2013 ets ce course materials (stresa italy)

8 September 2013
14.00 – 17.30 STRESA, ITALY
41ST
ANNUAL
MEETING OF
THE
EUROPEAN
TERATOLOGY
SOCIETY
COURSE SPONSORED BY:
ETS Education Course: “Testing Strategies for
Biopharmaceuticals”

ETS Education Course:
“Testing Strategies for
Biopharmaceuticals”
8 S E P T E M B E R 2 0 1 3
PROGRAMME
14.00 - 15.00: INTRODUCTION INTO
BIOPHARMACEUTICALS
Manon Beekhuijzen, WIL Research Europe, the
Netherlands
15.00 – 16.00: THE NON-CLINICAL TESTING OF VACCINES
Paul Barrow, Roche, Switzerland
16.00 - 16.30: COFFEE/TEA BREAK
16.30 - 17.30: USE OF NONHUMAN PRIMATES FOR
BIOLOGICS DART
Gary Chellman, Charles River Laboratories, USA
SESSION CHAIRS:
Alan M. Hoberman
Manon Beekhuijzen
Note: Each topic will include
~15 minutes for questions and
discussion

ETS Education Course: “Testing Strategies for Biopharmaceuticals”
Page 1
SPEAKER INFORMATION AND ABSTRACTS
INTRODUCTION INTO BIOPHARMACEUTICALS
Speaker: Manon Beekhuijzen
Affiliation: WIL Research Europe, the Netherlands
After graduating as doctorandus in Medical Biology at the University of Utrecht and performing her
traineeships and master thesis in the field of reproduction and developmental toxicology (DART), Manon
started at the Toxicology department of WIL Research (then named NOTOX B.V.) in 1999. In 2001 she was
appointed study director at WIL Research for all types of DART studies. She obtained her Masters degree in
Applied Toxicology at the University of Surrey (England) in 2004. In 2007 she started a working group for
Belgium and Dutch companies to discuss the technical aspects of non-clinical DART testing. From 2011,
onwards she is a council member of the NVT (Nederlandse Vereniging voor Toxicologie) reprotox section
and the ETS (European Teratology Society).
ABSTRACT
The principles of DART (developmental and reproductive toxicity) testing are the same as those for all
preclinical toxicology studies: to identify potential hazards to patients. However, DART studies are somewhat
unique in that controlled human clinical trials, which assess reproduction and development, are rarely
performed. Therefore, preclinical DART testing forms a fundamental basis for risk assessment.
A segmented approach to DART testing is most frequently conducted in which fertility, embryo/fetal
development (EFD), and pre/postnatal development are assessed in three separate studies in the rat; an EFD
study will also be performed in the rabbit. This most common option is described in ICH S5 (R2). However,
the guidance document also gives suggested options for combining the three segments into one or two
studies.
These principles, as described in the ICH S5 guideline, are also applicable for biopharmaceuticals. However,
because of species- specificity alternate approaches may be needed. And in that case you can follow the ICH S6
guideline. In addition to these ICH guidelines, there is an FDA guidance for developmental toxicity testing of
preventive and therapeutic vaccines.
In May 2012, the ICH S6 guidance for industry was updated with an addendum to clarify several subjects
discussed in the guidance of 1997, including DART. The need for DART testing depends on the type of
product, clinical indication and the intended patient population. The specific study design and dosing schedule
may be modified based on issues related to species specificity, immunogenicity, nature of the product,
mechanism of action, pharmacokinetic behaviour, and embryo-fetal exposure. The ICH S6 guidance mentions
when the clinical candidate is pharmacologically active in rodents and rabbits, both species should be used for
EFD studies. Developmental toxicity studies should only be conducted in NHP when they are the only
relevant species. When no relevant animal species exists for testing the clinical candidate, the use of
transgenic mice expressing the human target or homologous protein can be considered.
A stepwise approach to selecting the optimal preclinical DART package for a biopharmaceutical will be
discussed. First, the most relevant test species must be selected. The relevant test species needs to fulfill four
criteria: acceptable species cross-reactivity, appropriate pharmacology/efficacy, acceptable immunogenicity
and acceptable exposure. These criteria apply to the overall preclinical package, so it is not only applicable for

