tubing (polyethylene, PE50) with the testcompound.

Toxicokinetics

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A Luer-lok syringe should be used for dosing to avoid spillage of the radioactive testcompound that may occur as a result of high back pressure caused by a regular (slip-tip)syringe.

For aqueous solutions, the volume of the dose is maintained at ∼2 ml/kg and for suspen-sions/emulsions, ∼1 ml/kg dose is used.

A very rapid injection, even of the animal’s own blood, can cause cardiovascular failureand can be lethal (Waynforth and Flecknell, 1992).

Animals are not fasted prior to dosing.

Injection of the test compound via JVC is easy and non-invasive; however, care mustbe taken to ensure that no residual test compound remains in the cannula. Intraveneousinjection through the cannula can be performed while the rat is in the metabolism cageor, if needed, inside a restrainer.

9. Attach a 23-G Luer-stub adapter to a 1- to 3-ml Luer-lok glass or plastic syringecontaining the weighed dose medium.

10. Measure the amount of test compound and radioactivity administered to each rat byweighing the dosing medium in a tared syringe with needle attached.

Injection through JVC

11a. Remove the cannula plug using a hemostat and forceps. First remove the cannulalock solution using an empty syringe with a stub adapter inserted into the cannulaand draw the liquid until blood becomes visible in the stub adapter hub.

12a. Discard the syringe and stub adapter.

13a. Insert the stub adapter of the syringe containing the dosing medium into the cannulaand slowly inject test compound (over 1 to 2 min).

14a. Once the injection is complete, aspirate blood up to the hub of the stub adapterand push it back to clean the cannula of any remaining test compound. Repeat thisprocess two to three times.

15a. Remove the syringe and stub adapter and place it for extraction into a suitablesolvent to determine the leftover radioactivity.

16a. Following injection, rinse the cannula with ∼0.1 ml of cannula flush solution andthen plug the cannula.

17a. After completion of dose administration, rinse/extract the remaining dosing mediumin the syringe and stub adapter (and extension of cannula, if used) in a suitablesolvent of known weight and determine radioactivity in weighed aliquots by LSC.

18a. Subtract the leftover total radioactivity and test compound from the original syringetotal (step 10) to determine the actual injected dose of the test compound andradioactivity. Proceed to step 21.

Injection through tail vein

Dose injection into the tail vein requires skill and practice to ensure that 100% of the doseis injected into the blood vessels and that none ends up in a perivascular site, as this willhave a huge impact on the kinetics of the compound. The lateral veins of the tail are themost frequently used veins for injection of test compounds in rats. The principal functionof the tail veins in rats is thermoregulation; veins dilate when the rat’s body temperaturerises to disseminate heat. Access to a vein can be made easier by vasodilation.

11b. For best results, warm the whole animal for 10 to 15 min in the cage or a box witha warming lamp with a 60- to 100-W bulb or for 2 to 3 min in an ∼45◦C incubator.

Mixed results are obtained by immersing tail in warm water for 5 to 10 sec.


Bioavailability

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12b. Lift the tip of the tail and rotate slightly in either direction to see the lateral tail vein.Restrain the rat (e.g., using a hard Plexiglas rat holder or a Decapicone) so that itstail is accessible.

One can use a rat holder; however, a Decapicone (tapered plastic film tubes) works better(for details, see Reigle and Bukva, 1984). The animal is placed inside the Decapiconetube with head facing the tapered end. The wide end is closed with a twist-tie.

13b. Then place the rat on a board on its side with tail facing the investigator. Restrictmovement of the rat by attaching Decapicone to the board with tape.

14b. Locate vein and sterilize the area around the dosing site using isopropyl alcohol.

15b. Insert a 0.5-in. long, precision glide 23-G needle attached to a 1- to 3-ml Luer-lokglass syringe with its bevel pointing upward at an ∼20◦ angle. Insert the needleslowly, observing the needle as it enters the vein.

Use of a glass syringe avoids the binding/absorption of the test compound to the syringe.

Preferably, the needle is inserted into the vein midway down the tail, allowing additionalattempts of venipuncture proximally if the initial attempt is unsuccessful.

16b. Confirm the proper insertion of needle into the vein by aspirating blood into thehub of the needle before carrying out injection.

17b. Confirm proper test compound administration into the vein by observing blanchingof the vein during administration and no detection of material or swelling at theinjection site.

18b. Slowly administer test compound to avoid vascular overload or rupture of the veinfrom excess pressure. After completion of injection, apply pressure over injectionsite by gently holding a cotton pledget or piece of gauze over the injection site for∼30 sec to prevent the formation of a hematoma.

19b. After completion of dosing, rinse/extract the leftover dosing medium in the sy-ringe/needle and pledget/gauze in a suitable solvent of known weight and determineradioactivity in weighed aliquots by LSC.

20b. Subtract the leftover radioactivity and test compound from the total in the sy-ringe to determine the actual injected dose of the test compound and radioactiv-ity. Also, at the end of the experiment, collect the area around the dosing site,digest it, determine the radioactivity, and subtract it from the dose. Proceed tostep 21.

Observe rats and collect required samples21. After completion of dosing, place rats individually in clean metabolism cages whose

joints have been sealed with silicone grease. Draw air through a tube and pass itthrough trapping agents, e.g., activated charcoal and a 3:7 monoethanolamine/1-methoxy-2-propanol solution.

Silicone grease is applied to the joints of the metabolism cage to create an air-tightenvironment.

Activated charcoal is used to trap exhaled volatile organics. The 3:7 (v/v)monoethanolamine/1-methoxy-2-propanol solution is used to trap expired CO2.

