Guideline for Industry

Toxicokinetics: The

Assessment of Systemic

Exposure in Toxicity

Studies
ICH - S3A

March 1995
i

TABLE OF CONTENTS

I. INTRODUCTION ................................................ 1

II. THE OBJECTIVES OF TOXICOKINETICS AND THE PARAMETERS WHICH

MAY BE DETERMINED .......................................... 3

III. GENERAL PRINCIPLES TO BE CONSIDERED ....................... 4

A. Introduction ............................................... 4

B. Quantification of exposure ................................... 5

C. Justification of time points for sampling ......................... 5

D. Contribution to the setting of dose levels in order to produce adequate

exposure ................................................. 5

1. Low dose levels ...................................... 5

2. Intermediate dose levels ............................... 5

3. High dose levels ...................................... 5

E. Extent of exposure assessment in toxicity studies ................. 6

F. Complicating factors in exposure interpretation ................... 6

G. Route of administration ...................................... 7

H. Determination of metabolites ................................. 7

I. Statistical evaluation of data .................................. 8

J. Analytical methods ......................................... 8

K. Reporting ................................................ 9

IV. TOXICOKINETICS IN THE VARIOUS AREAS OF TOXICITY TESTING -

SPECIFIC ASPECTS ............................................ 9

A. Introduction ............................................... 9

B. Single dose toxicity studies ................................... 9

C. Repeated dose toxicity studies ............................... 10

D. Genotoxicity studies ....................................... 10

E. Carcinogenicity (Oncogenicity) studies ........................ 10

1. Sighting or dose-ranging studies ........................ 10

2. The main studies .................................... 11

F. Reproductive toxicity studies ................................ 12

1. Introduction (4.6.1) ................................... 12

2. Fertility studies ...................................... 12

3. Studies in pregnant and lactating animals ................. 12

V. REFERENCES ................................................ 13

SUPPLEMENTARY NOTES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . APPENDIX A
This guideline was developed within the Expert Working Group (Safety) of the †

International Conference on Harmonisation of Technical Requirements for Registration

of Pharmaceuticals for Human Use (ICH) and has been subject to consultation by the

regulatory parties, in accordance with the ICH process. This document has been

endorsed by the ICH Steering Committee at Step 4 of the ICH process, October 27,

1994. At Step 4 of the process, the final draft is recommended for adoption to the

regulatory bodies of the European Union, Japan and the USA. This guideline was

published in the Federal Register on March 1, 1995 (60 FR 11264) and is applicable to

drug and biological products. In the past, guidelines have generally been issued under

§ 10.90(b) [21 CFR 10.90(b)], which provides for the use of guidelines to state

procedures or standards of general applicability that are not legal requirements but that

are acceptable to FDA. The agency is now in the process of revising §10.90(b). 

Therefore, this guideline is not being issued under the authority of §10.90(b), and it

does not create or confer any rights, privileges or benefits for or on any person, nor

does it operate to bind FDA in any way. For additional copies of this guideline contact

the Consumer Affairs Branch (formerly the Executive Secretariat Staff), HFD-210,

Center for Drug Evaluation and Research, 7500 Standish Place, Rockville, MD, 20855,

301-594-1012. An electronic version of this guideline is also available via Internet by

connecting to the CDER file transfer protocol (FTP) server

(CDVS2.CDER.FDA.GOV).

GUIDELINE FOR INDUSTRY†

TOXICOKINETICS:

THE ASSESSMENT OF SYSTEMIC EXPOSURE 

IN TOXICITY STUDIES

I. INTRODUCTION (1)

This Note for Guidance concerns toxicokinetics only with respect to the

development of pharmaceutical products intended for use in human subjects.

In this context, toxicokinetics is defined as the generation of pharmacokinetic

data, either as an integral component in the conduct of nonclinical toxicity

studies or in specially designed supportive studies, in order to assess systemic

exposure. These data may be used in the interpretation of toxicology findings

and their relevance to clinical safety issues (see Note 1 for definitions of other

terms used in this document).1

The Note for Guidance has been developed in order to provide an
2

understanding of the meaning and application of toxicokinetics and to provide

guidance on developing test strategies in toxicokinetics. The guidance

highlights the need to integrate pharmacokinetics into toxicity testing, which

should aid in the interpretation of the toxicology findings and promote rational

study design development.