Page 2
DART. However, there are certain specifics for DART that need to be taken into consideration when going
through these criteria.
Based on the most relevant animal species, there are five possible approaches for DART testing of a
biopharmaceutical: use of traditional animal species, non-traditional animal species, genetically modified
animals or surrogate molecules, or to do no DART testing.
There is one additional approach which was developed especially for preventive and therapeutic vaccines. The
EMEA guidance of 1997 mentions that embryo-fetal and/or perinatal studies may be necessary for vaccines
that will be given to WOCBP or during pregnancy, but it gives no guidance on study design. Guidance was
issued by the FDA in 2006, and is currently used as standard study design for vaccines. The reasons for the
need of this adjusted study type are that vaccines are intended to give an immune response, vaccines are
usually administered in limited, episodic dosages with months or even years between doses, and these may be
adjuvanted.
In conclusion, a scientific-based case-by-case approach is considered the most appropriate strategy for
preclinical safety assessment of biopharmaceuticals. There is not one standard approach applicable per
biopharmaceutical subclass. While the stepwise approach for non-clinical safety assessment for general
toxicity and DART is similar, there are DART specifics that need to be considered for safety testing.
THE NON-CLINICAL TESTING OF VACCINES
Speaker: Paul Barrow
Affiliation: Roche, Switzerland
Paul Barrow has just started work in his fourth European country, having been recruited recently by Roche in
Switzerland to take over as Global Head of Reproductive Toxicology when the current occupant retires in a
few months. He previously worked as a regulatory toxicologist at Beecham Pharmaceuticals (UK), Roma
Toxicology Centre (Italy), MDS (France) and CiToxLAB (France). Currently Vice President of the ETS, he
will assume presidential office in 2014. Paul’s latest book on Teratogenicity Testing was published this year;
he will probably try to sell you a copy (beware).
ABSTRACT
Preventative and therapeutic vaccines are increasingly used during pregnancy and present special
considerations for developmental toxicity testing. The various components of the vaccine formulation (i.e.
protein or polysaccharide antigen, adjuvants and excipients) need to be assessed for direct effects on the
developing conceptus. In addition, possible adverse influences of the induced antibodies on fetal and/or post-
natal development need to be evaluated. A guidance document on the preclinical testing of preventative and
therapeutic vaccines for developmental toxicity was issued by the FDA in 2006. Preclinical studies are
designed to assess possible influences of vaccines on pre- and post-natal development. The choice of model
animal for these experiments is influenced by species differences in 1) immunogenicity and 2) the timing and
extent of transfer of the induced maternal antibodies to the fetus. The cross-placental transport of maternal
immunoglobulins generally only occurs in late gestation and tends to be greater in humans and monkeys than
in non-primate species. For many vaccines, the rabbit shows a greater rate of prenatal transfer of the induced
antibodies than rodents. For biotechnology-derived vaccines that are not immunogenic in lower species, non-
human primates may be the only appropriate models. It may be advisable to test new adjuvants using the ICH

Page 3
study designs for conventional pharmaceuticals in addition to the developmental toxicity study with the final
vaccine formulation. Possible effects of vaccines on fertility may also need to be considered. At present, there
are no formal requirements for preclinical juvenile studies, even though the majority of vaccines are intended
for children.
Reference: Barrow (ed.) 2013, Teratogenicity Testing: Methods and Protocols, DOI 10.1007/978-1-62703-131-8_7.
USE OF NONHUMAN PRIMATES FOR BIOLOGICS DART
Speaker: Gary Chellman
Program Director, Developmental and Reproductive Toxicology
Affiliation: Charles River Nonclinical Services, Nevada USA
Gary earned his Ph.D. in toxicology from the University of Rochester in 1984, followed by postdoctoral
training at the Chemical Industry Institute of Toxicology. Over the next 20+ years Gary has worked in
pharmaceutical toxicology, with specialty in reproductive toxicology. From 1986-1996, he was employed by
Syntex Pharmaceuticals where he advanced to Department Head of Reproductive Toxicology. For the next 3
years, he was Director of Toxicology at Sanofi Pharmaceuticals. Gary joined Sierra Biomedical (now Charles
River Preclinical Services) in 1999, as Vice President of Toxicology. Since 2003, he has been the Program
Director for Developmental and Reproductive Toxicology, specializing in the design, conduct and
interpretation of nonhuman primate reproductive toxicology studies. Gary is a diplomat of the American
Board of Toxicology and a member of the Society of Toxicology, the American College of Toxicology, and the
Teratology Society. He has authored/co-authored numerous publications in reproductive toxicology, most
recently related to optimizing these studies in nonhuman primates to support biopharmaceutical drug
development.
ABSTRACT
Conduct of reproductive toxicology studies in nonhuman primates has increased in recent years, especially for
biopharmaceuticals based on considerations of pharmacologic responsiveness, immunogenicity, and the ability
to test the clinical drug candidate. Initially these studies reflected standard reproductive toxicology designs
and were conducted as stand-alone segmented studies. Recent changes in regulatory guidelines (ICH S6 (R1))
and growing emphasis on the 3Rs have resulted in new study designs that are improved scientifically while
substantially reducing the number of animals required. This presentation will review these various study
designs and discuss current trends in the conduct of NHP developmental and reproductive toxicology
(DART) studies. Challenges in interpretation of NHP DART data will be discussed, including fetal loss rates
and endpoints for which interpretation is complicated by background changes during pregnancy in
cynomolgus monkeys, the most commonly used species. Some key safety findings from NHP repro testing
will be presented. Specialty areas including developmental immunotoxicology (DIT) and juvenile toxicology
will be covered, including the advantages and disadvantages of working with NHPs for these types of studies.
The goal of this presentation will be to enhance the ability of course participants to approach NHP DART
study design and implementation in an effective, case-by-case manner.