22. Collect time-course blood, urine, feces, expired-CO2, and organic volatiles and ana-lyze them for radioactivity. Draw air through the metabolism cages at ∼800 ml/minto draw all exhaled CO2 and eliminate the chance of inhalation of the exhalents.For sample collection and analysis, see Support Protocols 1 to 5.

Toxicokinetics

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23. Continue experiments until >95% of the administered dose is recovered in urine,feces, and exhaled air or up to a maximum of 7 days.

24. At the end of the study (when >95% of the administered dose is recovered inexcreta and exhaled air to a maximum of 168 hr), anesthetize animals with CO2/O2

and sacrifice them by exsanguination. Following sacrifice, collect the followingsamples from each rat and rat cage: tissues (see Support Protocol 13) and final cagewash (see Support Protocol 14).

SUPPORTPROTOCOL 1

COLLECTION AND ANALYSIS OF PLASMA AND RED BLOOD CELLS

Time-course blood samples (0.1 to 0.2 ml) are collected using a 1-ml slip-tip syringefrom the jugular vein cannula at various times after dosing depending upon the natureof the test compound and the objectives of the study. The total volume of blood drawnfrom each rat should be <10% of the total blood volume (∼70 ml/kg; Waynforth andFlecknell, 1992). A typical blood sampling schedule may be 0.25, 0.5, 1, 2, 4, 8, 12,and 24 hr post-dosing, and every 24-hr interval thereafter or as long as the jugular veincannula remains patent. From the rats dosed through non-vascular routes, a few additionalearly time-course samples are routinely collected (e.g., 0.08 and 0.17 hr after dosing)for compounds that are rapidly absorbed. The need for these early time points can bedetermined with a pilot experiment with a few animals. After each blood collection,∼0.3 ml of cannula flush solution (see recipe) is injected into the rat to replace the fluidand clean the cannula for extended patency. When sampling times are more than 4 hrapart, 0.07 ml of cannula lock solution containing heparin and either glycerol or dextrose(see recipe) is used to keep the cannula patent. At each time-point, plasma and red bloodcells (RBC) may be separated from the whole blood by centrifugation (∼10 min at∼3000 rpm) to determine radioactivity. Weighed aliquots (∼25 μl) of whole blood orplasma at each collection time point may be placed in vials containing extraction and/orstabilizing solvents and stored at −80◦C for possible chemical analysis. Blood is alsocollected at sacrifice via cardiac puncture or from the dorsal aorta. The blood collected atterminal sacrifice may be centrifuged to obtain plasma and RBCs, and the radioactivitydetermined. Equal volume aliquots of whole blood or plasma from the terminal samplesmay be pooled (per group and sex), stabilized, and stored at −80◦C for possible chemicalanalysis.

A limited number of blood samples (e.g., two to three) may be taken from the jugular ortail vein over 12 hr from non-JVC rats exposed to test compound through diet (or otherroutes) for extended periods of time (e.g., 1, 3, 24 months; Waynforth and Flecknell,1992). These samples are analyzed for the test compound or metabolite(s) to determinethe dose-, age-, and duration-of-exposure-related changes (nonlinearity) in the dailysystemic dose as described by Saghir et al. (2006).

SUPPORTPROTOCOL 2

COLLECTION AND ANALYSIS OF URINE

All urine voided during the study is collected in dry ice–cooled traps. The urine trapsare changed at 12 and 24 hr post-dosing, followed by 24-hr intervals for the remainderof the study. The cages are rinsed with water at the time the traps are changed and therinse collected. Each urine specimen and urine/cage rinse is weighed, and a weighedaliquot of each sample analyzed for radioactivity by LSC. Equal volume aliquots ofurine samples from each animal at the 0-to-12-hr and 12-to-24-hr collection intervalsmay be pooled (per sex, time-point, and group) and stored at −80◦C for possible chemicalanalysis.


Bioavailability

5.8.8


SUPPORTPROTOCOL 3

COLLECTION AND ANALYSIS OF FECES

Feces are collected in dry ice–chilled containers at 24-hr intervals. Aqueous homogenates(∼25% w/w) are prepared, shaken for >4 hr, and weighed aliquots of homogenatesoxidized or digested (using a tissue oxidizer or solubilizing agent, e.g., sodium hydroxide,35% tetraethyl ammonium hydroxide in water), and quantitated for radioactivity by LSC.In addition, equal-volume aliquots of fecal homogenates from each animal may be takenfrom the 0-to-24-hr collection interval and pooled (per sex and group). These pooledsamples are stored at –80◦C for possible chemical analysis.

SUPPORTPROTOCOL 4

COLLECTION AND ANALYSIS OF EXPIRED VOLATILES

Air is drawn through the cages at ∼800 ml/min. Upon exiting each cage, the air ispassed through charcoal to trap organic volatiles. The charcoal traps are changed at24-hr intervals. Radioactivity trapped on the charcoal is determined by grinding anddetermining radioactivity in weighed aliquots that are oxidized. If an oxidizer (to combusttissues to 14CO2) is not available, representative weighed aliquots can be extracted in asuitable solvent and radioactivity determined in the aliquots of the solvent. If <1% ofthe administered dose is detected in the charcoal traps during the first 24 hr of trapping,no further trapping of expired air is needed.

SUPPORTPROTOCOL 5

COLLECTION AND ANALYSIS OF EXPIRED CO2

Following passage through the charcoal trap, expired air is passed through a 3:7 (v/v)monoethanolamine/1-methoxy-2-propanol solution to trap expired CO2, which is thenanalyzed for radioactivity. Volume of the trapping solution required is calculated on therate of the air passing through it and the maximum capacity of CO2 trapping (∼150 ml ofsolution for each 12-hr collection for 500 to 800 ml air/min). The CO2 trap is changed at12-hr intervals through 48 hr followed by 24-hr intervals for the remainder of the study.If <1% of the administered dose is detected in the CO2 traps of the dosed rats during thefirst 12-hr interval, no further trapping of CO2 is needed.

