S3A Guidance: Note for Guidance on 
Toxicokinetics: The Assessment of 
Systemic Exposure in Toxicity Studies: 
Focus on Microsampling 
         
Questions and Answers 
 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
 
May 2018 
ICH
 
 
 
 
   
 
 
S3A Guidance: Note for Guidance on 
Toxicokinetics: The Assessment of 
Systemic Exposure in Toxicity Studies: 
Focus on Microsampling 
         
Questions and Answers 
 
Guidance for Industry 
 
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Email: ocod@fda.hhs.gov 
 
https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
 
May 2018 
ICH
 
  Contains Nonbinding Recommendations 
 
 
 
TABLE OF CONTENTS 
 
 
 
 
I.      INTRODUCTION  SCOPE (1) .......................................................................................... 1 
II.    BASIC PRINCIPLE ON APPLICATION OF MICROSAMPLING (2) ........................... 2 
III.  EFFECT ON SAFETY EVALUATION (3) .......................................................................... 3 
IV.  ISSUES REGARDING THE BIOANALYTICAL METHOD (4) ...................................... 4 
V.   ANNEX: Q&As LINKED TO THE RESPECTIVE SECTIONS OF S3A GUIDANCE .. 6 
i 
  Contains Nonbinding Recommendations 
 
 
S3A Guidance: Note for Guidance on Toxicokinetics: The Assessment of 
Systemic Exposure in Toxicity Studies: Focus on Microsampling 
Questions and Answers 
Guidance for Industry1 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on this 
topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You can use an 
alternative approach if it satisfies the requirements of the applicable statutes and regulations.  To discuss an 
alternative approach, contact the FDA office responsible for this guidance as listed on the title page.   
 
 
 
The guideline for industry S3A Toxicokinetics: The Assessment of Systemic Exposure in Toxicity 
Studies2 (S3A guidance) has been successfully implemented since 1994, and in recent years, analytical 
method sensitivity has improved, allowing microsampling techniques to be used in toxicokinetic (TK) 
assessment.  This question-and-answer (Q&A) document focuses on points to consider before 
incorporating the microsampling method in TK studies, acknowledges the benefits (and some 
limitations) of using microsampling to assess toxicokinetics in main study animals, and acknowledges 
the overall important contribution of microsampling to the 3Rs benefits (replacement, reduction and 
refinement) by reducing or eliminating the need for TK satellite animals. 
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities.  Instead, 
guidances  describe  the  Agencys  current  thinking  on  a  topic  and  should  be  viewed  only  as 
recommendations, unless specific regulatory or statutory requirements are cited.  The use of the word 
should in Agency guidances means that something is suggested or recommended, but not required. 
 
 
I.  INTRODUCTION  SCOPE (1)3 
 
1.1:  What is the definition of microsampling? 
 
                                                            
1 This guidance was developed within the Safety Implementation Working Group of the International Council for 
Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) and has been subject to consultation 
by the regulatory parties, in accordance with the ICH process. The Q&As in this document have been endorsed by the ICH 
Steering Committee at Step 4 of the ICH process, November 2017. At Step 4 of the process, the final draft is recommended 
for adoption to the regulatory bodies of the European Union, Japan, and North America. 
2 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the FDA Drugs 
guidance web page at 
https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm. 
3 Arabic numbers reflect the organizational breakdown of the document endorsed by the ICH Steering Committee at Step 4 
of the ICH process, November 2017. 
1 
  Contains Nonbinding Recommendations 
 
 
For the purpose of this document, microsampling is a method to collect a very small amount of 
blood (typically 50 microliters) that is generally used to measure concentrations of a drug 
and/or its metabolites and subsequently calculate the appropriate TK parameters.  The 
appropriate matrices used for microsampling techniques include blood and its derived plasma or 
serum, which can be used in liquid or dried form for transportation, storage, and subsequent 
analysis.  Microsampling for toxicokinetics can be used in rodents and nonrodents.  
Microsampling in non-blood-derived matrices is outside the scope of this Q&A document. 
 
1.2:  What are the benefits/advantages of microsampling? 
 
Minimizing the volume of blood collection can reduce pain and distress in animals and improve 
the animal welfare (refinement) of rodents and nonrodents.  It can also eliminate the TK 
satellite group in which the TK assessment is conducted in main study animals or reduce the 
number of required animals in a TK satellite group when used in rodent studies (reduction).  
The benefit is particularly notable for mice, because a significant number of these animals are 
generally used in satellite groups in TK studies using conventional sample volumes.  The main 
scientific advantage of microsampling is that the relationship between the safety data and drug 
exposure can be directly evaluated in the same animal.  
 