Introduction into
biopharmaceuticals and DART
Manon Beekhuijzen
ETS EDUCATION COURSE 2013

Aim of presentation
Overview of stepwise approach to follow on
preclinical DART testing for biopharmaceuticals
 Regulations and considerations

Introduction
Developmental
And
Reproductive
Toxicity
testing
→ Basis for risk assessment

Introduction DART
One complete life cycle

Premating to
Conception
Conception
to
Implantation
Implantation
to Closure
of hard
palate
Hard palate
closure to
End of
pregnancy
Birth to
Weaning
Weaning to
Sexual
maturity
DOSING
DOSING
DOSING

Traditional ‘segmented’ approach (ICH S5 (R2))
I. Fertility and early embryonic development (FEED)
II. Embryo-fetal development (EFD)
III. Pre- and postnatal development (PPND)

Introduction
biopharmaceuticals

Definition biopharmaceuticals
 Biotechnology-derived pharmaceuticals
 Biotechnology is:
 the use of living systems and organisms to develop useful products,
or
 any technological application that uses biological systems, living
organisms or derivatives thereof, to make or modify products or
processes for specific use

Range of biopharmaceutical classes
Biopharmaceutical class Example
Hormones Insulin
Blood products Albumin
Cytokines and growth factors Interferons
Antagonists/inhibitors Soluble receptors
Monoclonal antibodies Mouse, chimeric or humanized
Modified human proteins PEGylation
Vaccines Recombinant proteins
Gene-transfer products Viral delivery systems
Cell-based therapies Autologous, allogeneic and xenogenic
Tissue-engineered products Long-term implants

Advantages of biopharmaceuticals
High degree of specificity:
 Highly effective and potent action
 Fewer side effects
 Cure disease rather than treat
symptoms

Small molecules versus Biopharmaceuticals
 Species independent
 Non-immunogenic
 Metabolized
 Short acting
 Chronic daily dosing
 Toxicity
 Linear dose-response curve
 Direct effects
 Complex formulations
 Oral route
 Species specific
 Immunogenic
 Degraded
 Long acting
 Intermittent dosing
 Exaggerated pharmacology
 Bell-shaped dose-response curve
 Complex temporal relationships
 Simple formulations
 Parenteral routes

Small molecules versus Biopharmaceuticals

ICH S6 update:
special DART considerations

Principles of DART testing
Follow ICH S5(R2) for biopharmaceuticals.
However, because of species specificity, alternate
approaches may be needed → follow ICH S6(R2).
FDA guidance (2006) for preventive and therapeutic
vaccines for infectious disease indications

ICH S6 addendum (2012)
Need for DART dependent upon:
 Product
 Clinical indication
 Intended patient population

 Specific study design and dosing schedule:
 Species specificity
 Immunogenicity
 Nature of the product
 Mechanism of action
 Pharmacokinetic behavior
 Embryo-fetal exposure

 When clinical candidate
pharmacologically active in
rodents and rabbits
 When only NHP relevant
species
 When no relevant species
Use both species in EFD
Use NHP
Use genetically modified animals
or homologous protein

Selection of relevant species
Four criteria:
A. Species cross-reactivity
B. Pharmacology/efficacy
C. Immunogenicity
D. Exposure

A. Species cross-reactivity
Acceptable level of reactivity
Similar tissue distribution

B. Pharmacology/efficacy
Theoretical hazards with established pharmacology

C. Immunogenicity
Anti-drug antibody (ADA) formation:
 Clearing antibodies
 Neutralizing antibodies
 Cross-reactive neutralizing antibodies
Collection of ADA data:
 Titer
 Number of responding animals
 Neutralizing activity

C. Immunogenicity
Successful strategy: ‘staggered window’ approach
 May change dose response
or pattern of developmental
effects
Premating to
Conception
Conception
to
Implantation
Implantation
to Closure
of hard
palate
Hard palate
closure to
End of
pregnancy
W
DOSING
DOSING

D. Exposure
ICH S6 addendum → differences in placental transfer
High molecular weight proteins do not cross the
placenta by simple diffusion
Monoclonal antibodies use specific transport
mechanism (FcRn) → varies across species

D. Exposure – placental transfer IgG
DeSesso, 2012

Non-human Primates:
 Comparable to humans
 It might be questioned if EFD studies are of added value
 Important to evaluate indirect result of maternal or
placental effects

Five possible approaches
 When relevant species:
1. Traditional animal species
2. Non-traditional animal species
 When no relevant species:
3. Genetically modified animals
4. Use of surrogate molecule
5. No DART testing

Ensure adequate exposure during developmentally-
sensitive periods

Mouse may not be appropriate for human mAbs
 Evaluation of a mixture of nonspecific source of human
IgGs by Janssen R&D → loss of conceptuses during pre-
or very early post-implantation period
Implants Live young Implants Live young
Range finding study
(N = 4-7)
Main study
(N = 21-25)
Control 13.4 12.0 13.0 11.6
50 mg/kg 13.2 12.8 12.4 11.3
200 mg/kg 11.7 11.4 11.6 10.4

ICH S6 addendum → Fertility studies in NHP:
 Mating studies are not practical
 Evaluation reproductive tract in general toxicity study
 If specific concern, add specialized assessments
 If potential effect on conception/implantation,
then homologous product or transgenic model