ALTERNATEPROTOCOL 1

CONDUCTING AN ORAL ADME STUDY

The most likely intake route for humans is by oral ingestion of therapeutic drugs and foodscontaining trace levels of chemicals (e.g., pesticides). Determining the oral bioavailabilityof both pharmaceuticals and environmental contaminants is important for the design oftherapeutic drugs to determine optimal absorption from the gastrointestinal tract (GI) andto better assess the risk of environmental chemicals and consumer products that come incontact with food products. Oral ADME of a test compound is usually determined by asingle bolus gavage (which is the introduction of a solution into the stomach by meansof a tube) of a known amount of the test compound. The steady-state systemic doseand some estimates of kinetic parameters can also be determined in animals exposed totest compound through diet (or other routes) for an extended period of time (e.g., 1, 3,24 months) as part of toxicokinetic analysis described by Saghir et al. (2006).

Additional Materials (also see Basic Protocol 1)

Ball-tipped fixed or flexible feeding needle (16- to 18-G curved or straight)

Prepare and analyze dose1. Prepare the oral dose solutions/suspensions in an appropriate vehicle (water, 0.5%

methylcellulose ether suspending agent, corn oil, or PEG-400) at the target concen-trations.

Be aware that the use of corn oil and PEG-400 as vehicles may alter the absorption oftest compound from the GI tract.

Toxicokinetics

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2. Administer the oral dose in a volume of ∼5 ml/kg using a ball-tipped fixed orflexible feeding needle. Target the radioactivity of the dose solutions/suspensions at∼500 μCi/kg.

Total radioactivity administered to each rat may vary depending upon the needs of thestudy and availability of the test compound.

3. Confirm stability of the test compound in the vehicle used at least for the durationof its use.

4. Determine homogeneity and concentration of the dose solutions/suspensions as de-scribed in Basic Protocol 1—briefly, take at least three weighed aliquots from dif-ferent regions of the dose solution/suspension containers and analyze by LSC andchemical analysis.

Care for animals, dosing, and sampling5. House JVC rats individually in glass metabolism cages and acclimate for a minimum

of ∼3 days (prior to their assignment to the study) under controlled conditions (22◦± 3◦C, 40% to 70% relative humidity, 12-hr light/dark photocycle, air exchange 12to 15 times/hr).


6. Following acclimation, randomly select animals for dosing based on body weight.

7. Attach the ball-tipped feeding needle to a 3- to 5-ml Luer-lok glass syringe and drawa measured volume of dose solution/suspension into the syringe and weigh.

Use of a glass syringe avoids absorption of the test compound by the syringe. Non-glasssyringes can also be used if test compound does not absorb to plastic.

8. Prior to dosing, measure and mark the distance that the needle needs to be insertedinto the rat (usually from the nose to the first rib).

9. Restrain the rat firmly by placing its head between index and middle fingers andsecure fore legs by the use of the other three fingers.

JVC rats should not be grasped by the loose skin of the neck and back as this may causethe JVC to dislodge from the vein.

10. During dosing, maintain rat in an upright (vertical) position and pass the gavageneedle along the inner side of the mouth (between left incisors and molars) andgently advance into the esophagus following along the roof of the mouth.

Rats do not struggle and will swallow the feeding needle when inserted properly. Gravityalone should be used to move the feeding needle down the esophagus. If an oral gavageerror does occur, the animal should be euthanized immediately.

11. After the needle is passed to the correct length, slowly inject the test compound toprevent fluid from coming back up into the oral cavity or rupturing the esophagus,and then withdraw the syringe.

If the animal coughs, chokes, or begins to struggle vigorously during the administration,the test compound may have been injected into the lungs. If this occurs, administration ofthe test compound should be stopped and the needle withdrawn immediately. If it appearsthat material has been injected into the lungs, the animal should be euthanized.

12. Monitor the rat after the completion of the dosing procedure to ensure that there areno adverse effects.

13. After completion of dosing, place rats individually in clean metabolism cages whosejoints have been sealed with silicone grease. Draw inlet air into the chamber throughan appropriate flow meter and pass air exiting the chamber through trapping agents.


Bioavailability

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14. Collect time-course blood, urine, feces, expired-CO2, and organic volatiles andanalyze for radioactivity. Draw air through the metabolism cages at ∼800 ml/minto ensure capture of all exhaled CO2 and to eliminate the chance of inhalation ofexhalents.

15. Continue the experiment until >95% of the administered dose is recovered in urine,feces, and exhaled air or to a maximum of 7 days. For sample collection and analysis,see Support Protocols 1 through 6.

16. At the end of the study (when >95% of the administered dose is recovered in excretaand exhaled air to a maximum of 168 hr), anesthetize animals with CO2/O2 andsacrifice by exsanguination. Following sacrifice, collect the following samples fromeach rat and rat cage: tissues (see Support Protocol 13), and final cage wash (seeSupport Protocol 14).

SUPPORTPROTOCOL 6

DETERMINING ORAL ABSORPTION

The total oral absorption of the test compound is determined by the amount of radioac-tivity recovered from urine, organic and CO2 traps, tissues, carcass, and material in thefeces that arises from biliary elimination. Since the radioactivity found in 0-to-24-hrfeces usually represents the unabsorbed test compound, as the GI transit time in rats is∼21 hr (Bungay et al., 1981), a minimum estimate of absorbed material present in thefeces would be to include radioactivity excreted after 24 hr. However, if significant fecalelimination is evident from other routes of exposure, an additional study may be war-ranted (e.g., oral ADME in bile duct–cannulated rats, or i.v. administration) to determinethe fraction of the dose remaining unabsorbed in the GI tract relative to the biliary elimi-nation of the absorbed dose. In most cases, comparison of the fecal elimination betweeni.v. and oral routes of administration is considered sufficient to answer this question.

