S3A Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies

Administrative guidance, not enacted legislation. This captured publisher edition preserves the listed document and attachments; later developments are not independently consolidated.

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 nonclinical1
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 time2
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, C and C are the most	max (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 time4
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 , the5
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) , the6

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, nonlinear3
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 for8
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 C ) in humans	max
generated by the existing and proposed routes of administration. If the
new route results in increased AUC and/or C , or a change in metabolic	max
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 whenever11
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 appropriate1
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 will12
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 milk14
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
C , or at some other selected time C ,are the most commonly used parameters.	max (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 C and/or C and area under matrix concentration time curve (AUC).	max (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:
C Maximum (peak) concentration.	max
C Maximum concentration at a specified time after administration of a	(time)
given dose.
t Time to reach peak or maximum concentration following administration.	max
AUC Area concentration-time curve from zero to time t. It should be noted that	(0-t)
AUC is a special case of AUC	(0-infinity) (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 C values	max
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

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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."