ICH S6 addendum → EFD and PPND studies in NHP:
 Combine these into an enhanced PPND (ePPND)
Stewart, 2009

Knock-out (KO): lack an endogenous gene and
therefore fail to express the related protein(s)
Transgenic (Tg): overexpress a target protein
“Humanized” knock-in (KI): a human gene is inserted
(independently or with endogenous KO)

Scientific justification:
 Lack of available ‘normal’ relevant animal species
 Ability to mimic disease and/or to assess worst-case
scenario
Can only identify potential hazards (not useful for
quantitative risk assessment)

Evaluate available data
 Worst-case
 Safety margins
 Compensatory mechanisms
Humanized KI mice
 Consider mouse-human
protein interactions

Scientific justification:
 Lack of available ‘normal’ relevant animal species
 Availability of comparable surrogate molecule

High demand on resource requirements:
 Thoroughly evaluation for similarity
→ target affinity, pharmacodynamics,
undesirable cross-reactivity
 Development of new assays
→ biochemical characterization,
bioanalysis, immunogenicity
 Separate manufacturing processes
→ characterization of purity and stability

Can also be used to replace NHP testing:
 Minimize use of NHP
 Allow for evaluation of DART endpoints not easily
addressed in NHP
 Well-established protocols and HCD
 Improved statistical power
 More experienced labs
 More favorable fertility rates
 Low spontaneous abortion rates

5. No DART testing
ICH S6 addendum (2012):
 Products directed at foreign target
 Suggestion adverse effect on fertility or pregnancy
outcome
More reasons:
 Based on intended use
 Life-saving therapy
 Combination with other molecule with DART liability
 Sufficient information available about product class

The ‘6th approach’ for
preventive and therapeutic vaccines

Vaccines – guidelines for DART
EMEA (1997).
Note for guidance on
preclinical pharmacological
and toxicological testing of
vaccines:
 Embryo-fetal and/or perinatal
studies may be necessary for
vaccines that will be given to
WOCBP.

Vaccines - Developmental toxicity study
Distinguishable factors relevant to vaccines:
 Intended vaccine-induced immune response
 Administered in limited, episodic dosages
 May be adjuvanted

Vaccines - Developmental toxicity study
Premating to
Conception
Conception
to
Implantation
Implantation
to Closure
of hard
palate
Hard palate
closure to
End of
pregnancy
Birth to
Weaning
Weaning to
Sexual
maturity

Summary (1)
DART ICH S5(R2): FEED, EFD, PPND
Species specificity biopharmaceuticals → ICH S6(R2)
Selection of relevant test species
 Species cross-reactivity
 Pharmacology/efficacy
 Immunogenicity
 Exposure

Summary (2)
Five approaches
 Traditional animal species
 Non-traditional animal species
 Genetically modified animals
 Use of surrogate molecule
 No DART testing
Sixth approach for preventive and therapeutic
vaccines (FDA, 2006)

Conclusion
A scientific-based case-by-case approach is
considered the most appropriate for nonclinical safety
assessment of biopharmaceuticals.
 There is not one standard approach applicable per
biopharmaceutical subclass
 The stepwise approach is similar for general toxicity as for
DART, however DART specifics need to be considered

manon.beekhuijzen@wilresearch.com

References
 Bailey G.P. et al. The mouse may not be an appropriate species for evaluation of
reproductive and developmental toxicity for human monoclonal antibodies. Birth
Defects Research (Part A) 97:339 P30 (2013).
 Barrow P. Developmental and reproductive toxicity testing of vaccines. Journal of
Pharmacological and Toxicological Methods 65: 58-63 (2012).
 Baumann A. Foundation review: nonclinical development of biopharmaceuticals.
Drug Discovery Today 14: 1112-1122 (2009).
 Bowman C.J. et al. Embryo-fetal developmental toxicity of Figitumumab, an anti-
insulin-like growth factor-1 receptor (IGF-1R) monoclonal antibody, in cynomolgus
monkeys. Birth Defects Research (Part B) 89: 326-338 (2010).
 Bowman C.J. et al. Embryo-fetal distribution of a biopharmaceutical IgG2 during rat
organogenesis. Reproductive Toxicology 34: 66-72 (2012).
 Bugelski P.J. and Martin P.L. Concordance of preclinical and clinical pharmacology
and toxicology of monoclonal antibodies and fusion proteins: cell surface targets.
British Journal of Pharmacology. 166: 823-846 (2012).
 Bussiere J.L. et al. Alternative strategies for toxicity testing of species-specific
biopharmaceuticals. International Journal of Toxicology 28 (3): 230-253 (2009).
 Cavagnaro J.A. Preclinical safety evaluation of biotechnology-derived
pharmaceuticals. Nature reviews drug discovery. Volume 1, June, 469-475 (2002).
 Chaible L.M. et al. Genetically modified animals for use in research and
biotechnology. Genetics and Molecular Research 9(3): 1469-1482 (2010).