Toxicokinetic measurements are normally integrated within the toxicity studies

and as such are described in this document as "concomitant toxicokinetics."1

Alternatively, data may be generated in other supportive studies conducted by

mimicking the conditions of the toxicity studies.

Toxicokinetic procedures may provide a means of obtaining multiple dose

pharmacokinetic data in the test species, if appropriate parameters are

monitored, thus avoiding duplication of such studies; optimum design in

gathering the data will reduce the number of animals required.

Various components of the total nonclinical pharmacokinetics and metabolism

program may be of value in contributing to the interpretation of toxicology

findings. However, the toxicokinetic data focus on the kinetics of a new

therapeutic agent under the conditions of the toxicity studies themselves.

Toxicokinetics is thus an integral part of the non-clinical testing program; it

should enhance the value of the toxicological data generated, both in terms of

understanding the toxicity tests and in comparison with clinical data as part of

the assessment of risk and safety in humans. Due to its integration into toxicity

testing and its bridging character between non-clinical and clinical studies, the

focus is primarily on the interpretation of toxicity tests and not on characterizing

the basic pharmacokinetic parameters of the substance studied.

As the development of a pharmaceutical product is a dynamic process which

involves continuous feedback between nonclinical and clinical studies, no rigid

detailed procedures for the application of toxicokinetics are recommended. It

may not be necessary for toxicokinetic data to be collected in all studies and

scientific judgment should dictate when such data may be useful. The need for

toxicokinetic data and the extent of exposure assessment in individual toxicity

studies should be based on a flexible step-by-step approach and a

case-by-case decision making process to provide sufficient information for a risk

and safety assessment.

II. THE OBJECTIVES OF TOXICOKINETICS AND THE PARAMETERS
3

WHICH MAY BE DETERMINED (2)

The primary objective of toxicokinetics is:

To describe the systemic exposure achieved in animals and its

relationship to dose level and the time course of the toxicity study. 

Secondary objectives are:

To relate the exposure achieved in toxicity studies to toxicological

findings and contribute to the assessment of the relevance of these

findings to clinical safety.

To support the choice of species and treatment regimen in nonclinical 1

toxicity studies.

To provide information which, in conjunction with the toxicity findings,

contributes to the design of subsequent nonclinical toxicity studies.

These objectives may be achieved by the derivation of one or more

pharmacokinetic parameters from measurements made at appropriate time 2

points during the course of the individual studies. These measurements usually

consist of plasma (or whole blood or serum) concentrations for the parent

compound and/or metabolite(s) and should be selected on a case-by-case

basis. Plasma (or whole blood or serum) AUC, Cmax and C are the most (time)

2

commonly used parameters in assessing exposure in toxicokinetics studies. 

For some compounds it will be more appropriate to calculate exposure based on

the (plasma protein) unbound concentration.

These data may be obtained from all animals on a toxicity study, in

representative subgroups, in satellite groups [see III.E. (3.5) and Note 1) or in

separate studies.

Toxicity studies which may be usefully supported by toxicokinetic information

include single and repeated dose toxicity studies, reproductive, genotoxicity and

carcinogenicity studies. Toxicokinetic information may also be of value in

assessing the implications of a proposed change in the clinical route of

administration.

III. GENERAL PRINCIPLES TO BE CONSIDERED (3)
4

A. Introduction (3.1)

In the following paragraphs some general principles are set out which

should be taken into consideration in the design of individual studies.

It should be noted that for those toxicity studies whose performance is

subject to Good Laboratory Practice (GLP) the concomitant toxicokinetics

must also conform to GLP. Toxicokinetic studies retrospectively

designed to generate specific sets of data under conditions which closely

mimic those of the toxicity studies should also conform to GLP when they

are necessary for the evaluation of safety.