ALTERNATEPROTOCOL 2

CONDUCTING A DERMAL ADME STUDY

The major route of human exposure to chemicals, especially occupational and environ-mental chemicals and, to a lesser extent, therapeutic drugs, is through dermal contact;therefore, determining kinetics and dermal bioavailability of chemicals is of paramountimportance. The current U.S. EPA guideline for evaluation of in vivo dermal penetrationis quite extensive in scope, as it requires numerous groups of animals sacrificed at multi-ple time-points to evaluate the uptake and distribution of the test compound from the siteof application (U.S. EPA, 1982, 1996; Zendzian, 1989, 1994, 2000). A new approach todetermine dermal penetration has been developed by Saghir et al. (2008). This new studydesign includes a dermal absorption group, in addition to the standard oral-administrationgroups. The blood and excreta time-course data are used, in conjunction with the ap-plication site residue results at sacrifice, to characterize the amount and rate of dermalpenetration. This approach has significantly reduced the number of animals required tocomplete the study from ∼25/dose to ∼4/dose. This study design is also more consistentwith the fact that the total bound skin residue is not systemically absorbed and thereforenot bioavailable (Thongsinthusak et al., 1999).

Additional Materials (also see Basic Protocol 1)

IsofluraneElectric hair clippersTeflon frames (∼1.5-mm thick; 4 × 5–cm with a 3 × 4–cm cut-out opening)Adhesive Velcro fastenerRodent jackets (Braintree Scientific)Ball-tipped feeding needle (∼20-G) and all-glass Luer-lok syringeTeflon spectra/mesh macroporous filter (Spectrum Laboratories)

Toxicokinetics

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Prepare dose and analyze1. Prepare the dermal dose solutions/suspensions in an appropriate vehicle (water or

PEG 400) at the target concentrations. Apply the dose at a volume of 10 μl/cm2 to12 cm2 rat skin.

Use of corn oil and DMSO as dosing vehicles should be avoided.

2. Target the radioactivity of the dose solutions/suspensions at ∼800 μCi/ml (∼100 μCiper rat or ∼500 μCi/kg).

Total radioactivity applied to each rat may vary depending upon the study needs andavailability of the test compound.

3. Confirm stability of the test compound in the vehicle over a period that is at least aslong as the intended duration of its use.

4. Determine homogeneity and concentration of the dose solutions/suspensions as de-scribed in Basic Protocol 1. Briefly, take at least three weighed aliquots from differentregions of the dose solution/suspension containers and analyze by LSC and chemicalanalysis.

Care for animal and dose5. House rats with jugular vein cannulas individually in glass metabolism cages and

acclimate for a minimum of 3 days (prior to their assignment to the study) undercontrolled conditions (22 ± 3◦C, 40% to 70% relative humidity, 12 hr light/darkphotocycle, air exchange 12 to 15 times per hr).


6. Following acclimation, randomly select animals for dosing.

7. Anesthetize animals with isoflurane and carefully clip the hair on the back of eachrat with electric clippers ∼18 hr prior to dosing, taking great care to avoid nicks onthe skin, which would preclude use of the animal.

8. Position the ∼1.5-mm thick Teflon frames (4 × 5–cm with a 3 × 4–cm cut-outopening; see Fig. 5.8.3), shaped into a saddle, intrascapularly as far anteriorly aspossible and attach to the animal using appropriate adhesive (e.g., Dermabond,Ethicon).

The region of exteriorization of JVC should be avoided.

9. In addition, fit each rat with a rodent jacket to prevent the animals from groomingthe dose site.

The Teflon frames are etched with a chemical etching agent (e.g., sodium naphthalene) orpurchased etched for securing properly to the skin and adhesive Velcro.

10. Anesthetize animals again with isoflurane for dosing.

11. Apply a measured (weighed) dose of 14C-test compound topically from a ball-tippedfeeding needle attached to a 1-ml Luer-lok glass syringe to an ∼12-cm2 area. Applythe dose solution/suspension (∼120 μl per rat) evenly to the skin in a volume of10 μl/cm2.

12. Weigh the syringe and feeding needle used for application of the dose solution beforeand after dosing to determine the actual dose applied.

Animals are not fasted prior to dosing.

Non-glass Luer-lok syringes can be used given that test compound is not absorbed intothe syringe material.


Bioavailability

5.8.12


Teflon frame with Velcro

A

B

mesh screen

Velcro to secure screen

Figure 5.8.3 The Teflon frame and mesh covering (A) used for demarcation of the dermal dosingsite and restriction of the site from grooming (oral ingestion) by the animal (B).

Teflon frame and mesh screen

flap to restrict grooming

cannula with plug

rodent jacket

Figure 5.8.4 A rat fitted with a rodent jacket after the dermal application; a plastic flap is alsoattached to the jacket, for the duration until the application site is washed, to further restrict theanimal from reaching the dose site skin.

13. Semi-occlude the dose site by covering it with the Teflon spectra/mesh macroporousfilter attached to the Teflon frame (see Fig. 5.8.3), using a Velcro fastener, to preventthe animal from grooming the dose site. Further restrict access to the dose site byusing a rodent jacket and plastic flap (Fig. 5.8.4).

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5.8.13


Collect samples14. After completion of dosing, place rats individually in clean metabolism cages with

joints sealed with silicone grease. Draw inlet air into the chamber through an appro-priate flow meter and draw air exiting the chamber through a tube, passing it throughtrapping agents.