References
 Chapman K. et al. Preclinical development of monoclonal antibodies: considerations
for the use of non-human primates. mAbs 1:5, 505-516 (2009).
 DeSesso J.M. et al. The placenta, transfer of immunoglobulins, and safety
assessment of bipharmaceuticals in pregnancy. Critical Reviews in Toxicology 42
(3): 185-210 (2012).
 EMEA. Note for guidance on preclinical pharmacological and toxicological testing of
vaccines (1997).
 FDA, Guidance for Industry. Considerations for developmental toxicity studies for
preventive and therapeutic vaccines for infectious disease indications (2006).
 Herzyk D.J. et al. Practical aspects of including functional endpoints in
developmental toxicity studies. Case study: immune function in HuCD4 transgenic
mice exposed to anti-CD4 MAb in utero. Human & Experimental Toxicology 21: 507-
512 (2002).
 ICH S5 (R2), Guideline. Detection of toxicity to reproduction for medicinal products
& toxicity to male fertility (2005).
 ICH S6, Guidance for Industry. Preclinical safety evaluation of biotechnology-
derived pharmaceuticals (1997).
 ICH S6 (R2), Guidance for Industry. Addendum to preclinical safety evaluation of
biotechnology-derived pharmaceuticals (2012).
 Kooijman M. et al. 30 Years of preclinical safety evaluation of biopharmaceuticals:
did scientific progress lead to appropriate regulatory guidance? Expert Opin Drug
Saf 1-5 (2012).

References
 Lebrec H. et al. Overview of the nonclinical quality and toxicology testing for
recombinant biopharmaceuticals produced in mammalian cells. Journal of Applied
Toxicology 30: 387-396 (2010).
 Martin P.L. et al. Considerations in assessing the developmental and reproductive
toxicity potential of biopharmaceuticals. Birth Defects Research (Part B) 86: 176-203
(2009).
 Martin P.L. and Weinbauer G.F. Developmental toxicity testing of
biopharmaceuticals in nonhuman primates: previous experience and future
directions. International Journal of Toxicology 29(6): 552-568 (2010).
 Pentšuk N. and van der Laan J.W. An interspecies comparison of placental antibody
transfer: new insights into developmental toxicity testing of monoclonal antibodies.
Birth Defects Research (Part B) 86: 328-344 (2009).
 Ponce R. et al. Immunogenicity of biologically-derived therapeutics: assessment and
interpretation of nonclinical safety studies. Regulatory Toxicology and Pharmacology
54: 164-182 (2009).
 Stewart J. Developmental toxicity testing of monoclonal antibodies: an enhanced
pre- and postnatal study design option. Reproductive Toxicology 28: 220-225
(2009).
 Verdier F. et al. Reproductive toxicity testing of vaccines. Toxicology 185: 213-219
(2003).

The non-clinical testing of vaccines
ETS Education Course, Stresa
8 September 2013
Paul Barrow
paul.barrow.pb1@roche.com
Overview
• Why test vaccines for developmental toxicity?
– possible specific risks to reproduction
• Testing strategy
– methodology
– species selection
– FDA guidelines
– proposed study designs
– adjuvant testing
– biotech vaccines (primate studies)
– paediatric / juvenile studies
– fertility assessments
Disclaimer: Some views expressed herein are my own;
others I’ve shamelessly stolen from far cleverer people. My
employer really has no idea what I’m up to.

Advertising
ISBN 9781627031301

Current vaccine products
• Inactivated bacterial and viral vaccines
• Cholera, influenza…
• Live attenuated vaccine strains
• Measles
• Purified, recombinant or engineered proteins
• Engerix hepatitis B vaccine
• Polysaccharide and conjugated vaccines
• Bacterial meningitis
• DNA vaccines
• EquineWest Nile Virus
• Therapeutic vaccines
• Cancer, Alzheimer
• Veterinary vaccines
Adjuvants
• Inorganic salts
• alum
• Oil emulsions
• MF59, AS03
• Lipid A fractions of LPS
• MPL
• Saponin-based mixtures
• QS-21
• Oligonucleotides
• CpG sequences.

Why test vaccines for developmental toxicity?
• Arguments against:
– Infrequent administration
– Vaccines rarely used in pregnant women in the past
– No documented evidence to date of reprotoxic effects in
humans of approved vaccines
– Too complex!
Why NOT test vaccines for developmental toxicity?
www.cdc.gov
• Arguments for:
– Extremely long duration of action
• High likelihood of exposure to Abs or sensitised T-cells during
pregnancy (even following paediatric use)
– Possible specific risks to development
– Very low acceptable risk threshold for preventative vaccines
– Vaccines now more frequently used in pregnancy
• e.g. H1N1

POSSIBLE risks to development
• Changes in maternal immune system during
pregnancy
– shift towards Th2 (humoral) responses
• effects of immune stimulation on pregnancy not always predictable
• Potential immune targets during development
– e.g. cell adhesion molecules
wikispaces.psu.edu
Immune influences on pregnancy
• Cytokines of macrophage
origin may cause
pregnancy loss
– IL-1β, TNFα, IFNγ...
• BUT, non-specific immune
stimulation may also
protect against chemical-
induced teratogenesis
– influence on specific gene
expression