B. Quantification of exposure (3.2)

The quantification of systemic exposure provides an assessment of the

burden on the test species and assists in the interpretation of similarities

and differences in toxicity across species, dose groups and sexes. The

exposure might be represented by plasma (serum or blood)

concentrations or the AUC's of parent compound and/or metabolite(s). In

some circumstances, studies may be designed to investigate tissue

concentrations. When designing the toxicity studies, the exposure and

dose-dependence in humans at therapeutic dose levels (either expected

or established), should be considered in order to achieve relevant

exposure at various dose levels in the animal toxicity studies. The

possibility that there may be species differences in the

pharmacodynamics of the substance (either qualitative or quantitative)

should also be taken into consideration.

Pharmacodynamic effects or toxicity might also give supporting evidence

of exposure or even replace pharmacokinetic parameters in some

circumstances.

Toxicokinetic monitoring or profiling of toxicity studies should establish

what level of exposure has been achieved during the course of the study

and may also serve to alert the toxicologist to non-linear, dose-related

changes in exposure (Note 3) which may have occurred. Toxicokinetic

information may allow better interspecies comparisons than simple

dose/body weight (or surface area) comparisons.

C. Justification of time points for sampling (3.3)
5

The time points for collecting body fluids in concomitant toxicokinetic

studies should be as frequent as is necessary, but not so frequent as to

interfere with the normal conduct of the study or to cause undue

physiological stress to the animals. In each study, the number of time 4

points should be justified on the basis that they are adequate to estimate

exposure [see III.B. (3.2)]. The justification should be based on kinetic

data gathered from earlier toxicity studies, from pilot or dose

range-finding studies, from separate studies in the same animal model or

in other models allowing reliable extrapolation.

D. Contribution to the setting of dose levels in order to produce adequate

exposure (3.4)

The setting of dose levels in toxicity studies is largely governed by the

toxicology findings and the pharmacodynamic responses of the test

species. However, the following toxicokinetic principles may contribute to

the setting of the dose levels.

1. Low dose levels (3.4.1)

At the low dose, preferably a no-toxic-effect dose level , the 5

exposure in the animals of any toxicity study should ideally equal

or just exceed the maximum expected (or known to be attained) in

patients. It is recognized that this ideal is not always achievable

and that low doses will often need to be determined by

considerations of toxicology; nevertheless, systemic exposure

should be determined.

2. Intermediate dose levels (3.4.2)

Exposure at intermediate dose levels should normally represent an

appropriate multiple (or fraction) of the exposure at lower (or

higher) dose levels dependent upon the objectives of the toxicity

study.

3. High dose levels (3.4.3)

The high dose levels in toxicity studies will normally be determined

by toxicological considerations. However, the exposure achieved

at the dose levels used should be assessed.

Where toxicokinetic data indicate that absorption of a compound

limits exposure to parent compound and/or metabolite(s) , the 6
6

lowest dose level of the substance producing the maximum

exposure should be accepted as the top dose level to be used

(when no other dose-limiting constraint applies ). 7

Very careful attention should be paid to the interpretation of

toxicological findings in toxicity studies (of all kinds) when the dose

levels chosen result in nonlinear kinetics. However, nonlinear 3

kinetics should not necessarily result in dose limitations in toxicity

studies or invalidate the findings; toxicokinetics can be very helpful

in assessing the relationship between dose and exposure in this

situation.

E. Extent of exposure assessment in toxicity studies (3.5)

In toxicity studies, systemic exposure should be estimated in an

appropriate number of animals and dose groups to provide a basis for 8

risk assessment.

Concomitant toxicokinetics may be performed either in all or a

representative proportion of the animals used in the main study or in

special satellite groups (Notes 1 and 5). Normally, samples for the

generation of toxicokinetic data may be collected from main study

animals, where large animals are involved, but satellite groups may be

required for the smaller (rodent) species.

