ADDENDUM TO ICH S6: 

PRECLINICAL SAFETY EVALUATION OF 

BIOTECHNOLOGY-DERIVED 

PHARMACEUTICALS 

S6(R1) 

This draft guidance, when finalized, will represent the Food and Drug 

Administration’s (FDA’s) current thinking on this topic. It does not create or 

confer any rights for or on any person and does not operate to bind FDA or the 

public. You can use an alternative approach if the approach satisfies the 

requirements of the applicable statutes and regulations. If you want to discuss

an alternative approach, contact the FDA staff responsible for implementing this 

guidance. If you cannot identify the appropriate FDA staff, call the appropriate 

number listed on the title page of this guidance. 

For questions regarding this draft document contact (CDER) Anne M. Pilaro 301-796-

2320, or (CBER) Mercedes A. Serabian 301-827-5377. 

i 
 

 

INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL 


REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN
 

USE
 

ICH HARMONISED TRIPARTITE GUIDELINE
 

ADDENDUM TO ICH S6: PRECLINICAL SAFETY EVALUATION OF
 

BIOTECHNOLOGY-DERIVED PHARMACEUTICALS
 

S6(R1) 

Current Step 2 version 

Dated October 29 2009 

At Step 2 of the ICH Process, a consensus draft text or guideline, agreed by the 

appropriate ICH Expert Working Group, is transmitted by the ICH Steering 

Committee to the regulatory authorities of the three ICH regions (the European 

Union, Japan and the USA) for internal and external consultation, according to 

national or regional procedures. 

i 
 

 

ADDENDUM TO ICH S6: PRECLINICAL SAFETY EVALUATION
 

OF BIOTECHNOLOGY-DERIVED PHARMACEUTICALS 


S6(R1) 


Current Step 2 version 

Dated October 29 2009 

Having reached Step 2 of the ICH Process at the ICH Steering Committee 

meeting on October 29, 2009, this guideline is recommended for release for 

internal and external consultation, according to national or regional procedures 

of the three regulatory parties to ICH. 

TABLE OF CONTENTS 

1. INTRODUCTION ................................................................................... 5
 

1.1 Purpose of the Addendum ......................................................................5
 

1.2 Background.............................................................................................5
 

1.3 Scope of the Guideline............................................................................5
 

2. SPECIES SELECTION........................................................................... 5
 

2.1 General Principles ..................................................................................5
 

2.2	 Tissue Cross-Reactivity:.........................................................................6
 

2.3	 One or Two Species.................................................................................7
 

I2.4	 Use of Homologous Proteins, Transgenic Models, KOs and 

Disease Models: ......................................................................................7
 

3. STUDY DESIGN..................................................................................... 7
 

3.1 Dose Selection and application of PK/PD Principles ............................7
 

3.2 Duration of Studies ................................................................................8
 

3.3 Recovery..................................................................................................8
 

3.4 Exploratory Clinical Trials ....................................................................8
 

4. IMMUNOGENICITY .............................................................................. 8
 

5. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY..................... 9
 

5.1 General Comments.................................................................................9
 

ii 
5.2 Fertility .................................................................................................10
 

5.3 Embryo-fetal and Pre/Post-Natal Development..................................10
 

5.4 Timing of studies ..................................................................................11
 

6. CARCINOGENICITY ........................................................................... 11
 

7. ENDNOTES .......................................................................................... 13
 

8. REFERENCES...................................................................................... 14
 

iii 
LIST OF ABBREVIATIONS
 

ADA Anti-drug antibodies 

ADC Antibody drug conjugate 

ePPND Enhanced pre- and post-natal development 

EU European Union 

GD Gestational Day 

ICH International Conference on Harmonisation of Technical 

Requirements for Registration of Pharmaceuticals for Human Use 

i.v. Intravenous 

NHP Non Human Primate 

PK Pharmacokinetics 

PD Pharmacodynamics 

WOCBP Women of Childbearing Potential 

iv 
ADDENDUM TOICHS6: PRECLINICAL SAFETY EVALUATION OF 


BIOTECHNOLOGY-DERIVED PHARMACEUTICALS
 

S6(R1) 


1. INTRODUCTION 

1.1 Purpose of the Addendum 

The purpose of the addendum is to provide clarification on and an update of the 

following topics discussed in the original ICH S6 guidance: species selection, 

study design, immunogenicity, reproductive and developmental toxicity and 

assessment of carcinogenic potential. Scientific advances and experience gained 

since publication of the original ICH S6 guidance call for this addendum. 