15. Collect time-course blood, urine, feces, expired-CO2, and organic volatiles andanalyze for radioactivity. Draw air through the metabolism cages at ∼800 ml/min todraw all exhaled CO2 and eliminate the chance of inhalation of exhalents.

16. Continue the experiment for 7 days to determine the fate of the dose that may havepenetrated through the stratum corneum and remained in the deeper skin layers. Forsample collection and analysis, see Support Protocols 1 through 5 and 7 through 11.

SUPPORTPROTOCOL 7

SAMPLE COLLECTION BY DERMAL WASH

Animals are anesthetized with isoflurane at a specified time (e.g., 6, 8, or 24 hr post-dosing). The macroporous filter covering is removed and the skin at the dosing siteis washed five times with cotton-tipped applicators dipped in an aqueous solutionof detergent (i.e., ∼2% to 4% dish washing liquid in water). The dose site is alsorinsed several times with water-soaked gauze and the area blotted dry with dry cottongauze squares. Following washing, the Teflon spectra/mesh covering is replaced, and an-imals are returned to their cages wearing rodent jackets for the continuous collection oftime-course blood, urine, feces, and collection of terminal tissues. Radioactivity is mea-sured on the Teflon spectra/mesh coverings, the cotton tips, and gauze by extracting ina suitable solvent (e.g., water, acetone, hexane, methanol, acetonitrile) to determine thedose remaining at the site of application at specified times after application. At the end ofthe study (7 days post-dosing), anesthetize animals with CO2/O2 and sacrifice them byexsanguination. Following sacrifice, collect the following samples from each rat and ratcage: tissues (see Support Protocol 13) and final cage wash (see Support Protocol 14).

SUPPORTPROTOCOL 8

SAMPLE COLLECTION FROM DERMAL APPLICATION DEVICES

At the time of sacrifice, the Teflon frames, Teflon spectra/mesh coverings, and the dermaljackets are collected and extracted in a suitable solvent (e.g., water, methanol, hexane,acetone, acetonitrile) to determine the remaining radioactivity.

SUPPORTPROTOCOL 9

SAMPLE COLLECTION BY TAPE STRIPPING OF THE DERMALAPPLICATION SITE

At sacrifice, the dermal application site is tape stripped (10 to 15 times) to collect stratumcorneum (D-SQUAME skin surface sampling adhesive discs, CuDerm Corporation) andto determine radioactivity associated with it after extraction in acetone or other suitablesolvent.

SUPPORTPROTOCOL 10

SAMPLE COLLECTION OF THE DERMAL APPLICATION SITE

The full-thickness skin around the application site (0.25 to 0.5 in. around the Teflonframe) of each animal is excised and collected to determine radioactivity after oxidizingor digesting.

SUPPORTPROTOCOL 11

DETERMINING DERMAL ABSORPTION

Determination of dermal absorption is a complicated process that cannot be effectivelycharacterized by a single first-order rate constant (Flynn, 1985). The amount of the14C-test compound absorbed can be estimated in several ways (Wester and Maibach,


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1985a). One approach is to assume that all absorbed 14C-test compound is found in thetissues, carcass, excreta, and final cage wash (FCW). The trapped organics in charcoal areusually considered to be evaporated/volatized test compound and not exhaled materialafter penetration, unless proven otherwise. Similarly, the amount found at the applicationsite skin is apparently not bioavailable if it was not internalized to systemic circulation by162 hr post-washing, and should not be considered as part of the amount absorbed. A moreconservative method would be to assume that all 14C-test compound not found at the dosesite is absorbed. However, if the mass-balance of the applied dose is low, the potentialfor error associated with these estimates is large. Absorption can also be estimatedby methods that are independent of the overall recovery. For example, absorption isfrequently estimated from the fraction of the dose excreted in the urine following dermalversus i.v. administration and can be corrected by using urinary elimination in the i.v.-dosed rats. Finally, absorption can also be determined by comparing blood or plasmaAUCs (area under the time course blood concentration curves) of the i.v. and dermaldoses (see Saghir et al., 2007). The approach to calculate the rate of dermal penetrationis to use the elimination data after the steady-state blood levels are achieved (Saghiret al., 2007). When extrapolating results of the dermal penetration data generated in ratsto humans, differences in the skin of the two species should not be ignored; rat skin isapproximately eleven-fold more permeable to many chemicals than human skin (Westerand Maibach, 1985b; van Ravenzwaay and Leibolde, 2004).

SUPPORTPROTOCOL 12

14C ANALYSIS OF SAMPLES

Radioactivity in each sample collected is quantified by LSC. Counts per minute (cpm) arecorrected for quench and background, and converted to disintegration per minute (dpm)prior to data analysis. For the dermal ADME studies, samples, especially blood/plasma,are counted in an LSC for 2 hr once 14C decreases to below twice the background levelsin order to confirm that extremely low levels of the applied radioactivity are not movinginto the blood stream from deeper layers of the application site skin.

SUPPORTPROTOCOL 13

COLLECTION AND ANALYSIS OF TISSUES

After sacrifice, each animal is dissected and required tissues are collected. Large tissuesare homogenized (∼33% aqueous homogenate). A weighed aliquot of homogenate isthen oxidized or digested, neutralized with acetic acid, decolorized with oxidizing agent(e.g., H2O2), and analyzed for radioactivity. Small tissues are weighed and directly oxi-dized/digested without homogenization and analyzed for radioactivity by LSC. Plasma isdirectly analyzed for radioactivity, whereas RBCs are oxidized or digested before LSC.The skin is removed from the carcass and a representative skin sample (away from ap-plication site for dermally dosed rats) is oxidized/digested and analyzed for radioactivityby LSC. In some cases, whole skin may need to be solubilized in a strong base and aweighed aliquot analyzed for radioactivity.