The number of animals to be used should be the minimum consistent with

generating adequate toxicokinetic data. Where both male and female

animals are utilized in the main study it is normal to estimate exposure in

animals of both sexes unless some justification can be made for not so

doing.

Toxicokinetic data are not necessarily required from studies of different

duration if the dosing regimen is essentially unchanged [see also IV.C.

(4.3)].

F. Complicating factors in exposure interpretation (3.6)

Although estimating exposure as described above may aid in the

interpretation of toxicity studies and in the comparison with human

exposure, a few caveats should be noted.

Species differences in protein binding, tissue uptake, receptor properties

and metabolic profile should be considered. For example, it may be more
7

appropriate for highly protein bound compounds to have exposure

expressed as the free (unbound) concentrations. In addition, the

pharmacological activity of metabolites, the toxicology of metabolites and

antigenicity of biotechnology products may be complicating factors. 

Furthermore, it should be noted that even at relatively low plasma

concentrations, high levels of the administered compound and/or

metabolite(s) may occur in specific organs or tissues.

G. Route of administration (3.7)

The toxicokinetic strategy to be adopted for the use of alternative routes

of administration, for example by inhalation, topical or parenteral delivery,

should be based on the pharmacokinetic properties of the substance

administered by the intended route.

It sometimes happens that a proposal is made to adopt a new clinical

route of administration for a pharmaceutical product; for example, a

product initially developed as an oral formulation may subsequently be

developed for intravenous administration. In this context, it will be

necessary to ascertain whether changing the clinical route will

significantly reduce the safety margin.

This process may include a comparison of the systemic exposure to the

compound and/or its relevant metabolite(s) (AUC and Cmax) in humans

generated by the existing and proposed routes of administration. If the

new route results in increased AUC and/or Cmax, or a change in metabolic

profile, the continuing assurance of safety from animal toxicology and

kinetics should be reconsidered. If exposure is not substantially greater,

or different, by the proposed new route compared to that for the existing

route(s) then additional nonclinical toxicity studies may focus on local

toxicity.

H. Determination of metabolites (3.8)

A primary objective of toxicokinetics is to describe the systemic exposure

to the administered compound achieved in the toxicology species. 

However, there may be circumstances when measurement of metabolite

concentrations in plasma or other body fluids is especially important in

the conduct of toxicokinetics.9

When the administered compound acts as a "pro-drug" and the

delivered metabolite is acknowledged to be the primary active

entity.
8

When the compound is metabolized to one or more

pharmacologically or toxicologically active metabolites which could

make a significant contribution to tissue/organ responses.

When the administered compound is very extensively metabolized

and the measurement of plasma or tissue concentrations of a

major metabolite is the only practical means of estimating

exposure following administration of the compound in toxicity

studies.10

I. Statistical evaluation of data (3.9)

The data should allow a representative assessment of the exposure. 

However, because large intra- and inter-individual variation of kinetic

parameters may occur and small numbers of animals are involved in

generating toxicokinetic data, a high level of precision in terms of

statistics is not normally needed. Consideration should be given to the

calculation of mean or median values and estimates of variability, but in

some cases the data of individual animals may be more important than a

refined statistical analysis of group data.

If data transformation (e.g., logarithmic) is performed, a rationale should

be provided.

J. Analytical methods (3.10)

Integration of pharmacokinetics into toxicity testing implies early

development of analytical methods for which the choice of analytes and

matrices should be continually reviewed as information is gathered on

metabolism and species differences.

The analytical methods to be used in toxicokinetic studies should be

specific for the entity to be measured and of an adequate accuracy and

precision. The limit of quantification should be adequate for the

measurement of the range of concentrations anticipated to occur in the

generation of the toxicokinetic data.

The choice of analyte and the matrix to be assayed (biological fluids or

tissue) should be stated and possible interference by endogenous

components in each type of sample (from each species) should be

investigated. Plasma, serum or whole blood are normally the matrices of

choice for toxicokinetic studies.
9

If the drug substance is a racemate or some other mixture of enantiomers

additional justification should be made for the choice of the analyte

[racemate or enantiomer(s)] .