This harmonised addendum will help to define the current recommendations and 

reduce the likelihood that substantial differences will exist among regions. 

This guidance should facilitate the timely conduct of clinical trials, reduce the use 

of animals in accordance with the 3Rs (reduce/refine/replace) principles and 

reduce the use of other drug development resources. Although not discussed in 

this guidance, consideration should be given to use of new in vitro alternative 

methods for safety evaluation. These methods, if validated and accepted by all 

ICH regulatory authorities, can be used to replace current standard methods. 

This guidance promotes safe and ethical development and availability of new 

pharmaceuticals. 

1.2 Background
 

The recommendations of this addendum further harmonise the nonclinical safety 

studies to support the various stages of clinical development among the regions of 

European Union (EU), Japan, and the United States. The present guidance 

represents the consensus that exists regarding the safety evaluation of 

biotechnology-derived pharmaceuticals. 

1.3 Scope of the Guideline 


This addendum does not alter the scope of the original ICH S6 guideline. 

2. SPECIES SELECTION 

2.1 General Principles 

A number of factors should be taken into account when determining species 

relevancy. Comparisons of target sequence homology between species can be an 

appropriate starting point, followed by cell-based assays to make qualitative and

quantitative cross-species comparisons of relative target binding affinities and 

receptor/ligand occupancy and kinetics. Assessments of functional activity are 

also recommended. Functional activity can be demonstrated in species-specific 

5
 
cell-based systems and/or in vivo pharmacology or toxicology studies. Modulation 

of a known biologic response or of a pharmacodynamic marker can provide 

evidence for functional activity to support species relevance. 

Consideration of cross-species differences in target binding and functional

activity in the context of the intended dosing regime should provide confidence 

that a model is capable of demonstrating any potentially adverse consequences of 

target modulation. When the target is not constitutively expressed in typical 

preclinical species, binding affinity and activity in cell-based systems can be 

sufficient to guide species selection. 

For monoclonal antibodies and related products directed at foreign targets (i.e. 

bacterial, viral, etc.), it is desirable to evaluate safety in an animal model of 

disease. Where this is not feasible, a short-term safety study (see ICH S6) in one 

species (choice of species to be justified by the sponsor) can be considered and 

appropriate risk mitigation strategies should be adopted for clinical trials. No 

additional toxicity studies are appropriate. 

As described in ICH S6, when no relevant species can be identified because the

biopharmaceutical does not interact with the orthologous target in any species,

use of homologous molecules, transgenic models and/or animal models of disease 

can be considered. 

The potential for toxicity arising from a novel toxin or toxicant incorporated as an

antibody-drug/toxin conjugate (ADC) is likely to occur in a target-independent 

manner and should be assessed in two species (one rodent and one non-rodent). 

Where possible, preference should be given to species that exhibit target-specific 

binding. For toxins or toxicants which are not novel and for which there is a 

sufficient body of scientific information available, safety evaluation of ADC in a 

single relevant species should suffice. 

2.2 Tissue Cross-Reactivity: 

Based on recent scientific data, the text in ICH S6, Section 3.3 paragraph 2 is no

longer appropriate. Immunohistochemical examination of potential binding of 

monoclonal antibodies and related products to the target epitope (tissue cross 

reactivity) should not be used for selection of relevant species for safety

evaluation. Other techniques that assess target expression (e.g. in situ 

hybridization, flow cytometry) can provide supportive information for species 

selection. 

However, tissue cross-reactivity data with human tissues can provide useful 

information to supplement knowledge of target distribution and can provide 

information on unexpected epitope binding. Tissue cross reactivity studies in

nonclinical species are considered to have limited value and therefore are not 

generally recommended. 

Binding to areas not typically accessible to the biopharmaceutical in vivo i.e. 

cytoplasm might not be relevant. 