Alternatively, distribution of test compound radioactivity can be evaluated by whole-body/whole tissue autoradiography. This is conducted by administering radiolabeled testcompound to the rat, snap freezing the whole animal or dissected organ(s) in a hexane/dryice slurry at designated times, sectioning the frozen animal or organ(s), and conductingquantitative (or qualitative) image autoradiography.

SUPPORTPROTOCOL 14

COLLECTION AND ANALYSIS OF FINAL CAGE WASH

Following the terminal sacrifice of the animals, a final cage wash is performed usinga detergent (e.g., full-strength scrubbing bubbles as needed to cover entire surface) orin some cases solvent. The final cage wash (∼500 ml) is collected and shaken for>4 hr and a weighed aliquot of the final cage wash analyzed for radioactivity.

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SUPPORTPROTOCOL 15

METABOLIC PROFILE AND IDENTIFICATION

Pooled urine and/or fecal samples may be processed and analyzed for parent test com-pound and/or metabolite(s). Standard analytical procedures for the analyses of samplesare utilized. Major (e.g., liver, kidney) or target tissues may also be analyzed for parenttest compound and/or metabolites.

SUPPORTPROTOCOL 16

COLLECTION OF CONTROL SAMPLES

Control urine and feces are collected in dry-ice-cooled traps from one male and/or femalerat not dosed with 14C-test compound. At sacrifice, control blood is obtained. Controlsamples are used for matrix standards (test compound and/or metabolite(s) standardfor consistency, to detect possible interfering peaks on the instruments, e.g., HPLC,HPLC/MS, etc.) in chemical analysis.

BASICPROTOCOL 2

DATA ANALYSIS

Individual time-course blood (plasma/RBC) and possibly urine radioactivity concen-tration data, either total or split between parent and metabolite(s), are analyzed forpharmacokinetic parameters using one of the pharmacokinetic computer modeling pro-grams or the functions of Microsoft Excel. AUC is determined by the trapezoidal methodand the rates of absorption and elimination are calculated. Most of the time, a one- ortwo-compartmental model is considered sufficient to adequately describe the eliminationof the test compound from plasma. The total radioactivity recovered in excreta, exhaledair, final cage wash, carcass, and tissues is summed to determine the mass-balance of theadministered radioactivity with that of the recovered radioactivity. Mass-balance of theadministered dose is important for the determination of the fate of the test compound andthe assessment of its bioavailability across routes.

Calculation of Absolute and Relative Bioavailability

Bioavailability is the extent to and rate at which the test compound—parent and/ormetabolite(s)—enters systemic circulation, thereby accessing the site of action. Bioavail-ability of a test compound is influenced by the properties of the dosage form and the routeof exposure. Differences in bioavailability of a test compound, especially a therapeuticdrug, can have major impact on pharmacological effects.

For an orally administered test compound, a lower bioavailability is largely due toits passage through the intestinal wall and then through the portal circulation to theliver, both of which are common sites of first-pass metabolism. Other factors of low oralbioavailability are insufficient residence time of the test compound in the GI tract and highionization, limiting absorption. For dermally applied test compound, low bioavailabilityis mostly due to the stratum corneum, which limits the penetration of the test compoundinto the systemic circulation. The first-pass metabolism of test compound in dermis mayalso have some affect on bioavailability. Additionally, age, sex, physical activity, geneticphenotype, stress, and disease can also affect test compound bioavailability. Furtherdiscussion on dermal absorption can be found in the EPA review of dermal assessmentmethods (U.S. EPA, 1992).

Bioavailability is usually determined by comparing blood/plasma Cmax (maximum con-centration), tmax (time to Cmax), and AUC. Blood/plasma concentrations of a test com-pound increases with higher absorption; however, determination of bioavailability basedon Cmax or tmax can be misleading as distribution and elimination rates can affect Cmax.The most reliable measure of bioavailability is the AUC. The AUC is proportionalto the total amount of a test compound and/or metabolite(s) that reach the systemic


Bioavailability

5.8.16


circulation. A test compound is considered bioequivalent in the extent of absorption iftheir blood/plasma concentration AUC values are equal.

Absolute bioavailabilityAbsolute bioavailability is the fraction of the test compound and/or metabolite(s) ab-sorbed through non-i.v. routes of administration compared with the corresponding i.v.administration of the same test compound. Absolute bioavailability is determined bycomparing AUC of the test compound and/or metabolite(s) in systemic circulation afternon-i.v. (e.g., oral, dermal, etc.) routes with that of the bioavailability of the same testcompound and/or metabolite(s) after i.v. administration. The AUCs must be correctedfor the differences in the dose; therefore, each AUC is corrected by dividing with itscorresponding dose.

F = (AUCnon-i.v. × dosei.v.)/(AUCi.v. × dosenon-i.v.)

where, F is the fraction of the test compound absorbed by the non-i.v. dose, which iscorrected for the differences in the dose for the two routes of administration. A testcompound administered through i.v. route will have an absolute bioavailability of 1(F = 1); whereas, test compound administered via other routes usually have an absolutebioavailability of <1.

Relative bioavailabilityRelative bioavailability compares the AUC of a test compound with another formulationof the same or another test compound, usually via the same route of administration, orthrough a different route.

relative bioavailability = (AUCA × doseB)/(AUCB × doseA)

where, AUCA and AUCB are the systemic dose of the two formulations of the same orsimilar compounds administered via the same or different routes and doseA and doseB aretheir corresponding doses administered. Whenever, an i.v. route is used for comparison,the term absolute bioavailability is used instead of relative bioavailability.