 
II.  BASIC PRINCIPLE ON APPLICATION OF MICROSAMPLING (2) 
 
2.1:    For what types of pharmaceuticals and for what types of safety studies can we use 
microsampling? 
 
Generally, microsampling is applicable to the majority of pharmaceuticals, including 
biopharmaceuticals.  However, for all types of analytes, consideration should be given on a 
case-by-case basis as to whether the sensitivity of the measurement method is appropriate for 
the small sample volumes available. 
 
Microsampling can be used in any type of toxicology study, including single-dose, repeat-dose, 
carcinogenicity, juvenile, and reproductive.  When microsampling is applied, sampling of a 
representative subgroup is acceptable, as mentioned in the S3A guidance.  There are published 
examples demonstrating no effects on key veterinary clinical pathology or pathological 
parameters when small volumes of blood are taken from adult animals.  However, 
microsampling is not warranted when the drug concentration is low and the majority or all 
samples are Below the Limit of Quantification (BLQ) (e.g., exposure after topical or inhaled 
administration).  However, when the bioanalytical method for microsampling has the same 
Lower Limit of Quantitation (LLOQ) as that for conventional sample volume, microsampling 
can be used even if the majority or all samples are BLQ. 
 
2.2:   What are the points to consider when applying microsampling to TK studies? 
 
As with other approaches to kinetic sampling, to adopt a microsampling technique 
appropriately, a bioanalytical method should be developed and qualified (or validated for Good 
Laboratory Practice studies, in accordance with regulatory guideline/guidance in each region) to 
2 
  Contains Nonbinding Recommendations 
 
 
ensure the reliability of analytical results.  Analytical characteristics, such as LLOQ, accuracy, 
precision, influence of matrix using dilution prior to storage, and the stability of the analyte(s) 
in the biological matrix for the entire period of sampling, storage, and processing conditions, 
should be carefully assessed to establish the microsampling method.  When conventional 
methods have already been used in some studies and microsampling is proposed for others, an 
assessment of the comparability of the exposure measurement, in the given matrix, between 
microsampling and conventional methods may be necessary. This comparison is particularly 
important if the conditions that the TK samples are presented in are substantially different (e.g., 
dried sample from microsampling vs. liquid sample from conventional sampling).  This 
comparability assessment can be done in a separate pharmacokinetic (PK) study to measure 
area under the matrix level concentration-time curve (AUC) and/or maximum drug 
concentration (C ),provided that the appropriate range of concentrations is evaluated and the 
max  
resulting parameters compared between the two methods. This PK study, if needed, should be 
completed prior to conducting definitive studies that will include the microsampling procedure. 
A separate PK study for comparison may be omitted on a case-by-case basis and with 
appropriate scientific justification, for example, when using similar assay conditions to test 
blood, plasma, or serum drawn from the same sampling site. 
 
During this comparison process, multiple blood collections at a few time points by 
microsampling from a few animals and their subsequent measurement of analyte concentrations 
can be considered to check the variability of sampling.  Ideally, the same matrix should be used 
throughout the TK studies and also in clinical studies for comparison of exposure.  When 
different matrices are used in different studies, the drug concentration relationship among 
matrices should be defined considering appropriate factors such as hematological parameters, 
plasma protein binding ratio, and blood/plasma (or serum) ratio of the drug, so that systemic 
exposure can be evaluated appropriately from each measurement using different matrices. 
 
2.3:   What types of blood collection are used for microsampling? 
 
Blood can be collected from the tail vein, saphenous vein, etc., using capillary tubes or any 
appropriate miniaturized collection devices. The collected blood, and its derived plasma or serum, 
can be used to measure drug concentrations in either liquid or dried form.  In some cases, liquid 
samples can be diluted with the appropriate vehicles or blank matrices before storage, shipment, 
and subsequent analysis.  Dried sample methods are also available, wherein the sample is usually 
applied directly onto cellulose-based or other types of materials and then dried.  A fixed-diameter 
subpunch or the whole quantity of the sample on the card/device can be extracted and analyzed.  
Recent and ongoing advancements in microsampling methods have demonstrated the ability to 
collect precise volumes of blood, such that the entire sample can be used for analysis without 
additional volumetric measurements.  In addition, newly developed techniques could also be 
considered with adequate validation. 
 