6
 
For bi-specific antibodies, evaluating each binding site separately in this assay is 

not called for. 

2.3 One or Two Species 

If there are two pharmacologically relevant species for the clinical candidate (one

rodent and one non-rodent), then both species should be used for short-term (up

to 1 month duration) toxicology studies. If the toxicological findings from these 

studies are similar in both species, then longer-term studies in one species are 

usually considered sufficient; the rodent species should be considered unless 

there is a rationale for using non-rodents. Studies in two non-rodent species are 

not appropriate. 

The use of one species is justified when the biological activity of the

biopharmaceutical is well understood or the clinical candidate is 

pharmacologically active in only one species. Studies in a second species with a

homologous product are not considered to add further value for risk assessment

and are not recommended. 

2.4 Use of Homologous Proteins, Transgenic Models, KOs and Disease Models: 

In some cases, alternative approaches to evaluating safety in a 

pharmacologically-relevant animal species should be used. ICH S6 section 3.3 

can be referenced for further information. 

Homologous proteins can be used for hazard detection and understanding the 

potential for adverse effects due to exaggerated pharmacology, not for exposurebased quantitative risk assessment. It can be possible to conduct safety

evaluation studies with homologous proteins using a control group and one dose 

group provided there is a scientific justification for the dose level selected. 

3. STUDY DESIGN 

3.1 Dose Selection and application of PK/PD Principles 

The toxicity of most biopharmaceuticals is related to their targeted mechanism of 

action; therefore, relatively high doses can elicit adverse effects which are 

apparent as exaggerated pharmacology. 

A rationale should be provided for high dose selection. PK-PD approaches can

assist in high dose selection by identifying i) a dose which gives the maximum 

intended pharmacological effect in the preclinical species and ii) a dose which 

gives an up to 10-fold exposure multiple over the maximum exposure to be 

achieved in the clinic. The highest of these two doses should be chosen as the 

high dose group in pre-clinical toxicity studies unless scientific data supports a

lower dose. 

Where PD endpoints are not available, then an up to 10-fold multiple over the 

highest anticipated clinical exposure is sufficient, provided that corrections are 

made for differences in target binding and in vitro pharmacologic activity 

7
 
between the nonclinical species and humans. For example, a large relative

difference in binding affinity and/or in vitro potency might suggest the need for

higher doses in the nonclinical studies. In the event that toxicity cannot be

demonstrated by this approach, then additional toxicity studies at higher

multiples of human dosing are unlikely to provide additional useful information. 

3.2 Duration of Studies 

For chronic use products, the adequacy of 6-month chronic studies is supported 

by the scientific experience with biopharmaceuticals to date. Studies of longer 

duration are not anticipated to provide useful information to change the clinical 

course of development. 

3.3 Recovery 

Recovery of pharmacological and toxicological effects with potential adverse

clinical impact should be understood. This information can be obtained by 

including a non dosing period in at least one study. The purpose of the non

dosing period is to examine reversibility of these effects, not to assess delayed 

toxicity. The demonstration of complete recovery is not considered essential. An 

evaluation of recovery is not warranted if there are no adverse effects at the end 

of the dosing period or sufficient scientific justification can be provided (e.g.

evidence that an adverse effect is generally reversible, or an adequate margin of

safety exists for the proposed clinical population). 

The addition of a recovery period just to assess for immunogenicity is not

appropriate. 

3.4 Exploratory Clinical Trials 

The flexible approaches to support exploratory clinical trials as outlined in ICH 

M3 (R2) can be applicable to biopharmaceuticals. It is recommended that these 

approaches be discussed and agreed upon with the appropriate regulatory

authority. 

4. IMMUNOGENICITY 

Immunogenicity assessments are conducted to assist in the interpretation of the

study results and design of subsequent studies. Such analyses in nonclinical 

animal studies are not relevant in terms of predicting potential immunogenicity 

of human or humanized proteins in humans. 