Effects of maternal immune stimulation
S Holladay et al.
Mouse
Valproic acid
500 mg/kg on GD8
Neural Tube Defects
In 18% of fetuses
Immune stimulation of
mother before mating
GM CSF or IFNγ
Valproic acid
500 mg/kg on GD8
NTD
In 3% of fetuses
Cell adhesion glycoproteins
• Polysialiated forms of NCAMS play a
crucial role throughout formation of the
CNS (and other tissues, e.g. testis)
– not normally prevalent in adult
• except in some pathological states and as a
consequence of developmental toxicity
– similar isoforms in polysaccharide MenB
vaccines
• risk of auto-immunity?
www.med.unc.edu/embryo_images/

Group B Neisseria meningitides
• Polysaccharide structure of capsule well preserved
between all strains of GBM
– Potential antigenic target
– Immunogenicity improved by chemical modification and
conjugation with tetanus toxoid.
– GBMP contains homopolimer of sialic acid
• Murine antibodies to GBMP cross react (weakly)
with human polysialiated NCAMS
– Coquillat et al., 2001
Vaccine safety data
- Immunogenicity
- Local tolerance and reactogenicity
- General (“repeat dose”) toxicity
- Safety pharmacology investigations ?
- Reproductive toxicity
- Biodistribution studies ?
- Reversion to virulence
- Part of product quality control
- Mutagenicity studies not required
- Pharmacokinetics studies not required
- Pharmacologic action mediated at injection sites or draining LN.

General toxicology: example study design
20♂, 20♀/group
10♂, 10♀/group
Early necropsy
10♂, 10♀/group
Late necropsy
Control + 1 human dose + adjuvant control (optional)
N vaccinations = human schedule + 1
Performed before first human exposure
Species selection for general toxicology
• Rodents and rabbits often preferred for vaccine safety studies:
• Rabbit is sufficiently large to allow administration of human dose.
• The mouse can be useful when Cytotoxic T-Lymphocyte (CTL)
responses are to be characterised, because of availability of
reagents.
• The monkey is sometimes the only species that meets criteria
for selection
• Genetically modified animals or alternative species are
sometimes appropriate
• Ferret, cotton rat.

ICH DART protocols not applicable
• Daily vaccination not feasible
– overdose
– desensitisation
• Several potential reproductive toxicants
– protein/polysaccharide component (+ adjuvant, excipients, etc)
• vaccinate at least once during organogenesis
• Time to mount antibody response
– vaccinate before mating
• Not limited to effects during organogenesis
GD0 GD6 GD17 GD20
4 groups of 24 mated females
Necropsy
Fetal examinations
Species selection
• Selected species should be responsive to the intended action of the
vaccine:
– develop an immune response similar to that expected in humans
• humoral and/or cell mediated
– demonstrate a similar effect of any adjuvant
– be susceptible to the pathogen or disease
• reflecting infection in man, permitting evaluation of viremia / bacteremia
• Practical considerations
– previous experience with the pathogen
– feasibility of route of administration
– availability of naïve animals
– practicability of reproductive investigations
– availability of reagents for immune analysis

Comparative development
• Rodents immature at birth with respect to humans
– may need to expose rat pups post-natally to cover
gesational events in human
• kidney development, Ig transfer...
• May need to evaluate entire development
Placentation
www.vivo.colostate.edu
Horse, pig Ruminants
Primates, rodents Dog, cat
Fetal endothelial cells
Fetal connective tissue
Chorionic epithelial cells
Endometrial epithelial cells
Maternal connective tissue
Maternal endothelial cells

Types of placenta & antibody transfer
Placenta Relation to
maternal tissue
IgG
transfer
Primates Chorioallantoic Hemochorial +++
Lagomorphs Yolk sac, then
Chorioallantoic
Endotheliochorial ++
Rodents Yolk sac, then
Chorioallantoic
Hemochorial +
Carnivors Chorioallantoic Epitheliochorial ~0
Timing of maternal Ig transfer
From
placenta
From colostrum
(FcRn in gut)
Primates 100% (IgG) 0
Rodents 10% (IgG) 90% (IgG, IgA, IgM)
Lagomorphs 100% (IgG, IgM) 0
Dog, cat 5% (IgG) 95%
Pig, sheep 0 100% (IgG, IgA, IgM)
More work need to investigate FcRn ontogeny in the
placenta/yolk sac across species (possible ILSI/HESI project)

Fetal:maternal IgG ratio
0
100
0.0 0.2 0.4 0.6 0.8 1.0
Proportion of gestation
%maternallogtitre
Human
Monkey
Rabbit
Rat
Redrawn from Pentsuk & van der Laan, 2009
Guidance for Industry
– Considerations for Developmental Toxicity Studies for Preventive and
Therapeutic Vaccines for Infectious Disease Indications. CBER February,
2006
• Single species acceptable
–expected to show Ab response
• Episodic dosing preferable
• Dosing before mating
• Single dose level acceptable
–1 human dose per animal if possible
• 20 females for caesarean, 20 for post-natal
• Sampling for Ab analysis
–maternal Ab transfer / persistence in pups
• Assess post-natal development