The analyte and matrix assayed in non-clinical studies should ideally be

the same as in clinical studies. If different assay methods are used in

non-clinical and clinical studies they should all be suitably validated.

K. Reporting (3.11)

A comprehensive account of the toxicokinetic data generated, together

with an evaluation of the results and of the implications for the

interpretation of the toxicology findings should be given.

An outline of the analytical method should be reported or referenced. In

addition, a rationale for the choice of the matrix analyzed and the analyte

measured [see 3.8 (H) and 3.10 (J)] should be given.

The positioning of the report within the application will depend upon

whether the data are specific to any one toxicity study or are supportive

of all toxicity testing.

IV. TOXICOKINETICS IN THE VARIOUS AREAS OF TOXICITY TESTING -

SPECIFIC ASPECTS

A. Introduction (4.1)

Based on the principles of toxicokinetics outlined above the following

specific considerations refer to individual areas of toxicity testing. The

frequency of exposure monitoring or profiling may be extended or

reduced where necessary.

It may be appropriate to take samples from some individual animals only,

where this may help in the interpretation of the toxicology findings for

these animals.

B. Single dose toxicity studies (4.2)

These studies are often performed in a very early phase of development

before a bioanalytical method has been developed and toxicokinetic

monitoring of these studies is therefore not normally possible. Plasma

samples may be taken in such studies and stored for later analysis, if

necessary; appropriate stability data for the analyte in the matrix sampled
10

would then be required.

Alternatively, additional toxicokinetic studies may be carried out after

completion of a single dose toxicity study in order to respond to specific

questions which may arise from the study.

Results from single dose kinetic studies may help in the choice of

formulation and in the prediction of rate and duration of exposure during a

dosing interval. This may assist in the selection of appropriate dose

levels for use in later studies.

C. Repeated dose toxicity studies (4.3)

The treatment regimen and species should be selected whenever 11

possible with regard to pharmacodynamic and pharmacokinetic

principles. This may not be achievable for the very first studies, at a time

when neither animal nor human pharmacokinetic data are normally

available.

Toxicokinetics should be incorporated appropriately into the design of the

studies. It may consist of exposure profiling or monitoring at appropriate 1

dose levels at the start and towards the end of the treatment period of the

first repeat dose study. The procedure adopted for later studies will 12

depend on the results from the first study and on any changes in the

proposed treatment regimen. Monitoring or profiling may be extended,

reduced or modified for specific compounds where problems have arisen

in the interpretation of earlier toxicity studies.

D. Genotoxicity studies (4.4)

For negative results of in vivo genotoxicity studies, it may be appropriate

to have demonstrated systemic exposure in the species used or to have

characterized exposure in the indicator tissue.

E. Carcinogenicity (Oncogenicity) studies (4.5)

1. Sighting or dose-ranging studies (4.5.1)

Appropriate monitoring or profiling of these studies should be

undertaken in order to generate toxicokinetic data which may

assist in the design of the main studies [see IV.E.2 (4.5.2.)]. 

Particular attention should be paid to species and strains which

have not been included in earlier toxicity studies and to the use of
11

routes or methods of administration which are being used for the

first time.

Particular attention should be paid to the establishment of

appropriate toxicokinetic data when administration is to be in the

diet.13

Toxicokinetic data may assist in the selection of dose levels in the

light of information about clinical exposure and in the event that

nonlinear kinetics may complicate the interpretation of the study. 3

In principle, the ideal study design would ensure that dose levels in

oncogenicity studies generate a range of systemic exposure

values that exceed the maximum therapeutic exposure for humans

by varying multiples. However, it is recognized that this idealized

selection of dose levels may be confounded by unavoidable

species-specific problems. Thus, the emphasis of this guidance is

on the need to estimate systemic exposure, to parent compound

and/or metabolite(s) at appropriate dose levels and at various

stages of an oncogenicity study, so that the findings of the study

may be considered in the perspective of comparative exposure for

the animal model and humans.