Measurement of anti-drug antibodies (ADA) in nonclinical studies is not 

routinely warranted if there is evidence of sustained pharmacodynamic activity, 

no unexpected changes in the pharmaco/toxicokinetics of the test article during 

the dosing or recovery phase, and/or no evidence of immune-mediated reactions 

(immune complex-related, vasculitis, anaphylaxis, etc.). However, it is difficult to 

predict whether such analysis will be called for prior to completion of the in-life 

8
 
phase of the study; therefore, it is often useful to obtain appropriate samples 

during the course of the study, which can subsequently be analyzed if needed to 

aid in interpretation of the study results. When study results suggest there is a

need to understand immunogenicity to interpret study data, potential for 

immunogenicity antibody detection assays should be conducted to evaluate the 

presence of ADAs. When ADAs are detected, the effect on the study results 

should be assessed, including effects on PK and drug clearance, pharmacology 

effects, and toxicity. Characterization, specifically of neutralizing potential, is 

generally not warranted, particularly if adequate exposure and pharmacological 

effect can be demonstrated by a pharmacodynamic marker of activity in the in

vivo toxicology studies. In the event that neutralizing antibody assessment is 

deemed appropriate to interpret the study findings, assessment of neutralizing 

activity can be addressed indirectly with ex-vivo bioactivity assay, a combination 

of assay formats for PK-PD, or directly in a specific neutralizing antibody assay. 

5. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY 

5.1 General Comments 

Reproductive toxicity studies should be conducted in accordance with the 

principles outlined in with ICH S5(R2); however, the specific study design and 

dosing schedule can be modified based on an understanding of species specificity, 

the nature of the product and mechanism of action, immunogenicity and/or

pharmacokinetic behaviour and embryo-fetal exposure. 

Note 2 of ICH S6 still applies with respect to products where there is public 

knowledge regarding toxicity to reproduction. 

The evaluation of toxicity to reproduction should be conducted only in 

pharmacologically relevant species. When the clinical candidate is 

pharmacologically active in rodents and rabbits, these species should be used 

unless there is a scientific reason to use a NHP. As per ICH S5 Note 5 (2.1), one

species can be sufficient to address effects on embryo-fetal development if there is 

more than one relevant species (see section on species selection). The sponsor 

should provide a scientific rationale for selection of species for assessment of 

effects on embryo-fetal development. 

When the clinical candidate is pharmacologically active only in NHP, an 

appropriate assessment of reproductive toxicity in NHPs is generally preferred 

over alternative approaches such as studies with homologous products in other 

species. However, the sponsor should provide a scientific justification when these

alternative approaches are proposed. When the weight of evidence (e.g.

mechanism of action, phenotypic data from KO mice, class effects) suggests that

there will be an adverse effect on pregnancy outcome, these data might provide

adequate information to communicate risk, and additional nonclinical studies 

might not be warranted. 

9
 
When no relevant animal species exists for the clinical candidate, the use of 

transgenic mice expressing the human target or homologous protein in a species 

expressing the human ortholog should be considered. 

5.2 Fertility 

For products where rodents are a relevant species, an assessment of fertility can

be made in a rodent species. The design of the study should be amended as

appropriate, for example, to address the nature of the product and potential for 

immunogenicity. 

When the NHP is the only relevant species, the potential for effects on male and 

female fertility can be assessed by standard histopathological evaluation and 

assessment of menstrual cyclicity in repeat dose toxicity studies of at least 3

months duration using sexually mature NHPs. If there is a specific cause for 

concern, specialized assessments such as sperm count, sperm 

morphology/motility, testicular volume, and male or female reproductive

hormone levels should be evaluated in the repeat dose toxicity study. 

It is recognized that mating studies are not practical for NHPs. If there is a 

specific concern from the pharmacological activity about potential effects on 

conception / implantation and the NHP is the only relevant species, the concern

should be addressed experimentally. A homologous product or transgenic model

could be the only practical means to assess potential effects on conception or 

implantation when those are of specific concern. However, it would not be 

appropriate to produce a homologous product or transgenic model only to conduct

mating studies in rodents. 

5.3 Embryo-fetal and Pre/Post-Natal Development 

The type of molecule and potential differences in placental transfer should be 

considered in the choice of species for testing – see note 1. 