 
III.  EFFECT ON SAFETY EVALUATION (3) 
 
3.1:   How to evaluate the effect of blood sampling on the toxicity data and well-being of animals 
in the main study group? 
3 
  Contains Nonbinding Recommendations 
 
 
 
When blood sampling is performed on the main study animals, it is important to consider the 
effect of blood collection on the physiological condition of the animals.  The main factors to 
consider include: (1) the volume and the number of samples taken in a given period; (2) the 
properties of the test drug (e.g., its effects on red blood cells, anticoagulant effect, or 
hemodynamic properties); (3) the test system (e.g., species, age, body weight, total blood 
volume); and (4) the site of collection.  Because frequent and repeated blood collection may 
affect physiological data, such as hematological parameters, sampling protocols should be 
appropriately established, even with microsampling.  It is prudent to record the relevant animal 
data, such as changes in body weight, food consumption, hematological parameters (e.g., red 
blood cell count, hemoglobin level, hematocrit value, mean corpuscular volume, electrolytes, 
total proteins), and any effect on the blood collection site (e.g., tissue damage, inflammation).  
Evaluation of these parameters compared to matching control animals, which have had the same 
number and volume of samples drawn as the test drug groups, will be important to establish 
whether any suspected effects are related to the test drug or to procedures, within the context of 
the specific study conditions.  If previous studies show that test-drug-related changes to 
hematological parameters could be exacerbated by frequent blood sampling or if it is suspected 
that the pharmacological action of the test drug may induce such effects, the use of satellite 
groups of animals for TK assessment would be warranted, even if microsampling techniques are 
used. Alternatively, sparse sampling4 can be used in conjunction with microsampling, if 
scientifically justified.   
 
 
IV.  ISSUES REGARDING THE BIOANALYTICAL METHOD (4) 
 
4.1:    What are important points to consider in bioanalytical method development and 
validation of treatment of liquid or dried samples? 
 
In addition to the analytical method validation stipulated in the bioanalytical guideline/guidance 
in each regulatory region, the following points should be considered when analyzing samples 
derived from microsampling: 
  For liquid sampling, the following issues should be considered: (1) ensuring sample 
homogeneity, for example by pipetting;  (2) small volume handling issues (e.g., 
potential freezing/drying effects during the storage and subsequent freeze/thaw process, 
as applicable);  (3) potential increase in the LLOQ due to the limited sample volume; (4) 
impact of addition of anticoagulants to small containers/capillaries, resulting in dilution 
of the sample; (5) potential for increased adsorption of the analyte to the collection 
container (i.e., increased surface area to volume ratio); (6) maintenance of the 
appropriate preservation conditions of the sample; (7) risk of contamination and 
difficulty of repeated sampling using some methods.  
                                                            
4 Sparse sampling in TK studies usually involves the collection of a few blood samples at specified time points from each 
animal in a treatment group. The samples are allocated at different time points for different animals, often allowing some 
replicates, and statistical inferences are then made about the concentration-time behavior of the test compound.  By using 
proper study designs, investigators can limit the number of samples and the amount of blood drawn, so as not to affect the 
animals' health status, yet still achieve the customary TK objectives in a study. 
4 
  Contains Nonbinding Recommendations 
 
 
  For dried sampling techniques (e.g., spotting onto cellulosic or noncellulosic card, 
polymer matrix, etc.), it is important to select the method with adequate and 
reproducible recovery and minimal matrix interference with detection of the drugs.  If 
the subpunch of the dried spot approach is used, it is important to ensure that detection 
of the analyte is not affected by different hematocrit values, especially for small 
molecule drugs. The effect of hematocrit on analyte detection can be measured by using 
blood with different hematocrit values and spiking with test drugs of known 
concentrations.  It is also important to confirm the uniformity of the spots by evaluating 
analyte levels from multiple samples punched out from one spot or by evaluation, for 
example, using radiolabels.  Both of these issues can be minimized if an accurate 
volume of blood is collected on the device and the entire sample is subsequently 
analyzed.  
 
Incurred sample reanalysis (ISR) should be conducted according to each regional 
guidance/guideline, if described.  When doing ISR, care should be taken to ensure sufficient 
sample volumes or numbers of replicates (e.g., spots, containers, or tips) are retained for ISR.   
5 
  Contains Nonbinding Recommendations 
 
 
V.  ANNEX:  Q&As LINKED TO THE RESPECTIVE SECTIONS OF S3A GUIDANCE 
 
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SSe3cAt iGonusi doaf n ce  : Introduction  : The objective of toxicokinearameters which may deter : General principles to be co : Toxicokinetics in the variooxicity testing  specific aspe : Supplementary notes  : References (other ICH gui
1 2p 3 4t 5 6
1.1 (Q1)  1    3.10    Note 1   
1.2 (Q2)  1     3.5    Note 1   
2.1 (Q3)    2    4     
 
2.2 (Q4)      3.1       
3.10 
2.3 (Q5)      3.10       
3.1 (Q6)      3.3       
3.5 
4.1 (Q7)      3.10    Note 1   
 
6