REAGENTS AND SOLUTIONSUse fresh Milli-Q-purified water or equivalent in all recipes and protocol steps (samples shouldnot be stored longer than 7 days from their preparation). For common stock solutions, seeAPPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Cannula flush solution

25 U of heparin per ml of saline

Fill in advance, one 1-ml slip-tip syringe with ∼0.3 ml of cannula flush solutionfor each blood sample and store at 4◦C. Bring syringes to room temperature beforeuse.

Cannula lock solution

250 U of heparin per ml of saline/glycerol (1:3 v/v) solution

If glycerol interferes with the chemical analysis of parent compound and/or metabolite(s),the lock solution can be made in saline (500 U of heparin per ml saline). Glycerol can alsobe replaced with dextrose (50 U of heparin per ml of 50% dextrose) when use of glycerol isnot suitable.

Fill one 1-ml slip-tip syringe with ∼0.07 ml of cannula lock solution in advance foreach blood sample that is >4 hr apart and store at 4◦C until needed. Bring syringesto room temperature prior to use.

Toxicokinetics

5.8.17


COMMENTARY

Background InformationTo determine the extent of exposure

to chemicals, the concentration of the ac-tive molecules—parent test compound and/ormetabolite(s)—is measured in a biologicalmedium (e.g., blood, urine, or other medium).The most common way of determining sys-temic exposure to the chemical is by mea-suring the concentration of active chemical(s)in whole blood and/or plasma. Not all of thechemical that a human may be exposed tois absorbed and becomes available at the siteof action. The systemic availability of chem-icals when exposed via different routes is de-termined by comparing the available activemolecule(s) in whole blood and/or plasma.The term bioavailability is used to describethe fraction of an administered dose of un-changed (for most therapeutic drugs) and/oractive (for pro-drugs, occupational, and envi-ronmental chemicals) molecule(s) that reachthe systemic circulation (blood).

Critical ParametersInvestigators performing these studies

should be adequately experienced and trainedin regulatory and safety requirements forworking with laboratory animals, radioac-tive chemicals—including the use of liquidscintillation counters—corrosive solubilizingagents, and animal metabolism cages.

If radiolabeled chemicals are used, the radi-olabel (routinely 14C) should be situated when-ever possible at the most metabolically stableposition(s) to best follow the fate of the ma-jor portion of the test chemical and to min-imize the formation of 14CO2. This is ex-tremely important for 3H-labeled compounds,as metabolic loss would create 3H2O, compro-mising the interpretation of the tissue distri-bution and elimination datasets. The preferredradiochemical purity for such studies is ≥97%,or as high as possible. The specific activity ofthe chemical administered to rats depends onthe dose level used and the objectives of thestudy. For example, for the concurrent analysisof metabolite(s) in the collected time-courseblood samples, each rat is dosed with at least100 μCi of the radioactive chemical to ensurethat blood and excreta samples have enough ra-dioactivity for chemical analysis using a suit-able radiochemical detector or analytical in-strument (e.g., HPLC/MS or GC/MS).

For studies determining bioavailability andADME, use of the same strain of adult

rats that are used in other toxicology stud-ies (e.g., acute oral, chronic oral, reproduc-tive/developmental, inhalation) is preferredto provide a more meaningful compari-son/extrapolation of the data. In some cases,use of only one sex may be sufficient; however,that will depend on the objectives of the studyand use of the generated data. Beyond the dis-cussion in this unit, specific guidance on theconduct of multiple group experiments, com-paring dose-dependence, route-dependence,and repeat-dose effects on ADME can befound in the following guidelines: U.S. EPA870-7485 (1998), OECD 417 (1984) andOECD 427 (2004), EEC 440/2008 (2008), andJMAFF (2000).

Anticipated ResultsBy definition, when a chemical is adminis-

tered intravenously, its bioavailability is 100%,as delivery is directly into the systemic cir-culation, bypassing the process of absorp-tion. However, when the same chemical is ad-ministered via other routes, its bioavailabilitymay decrease due in part to incomplete ab-sorption and possible first-pass metabolism.For pharmaceutical chemicals, determinationof bioavailability is essential in calculatingdosages for non-i.v. routes of administration.Determination of absolute bioavailability(comparison between vascular and non-vascular routes of administration) and/orrelative bioavailability (comparison of differ-ent non-vascular routes or different formu-lations by the same route of administration)of occupational and environmental chemi-cal exposure through different routes is crit-ical in understanding the risk to the generalpopulation of a low-level exposure tothese chemicals. Bioavailability of chemi-cals is determined as part of the preclinical(pharmaceuticals)/registration (occupationaland environmental chemicals) pharmacoki-netic/toxicokinetic studies conducted in theearly stages of the programmed pharmacol-ogy/toxicology studies in animals.

Almost all of the inhaled volatile chemicalsare rapidly and almost completely absorbedthrough the lungs and thus behave similarly tothe i.v. route of exposure. However, if needed,time-course blood samples can be collectedfrom the tail vein of the rat or by using anextension attached to the jugular vein cannulafrom an animal exposed through a nose-onlyinhalation chamber.


Bioavailability

5.8.18


Time ConsiderationsAdministration (dosing) and sample collec-

tion take up to 7 days to complete. Additionaltime is required for terminal sample collec-tions, analysis of the samples, chemical anal-ysis (if appropriate), and data analysis.

AcknowledgementThe author would like to acknowledge the

help of Amy Clark, David Rick, Bill Stott, andMichael Bartels for careful editing the drafts,Jane Lacher for reviewing IACUC issues, andDavid Clark and Saarah Saghir for editing thephotographs.

Literature CitedBungay, P.M., Dedrick, R.L., and Matthews, H.B.