For products pharmacologically active only in NHPs, one well-designed study in 

NHPs (stage C to E ICH S5a) which includes dosing from day 20 of gestation to 

birth can be considered. It is also possible for the sponsor to provide a scientific 

justification for the evaluation of effects on embryo-fetal and postnatal

development using alternative study designs or a homologous product in rodents. 

For the single NHP study design addressing ICH S5a stages C to E, no caesarian 

section group is warranted, but assessment of pregnancy outcome at natural

delivery should be performed. This study should also evaluate offspring viability 

and survival, external malformations, skeletal effects (e.g. by X-ray) and, 

ultimately, visceral morphology at necropsy. Ultrasound is useful to track 

maintenance of pregnancy but not for monitoring embryo-fetal development or 

detecting malformations. Other endpoints in the offspring can also be evaluated 

if relevant for the pharmacological activity (e.g. immune function or 

neurobehavioural assessment). The duration of the postnatal phase will be 

dependent on which additional endpoints are considered relevant for the 

pharmacological activity. 

10
 
The numbers of confirmed pregnant cynomolgus monkeys per group should be 

sufficient to detect 3-fold increase in pregnancy/parturition-associated losses with 

80% power with 95% confidence interval (Jarvis P et al, abstract European 

Teratology Society Meeting, 2009). The sponsor should justify the study design if 

other NHP species are used. 

Because the developmental toxicity study in NHP as outlined above is a hazard

identification study, it might be possible to conduct these studies using a control

group and one dose group, provided there is a scientific justification for the dose

level selected. An example of an appropriate scientific justification would be a 

monoclonal antibody which binds a soluble target and the clinical dosing regimen 

is intended to saturate target binding. If such a saturation of target binding can 

be demonstrated in the animal species selected and there is an up to 10-fold

exposure multiple over therapeutic drug levels, a single dose level and control 

group would provide adequate evidence of hazard to embryo-fetal development. 

These study design principles can also be more appropriate than separate ICH

S5a C and D to E studies for biopharmaceuticals other than monoclonal

antibodies. 

5.4 Timing of studies 

For monoclonal antibodies for which embryo-fetal exposure during organogenesis 

is understood to be low in humans based on current scientific knowledge, the

embryo-fetal development toxicity study can be conducted during Phase III (see

ICH M3 (R2)). The completed reports should be available to support submission 

of a marketing application. For other biological products where embryo-fetal

exposure is demonstrated to be low during organogenesis, the same timing for 

testing can be applicable. Where there is embryo-fetal exposure during 

organogenesis and the product is pharmacologically active only in NHPs and a 

sponsor elects to use the ePPND study design, an interim report (see note 2) for 

data to day 7 post-partum for all animals is called for to support Phase III. 

If the rodent or rabbit is a relevant species and embryo-fetal exposure is 

demonstrated, see ICH M3 (R2) for timing of reproductive toxicity studies. ICH 

M3 (R2) should also be followed for the timing of data on fertility for products 

where rodents are relevant species. 

For oncology products which fall within the scope of ICH S9, see this guidance for 

aspects relating to timing. 

6. CARCINOGENICITY 

The need for a product-specific assessment of the carcinogenic potential for 

biopharmaceutical should be determined with regard to the intended clinical

population and treatment duration (see ICH S1a). When an assessment is 

warranted, the sponsor should design a strategy to address the issue. 

11
 
This strategy could be based on a review of relevant data from a variety of 

sources. The data sources can include published data (e.g. information from 

transgenic, knock-out or animal disease models, human genetic diseases), 

information on class effects, detailed information on target biology, in vitro data, 

data from chronic toxicity studies and clinical data. 

The product specific assessment of carcinogenic potential is used to communicate

risk and provide input to the risk management plan along with labelling 

proposals, clinical monitoring, post-marketing surveillance, or a combination of

these approaches. 

In some cases, the available information can be considered sufficient to address 

carcinogenic potential and inform clinical risk without warranting additional 

nonclinical studies. For example, immunomodulators and growth factors pose a 

potential carcinogenic risk which can best be evaluated by post-marketing

clinical surveillance rather than further nonclinical studies. 