A highest dose based on knowledge of probable systemic

exposure in the test species and in humans may be an acceptable

end-point in testing for carcinogenic potential. Historically, a

toxicity end-point has been often used to select the top dose level.

2. The main studies (4.5.2)

The treatment regimen, species and strain selection should, as far

as is feasible, be determined with regard to the available

pharmacokinetic and toxicokinetic information. In practice, the

vast majority of these studies is conducted in the rat and mouse.

As mentioned in the "Introduction" to this section, it is

recommended that reassurance be sought from monitoring that the

exposure in the main study is consistent with profiles of kinetics

established in free-standing or specific dose-ranging studies. 

Such monitoring will be appropriate on a few occasions during the

study, but it is not considered essential to continue beyond six

months.
12

F. Reproductive toxicity studies (4.6) 2

1. Introduction (4.6.1)

It is preferable to have some information on pharmacokinetics

before initiating reproduction studies since this may suggest the

need to adjust the choice of species, study design and dosing

schedules. At this time the information need not be sophisticated

or derived from pregnant or lactating animals. At the time of study

evaluation further information on pharmacokinetics in pregnant or

lactating animals may be required depending on the results

obtained.

The limitation of exposure in reproductive toxicity is usually

governed by maternal toxicity. Thus, while toxicokinetic monitoring

in reproductive toxicity studies may be valuable in some instances,

especially with compounds with low toxicity, such data are not

generally needed for all compounds.

Where adequate systemic exposure might be questioned because

of absence of pharmacological response or toxic effects,

toxicokinetic principles could usefully be applied to determine the

exposures achieved by dosing at different stages of the

reproductive process.

A satellite group of female animals may be used to collect the

toxicokinetic data.

2. Fertility studies (4.6.2)

The general principles for repeated-dose toxicity studies apply [see

IV.C. (4.3)]. The need to monitor these studies will depend on the

dosing regimen used and the information already available from

earlier studies in the selected species.

3. Studies in pregnant and lactating animals (4.6.3)

The treatment regimen during the exposure period should be

selected on the basis of the toxicological findings and on

pharmacokinetic and toxicokinetic principles.

Consideration should be given to the possibility that the kinetics

will differ in pregnant and nonpregnant animals.
13

Toxicokinetics may involve exposure assessment of dams,

embryos, fetuses or newborn at specified days. Secretion in milk 14

may be assessed to define its role in the exposure of newborns. In

some situations, additional studies may be necessary or

appropriate in order to study embryo/fetal transfer and secretion in

milk.

Consideration should be given to the interpretation of reproductive

toxicity tests in species in which placental transfer of the

substance cannot be demonstrated.

V. REFERENCES

A. "Carcinogenicity: Guidance for Dose Selection for Carcinogenicity

Studies of Pharmaceuticals" (ICH Guideline S1C).

B. "Detection of Toxicity to Reproduction for Medicinal Products" (ICH

Guideline S5A).

C. Federal Register, Vol. 60, No. 40, Wednesday, March 1, 1995, pages 

11264-11268.
A-1

APPENDIX A

SUPPLEMENTARY NOTES

1. Definitions of expressions appearing in this "Note for Guidance:"

Analyte: The chemical entity assayed in biological samples.

Matrix: Blood, plasma, urine, serum or other fluid or tissue selected for assay.

Concomitant toxicokinetics: Toxicokinetic measurements performed in the toxicity

study, either in all animals or in representative subgroups or in satellite groups.

Exposure: Exposure is represented by pharmacokinetic parameters demonstrating the

local and systemic burden on the test species with the test compound and/or its

metabolites. The area under the matrix level concentration-time curve (AUC) and/or

the measurement of matrix concentrations at the expected peak-concentration time

Cmax, or at some other selected time C ,are the most commonly used parameters. (time)

Other parameters might be more appropriate in particular cases.

Monitor: To take a small number of matrix samples (e.g., 1 to 3) during a dosing

interval to estimate C or C .