1981. Enteric transport of chlordecone (Kepone)in the rat. J. Pharmacokinet. Biopharm. 9:309-341.

EEC (Europian Economic Communities) 2008.Official Journal of the European Communi-ties. Methods for the Determination of Toxic-ity, Part B 36 (Toxicokinetics) as specified bythe European Economic Communities, Direc-tive 440/2008/EEC.

Flynn, G.L. 1985. Mechanism of percutaneousabsorption from physico-chemical evidence.In Percutaneous Absorption: Mechanism-Methodology-Drug Delivery (R.L. Bronaugh,R.L. Bronaugh, and H.I. Maibach, eds.) pp. 17-42. Marcel Dekker, Inc., New York.

JMAFF (Japan Ministry of Agriculture, Forestryand Fisheries) 2000. Japan Requirements forSafety Evaluation of Agricultural Chemicals(Metabolism Study).

OECD (Organisation for Economic Co-Operationand Development) 1984. OECD Guidelinefor the Testing of Chemicals, Guideline 417(Toxicokinetics).

OECD (Organisation for Economic Co-Operationand Development) 2004. Guideline for the Test-ing of Chemicals, Guideline 427 (Skin Absorp-tion: in vivo Method), April 13, 2004.

Reigle, R.D. and Bukva, N.F. 1984. A method ofrestraining rats for intravenous injection usinga flexible plastic bag. Lab. Anim. Sci. 34:497-498.

Saghir, S.A., Clark, A.J., McClymont, E.L.,and Staley, J.L. 2008. Pharmacokinetics ofaminomethylpropanol in rats following oral anda novel dermal study design. Food Chem. Toxi-col. 46:678-687.

Saghir, S.A., Frantz, S.W., Spence, M.W., Nolan,R.J., Lowe, E.R., Rick, D.L., and Bartels, M.J.2007. Pharmacokinetics and bioavailability ofdiisopropanolamine (DIPA) in rats following in-travenous or dermal application. Food Chem.Toxicol. 45:2047-2056.

Saghir, S.A., Lebofsky, M., Pinson, D.M., andRozman, K.K. 2005. Validation of Haber’srule (dose × time = constant) in rats and

mice for monochloroacetic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin under conditions ofkinetic steady state. Toxicology 215:48-56.

Saghir, S.A., Mendrala, A.L., Bartels, M.J., Day,S.J., Hansen, S.C., Sushynski, J.M., and Bus,J.S. 2006. Strategies to assess systemic expo-sure of chemicals in subchronic/chronic diet anddrinking water studies. Toxicol. Appl. Pharma-col. 211:245-260.

Thongsinthusak, T., Ross, J.H., Saiz, S.G., andKrieger, R.I. 1999. Estimation of dermal ab-sorption using the exponential saturation model.Regul. Toxicol. Pharmacol. 29:37-43.

U.S. EPA (United States Environmental Protec-tion Agency) 1982. Dermal absorption studiesof pesticides (pesticide assessment guidelines,subdivision F-hazard evaluation; human and do-mestic animals). Office of Pesticide Programs,Series 85-3, Washington, D.C., report 540/09–82–025.

U.S. EPA (United States Environmental ProtectionAgency) 1992. Dermal exposure assessment:Principles and applications. Exposure Assess-ment Group, Office of Health and Environ-mental Assessment, Washington, D.C., ReportNo. EPA/600/8-91/011B. (http://www.epa.gov/oppt/exposure/presentations/efast/usepa 1992ddermalea.pdf).

U.S. EPA (United States Environmental ProtectionAgency) 1996. Health effects test guidelinesOPPTS 870.7600: Dermal penetration. Preven-tion, Pesticides and Toxic Substances (7101).Office of Pesticide Programs, Washington, D.C.,report 712–C–96–350.

U.S. EPA (United States Environmental Pro-tection Agency) 1998. Health Effects TestGuidelines, OPPTS 870.7485 (Metabolism andPharmacokinetics).

van Ravenzwaay, B. and Leibolde, E. 2004. A com-parison between in vitro rat and human andin vivo rat skin absorption studies. Hum. Exp.Toxicol. 23:421-430.

Waynforth, H.B. and Flecknell, P.A. 1992. Experi-mental and Surgical Technique in the Rat, 2nded. Academic Press Inc., San Diego.

Wester, R.C. and Maibach, H.I. 1985a. Invivo methods for percutaneous absorptionmeasurements. In Percutaneous Absorption:Mechanism-Methodology-Drug Delivery (R.L.Bronaugh, R.L. Bronaugh, and H.I. Maibach,eds.) pp. 245-249. Marcel Dekker, Inc.,New York.

Wester, R.C. and Maibach, H.I. 1985b. In vivoanimal models for percutaneous absorptionmeasurements. In Percutaneous Absorption:Mechanism-Methodology-Drug Delivery (R.L.Bronaugh, R.L. Bronaugh, and H.I. Maibach,eds.) pp. 251-256. Marcel Dekker, Inc.,New York.

Zendzian, R.P. 1989. Skin penetration method sug-gested for Environmental Protection Agency re-quirements. J. Am. Coll. Toxicol. 8:829-835.

Zendzian, R.P. 1994. Dermal Absorption ofPesticides. Pesticide assessment guideline,

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subdivision F, hazard evaluation, human anddomestic animals, series 85-3. Health EffectDivision, Office of Pesticide Programs, U.S.Environmental Protection Agency, Washington,D.C.

Zendzian, R.P. 2000. Dermal absorption of pesti-cides in the rat. Am. Ind. Hyg. Assoc. J. 61:473-483.

Documents

Current Protocols in Toxicology || Determination of ADME and Bioavailability Following Intravenous, Oral, and Dermal Routes of Exposure