The mode of action of some biopharmaceuticals might raise concern regarding 

potential for neoplasm induction or tumour promotion. Rodent bioassays are not

warranted if data from in vitro or chronic toxicity studies support the concern 

regarding carcinogenic potential. When in vitro and chronic toxicity studies do

not support this theoretical risk but the sponsor prefers not to label on this basis, 

the sponsor can propose additional studies that could mitigate the concern. 

For products where there is insufficient knowledge about specific product 

characteristics and mode of action in relation to carcinogenic potential, a more

extensive assessment might be appropriate. The sponsor should consider the 

inclusion of additional endpoints in toxicity studies. If the weight of evidence 

from the product-specific assessment (e.g. in vitro and chronic toxicity studies)

does not suggest carcinogenic potential, a rodent bioassay is not recommended. If 

the weight of evidence suggests a concern about carcinogenic potential, then the 

label should reflect the concern or the sponsor can propose additional nonclinical 

studies that could mitigate the concern. 

Rodent bioassays or short-term carcinogenicity studies with homologous products 

are generally of limited value to assess carcinogenic potential of the clinical 

candidate. 

There is clearly a need for better assessment tools. Alternative approaches can

be considered as new strategies / assays are developed. 

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7. ENDNOTES 

Note 1 

High molecular weight proteins (>5,000 D) do not cross the placenta by simple

diffusion. For monoclonal antibodies with molecular weight as high as 150,000 

D, there exists a specific transport mechanism, (FcRn) which determines fetal 

exposure and varies across species. 

In the NHP and human, IgG does not begin to cross the placenta until early 

second trimester and increases to higher levels late in the third trimester; thus, 

in NHPs and humans, IgG crosses the placenta only after organogenesis 

(Pentsuk and Van der Laan, 2009). Therefore, standard embryo-fetal studies in

NHPs, which are dosed from early pregnancy up to Gestation Day 50, are not 

representative of human fetal exposure throughout pregnancy for a parenterally 

administered therapeutic IgG and might assess only indirect effects on embryofetal development. Therefore, this might not be the optimal study design to 

assess indirect and direct effects of treatment throughout gestation. 

IgG crosses the yolk sac in rodent/rabbits by FcRn transport mechanism and 

exposure will occur during late organogenesis. In addition, offspring of rat/mouse

dams dosed during lactation will be exposed via the milk. 

Note 2 

Endpoints to be included in interim report of ePPND study in non human 

primates; 

Dam data: survival, clinical observations, bodyweight, gestational exposure data 

(if available), any specific PD endpoints; 

Pregnancy data: number of pregnant animals started on study, pregnancy status

at end of organogenesis (GD50) and at GD100 as a minimum, abortions and 

timing of abortions. There is no need for ultrasound observations of fetal size in 

the interim report; these are not considered essential since actual birth weight

will be available; 

Pregnancy outcome data: number of live births / still births, infant birth weight, 

infant survival and bodyweight at day 7 post-partum, qualitative external 

morphological assessment (i.e. confirming appearance is within normal limits), 

infant exposure data (if available), any specific PD endpoints in the infant if 

appropriate. 

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8. REFERENCES 

1.	 ICH Topic S6 Document “Safety Studies for Biotechnological Products” 

2.	 ICH Topic S5(R2) Document “Detection of Toxicity to Reproduction for 

Medicinal Products and Toxicity to Male Fertility” 

3.	 ICH Topic S1A Document “Guideline on the Need for Carcinogenicity 

Studies for Pharmaceuticals” 

4.	 ICH Topic M3(R2) Document “Guideline on Nonclinical Safety Studies for 

the Conduct of Human Clinical Trials and Marketing Authorisation for 

Pharmaceuticals” 

5.	 ICH Topic S9 Document “Nonclinical Evaluation for Anticancer 

Pharmaceuticals” 

6.	 Pentsuk N, Van der Laan JW. An Interspecies Comparison of Placental 

Antibody Transfer: New Insights Into Developmental Toxicity Testing of

Monoclonal Antibodies. Birth Defects Research (Part B) 2009; 86: 328-344 

7.	 Jarvis P et al, abstract European Teratology Society Meeting, 2009 

14