Possible strategy
• Preliminary studies of Ab transfer
– rat, mouse, rabbit
• Main developmental tox. study in most
pertinent species
Preliminary study
• Groups of 12 females
• Treat before and during gestation
– eg. 2 weeks before mating, G6, end of gestation
• Submit 6 females to caesarean
– maternal & fetal blood samples for Ab analysis
• Terminate 6 females 1 week after birth
– maternal & pup blood samples for Ab analysis

Example - HIV vaccine
IgG anti-gp 160 - % maternal titre
0
100
rat mouse rabbit
%
(log titre)
fetus
LD11
Species selected
Vaccine Species Reason
HIV Mouse Maternal Ab transfer
Tetanus/diptheria/pertussis Mouse Maternal Ab transfer
Rabies Rabbit Maternal Ab transfer
Meningitis Rabbit Immunogenicity

Main study
• Groups of 50 rodents or rabbits
– sub-group for caesarean
• routine embryotox
– sub-group for post-natal examinations
• routine developmental assessments up to weaning
– growth, physical development, behaviour...
• routine necropsy
Developmental toxicity study for vaccines
50 ♀ / group
25 ♀ / group post-natal
M-14d M-1d
GD6
GD28
GD29
PND35
25 ♀ / group caesarean

Rabbit – post-natal investigations
Rabbit background data
• Historical control data from 15 studies: Barrow and Allais 2012, In: Barrow, P.C.
(Ed). Teratogenicity Testing. 602p. Springer, Berlin. ISBN 9781627031301.
Number of females Mean Mean % of kits % of kits
Mated Pregnant With With kits gestation length number of liveborn surviving to
liveborn at weaning (days) kits born weaning
326 301 289 241 31.8 8,6 97 91
92% 89% 74%
Data: WIL Europe, Lyon

Adjuvants
• Separate study:
– Following ICH S5(R2) embryotox guidelines
• No need for dosing before mating
• 2 species preferable
• Or, add additional group to vaccine study
– Also covers post-natal development
• Biomarkers of systemic inflammatory response?
– e.g. serum IL-6, CRP, fibrinogen…
• Possible exaggerated pharmacology
– e.g. TLR-9 agonists in rodents
Primates - considerations
• Poor availability of animals
• Poor mating performance
– Vaccination prior to mating is impractical
– Ab exposure will ensue during pregnancy thanks to long
gestation period
• Very limited number of offspring examined
– Only one fetus per pregnancy
– Frequent fetal loss
• Study duration of 1.5 to 2 years
– ~4 months necessary to mate all females
– 160 days of gestation, several months of post-natal
development
• Long maturation period of offspring (4 to 5 years)

Proposed design - primates
Based on “Enhanced pre- and post-natal study
design” proposed for Mabs.
Stewart, 2009, Reproductive Toxicology 28 220-5.
12-14 ♀ / group
PND160~GD160
Birth
GD20
Ultrasound
scan
ePPND study - limitations
• Disadvantages
– Few animals
• Even less offspring (~10/group)
• Little or no statistical power
– No exposure before GD20 (pregnancy confirmed by
ultrasound scan)
• Not a major problem for mAbs
– No caesarean exams
• Fetal examination by X-ray
• Examination of babies at birth.

Assessment of immune system
• Clinical pathology
– e.g. lymphocyte subsets
• Organ weights & histopathology
– liver, spleen, lymph nodes...
• Functional tests of cellular & humoral immunity
– e.g. TDAR
Paediatric evaluations
• A paediatric evaluation (or waiver) is now mandatory for all
New Drug Applications in North America or Marketing
Authorisation Applications in Europe
– FDA, EMA and MHLW (Japan) have issued guidelines on juvenile toxicity
studies
• Developmental toxicity studies are usually not necessary for
vaccines indicated for immunization during childhood (yet!).
– Even though most vaccines are given to children

Suggestion
• Perform repeat dose studies in juvenile animals
– Examples of current RDS:
• Rats starting at 6 weeks old, rabbits starting at 12-14 weeks
• 3 vaccinations 2 weeks apart
• Half of animals retained for 2 weeks recovery
– Juvenile / RDS:
• Start dosing 1 week after weaning (4 weeks for rat, 8 for rabbit)
• Perform all examinations currently included in RDS
• Both species will be adult at time of necropsy
–10.5 weeks for rat, 14.5 weeks for rabbit (recovery animals)
• Covers all developmental periods for vaccines given to infants
or older
– No loss of sensitivity for effects in adult
POSSIBLE effects on fertility
• Effects on reproductive organs should be detected in repeat
dose studies
– Provided that animals are adult at time of necropsy
– No test of mating ability
• Mating performance of the female can be assessed during
developmental toxicity study
– Not the main objective of the study
• E.g.: what if copulation is delayed by a few days in the vaccinated group, but
females later prove to be fertile?