(time) max

Profile: To take (e.g., 4 to 8) matrix samples during a dosing interval to make an

estimate of Cmax and/or C and area under matrix concentration time curve (AUC). (time)

Satellite: Groups of animals included in the design and conduct of a toxicity study,

treated and housed under conditions identical to those of the main study animals, but

used primarily for toxicokinetics.

Support: In the context of a toxicity study - to ratify or confirm the design of a toxicity

study with respect to pharmacokinetic and metabolic principles. This process may

include 2 separate steps:

a. confirmation using toxicokinetic principles that the animals on a study were

exposed to appropriate systemic levels of the administered compound [see III.D.

(3.4)] and or its metabolite(s).

b. confirmation that the metabolic profile in the species used was acceptable;

data to support this will normally be derived from metabolism studies in animals

and in humans.

Validate: In the context of an analytical method -- to establish the accuracy, precision,
A-2

reproducibility, response function and the specificity of the analytical method with

reference to the biological matrix to be examined and the analyte to be quantified.

Note 2 Symbols and definitions according to "Manual of Symbols, Equations and

Definitions in Pharmacokinetics," Committee for Pharmacokinetic Nomenclature of the

American College of Clinical Pharmacology, Philadelphia, PA, May 1982:

Cmax Maximum (peak) concentration.

C(time) Maximum concentration at a specified time after administration of a

given dose.

tmax Time to reach peak or maximum concentration following administration.

AUC Area concentration-time curve from zero to time t. It should be noted that (0-t)

AUC(0-infinity) is a special case of AUC(0-t)

Other measurements, for example urinary excretion, may be more appropriate for

some compounds. Other derived parameters, for example bioavailability, half-life,

fraction of unbound drug and volume of distribution may be of value in interpreting

toxicokinetic data. Thus, the selection of parameters and time points has to be made

on a case-by-case basis considering the general principles as outlined in Section III

(3).

3. Increases in exposure may arise unexpectedly as a result of nonlinear kinetics due

to saturation of a clearance process. Increasing exposure may also occur during the

course of a study for those compounds which have a particularly long plasma half-life. 

Careful attention should also be paid to compounds which achieve high Cmax values

over comparatively short time periods within the dosing interval. Conversely,

unexpectedly low exposure may occur during a study as a result of auto-induction of

metabolizing enzymes.

4. If samples are taken from main study animals it should be considered whether

samples should be taken from all the dosed animals and the controls in order to treat

all animals on the study in the same way, or whether samples should be taken from

representative subgroups of the same size.

5. In this context, a "no-toxic-effect dose level" (deemed to be the same as

"no-observed-adverse-effect dose level") is defined as a dose level at which some

pharmacological response may be observed, but at which no adverse effect is found.

6. In these circumstances it should be established that absorption is the rate limiting

step and that limitations in exposure to the administered substance are not due to an
A-3

increased clearance.

7. The limits placed on acceptable volumes which can be administered orally to

animals may constrain the dose levels achievable for comparatively non-toxic

compounds administered as solutions or suspensions.

8. It is often considered unnecessary to assay samples from control groups. Samples

may be collected and then assayed if it is deemed that this may help in the

interpretation of the toxicity findings, or in the validation of the assay method.

9. Measurement of metabolite concentrations may be especially important when

documentation of exposure to human metabolite(s) is needed in the non-clinical toxicity

studies in order to demonstrate adequate toxicity testing of these metabolites.

10. It is recognized that measurement of metabolite(s) as a part of toxicokinetic

evaluation serves only to assess exposure and cannot account for possible reactive

intermediate metabolites.

11. Treatment regimen encompasses dosage, formulation, route of administration and

dosing frequency.

12. The first repeat dose study incorporating toxicokinetic data for each species is

normally of 14 days duration or longer.

13. Additional studies may be required in order to compare exposure to the compound

administered in diet and by gavage or by routes different from the intended clinical

route.

14. It should be noted that while it is important to consider the transfer of substances

entering the embryo-fetal compartment, fetal exposure is the parameter which is most

often assessed in practice in separate studies and expressed as "placental transfer."