Effects on early embryo?
• Preimplantation embryo has no protection from
immunoglobulins present in intrauterine or fallopian fluid.
• Toxic insult at this stage may result in embryonic death, but
not normally in dysmorphogenesis.

Vaccines and Medications
in Pregnancy Surveillance
System (VAMPSS)
“Women are also asked about all
vaccines they may have received,
including those given in non-
traditional settings such as health
fairs or at the supermarket.”
Summary
• Most vaccines present a theoretical risk of developmental
toxicity
– no proven causal links demonstrated with marketed vaccines to date
• Classical testing strategies (ICH) are not applicable
– species, dosing regime, post-natal evaluations
– primate studies will be necessary for some biotech vaccines
• Potential hazards are not limited to use during pregnancy
– need to think about paediatric studies
– fertility?
• Pharmacosurveillance in humans is important

Use of Nonhuman Primates for
Biologics DART
Gary J. Chellman, PhD, DABT
Program Director, DART
Charles River Preclinical Services, Reno, Nevada USA
European Teratology Society Meeting
Stresa, Italy
08 Sep 2013

Introduction of Topics
• Advantages and Disadvantages of NHP model
• Study Designs
• Current Trends
• Data Interpretation Challenges
• Developmental Immunotoxicology (DIT)
• Juvenile Toxicology

Advantages of NHP Model
• Biologics: pharmacology w/ minimal immunogenicity
• Allows testing of the clinical candidate (drug)
• Embryology/reproductive physiology similar to man
• Similarity in response to known human teratogens
• Pregnancy can be monitored by ultrasound
• Comprehensive endpoints can be evaluated

Disadvantages of NHP Model
• Small sample size (n = 12-20/group)
• Low conception rate (30-40%)
• Long duration (gestation = 5.5 months)
• High abortion rate (15-20% full term)
• Single offspring
• Limited availability of CROs with expertise
• Cost of studies

“Typical” NHP DART Study Designs
• Mating Studies
– Embryo-fetal development (EFD)
– Pre and postnatal (PPND)
• Non-mating Studies
– Male and/or female reproductive toxicity
– Juvenile
Think outside the box for study design!
Chellman et al. (2009) BDR 86:446-462

Reproductive Life Cycle – ICH S5 (Stages A to F)
Senescence
Embryo
Juvenile
Mating/fertility
Adolescence/puberty
Zygote
Fetus
Gametes (M/F)
A. Premating to
Conception
F. Weaning to
Sexual Maturity
B. Conception to Implantation
C. Implantation to Closure of Hard Palate
D. Hard Palate Closure to End of Pregnancy
E. Birth to Weaning
Source: Christian, M.S. (2001). In: Principles and
Methods of Toxicology (4th Edition, A. Wallace
Hayes, editor), pp. 1301-1381.

Male Reproduction Stand-Alone Study
(non-mating, sexually mature)
Treatment
Usually include:
• Sperm count, motility
and morphology
• Testes volume
• Testosterone
Duration spermatogenesis ~45d
Recovery
Necropsy
Organ weights
Histopathology (option for
spermatogenesis eval.)
Week -3 Day 1 Month 3 Month 4

Female Reproduction Stand-Alone Study
(non-mating, sexually mature)
Observation
Cycles
1
Treatment
Cycles
2 3 6 74 5
Recovery
(1+ cycle)
Synchronize dosing
to menstrual cycle?
Average NHP cycle
length 28-32 days
Necropsy
Organ weights
Histopathology
Usually include:
• Menstrual cycles (vaginal swabs)
• Hormones:
Blood samples every 2-3 days, 3 cycles
(Pre, EOD, EOR)
Measure E/P (LH/FSH optional)

Embryo-Fetal Developmental Study (EFD)
Cycle
check
2 months
GD 0 GD 18-20 GD 50 GD 100
Treatment
Pregnancy Monitoring
Confirm
pregnancy
(ultrasound)
Mating C-section
• Placenta
• Maternal blood
• Cord blood
• Amniotic fluid
Fetal exams
• External
• Visceral
• Skeletal
End of
major
organogenesis
Biologics: Immunologic Testing
• Flow cytometry (mother)
• Immunoglobulins (mother)
• Immunohistochemistry (fetus)
Note: extend dosing?
(e.g., MAb’s)

Placental Transfer NHP (FcRn)
Although fetal IgG exposure is represented as being at or
below baseline prior to the second trimester, recent
information suggests some IgG may be present during the 1st
trimester at levels that could be pharmacologically or
toxicologically active (Dybdal,2010; Wang et al., 2011).

Pre-Postnatal Development Study (PPND)
Cycle
checks
2-3 months
GD0 GD18-20 GD160 PP 3-12M
Treatment/
Confirm
pregnancy
(ultrasound)
Mating Infant necropsyDelivery
• Mothers: blood/milk
• Infants: blood,
physical exams,
neurobehavior
Nursing/Behavior
• Standard tox: clinical signs, BW, TK, clin path, necropsy/histo
• Immunologic: flow cytometry, Ig’s, TDAR (KLH), NK cell, IHC
• Specialized: fetal measures, infant behavior/learning

Juvenile NHP Toxicology Study
(13-week duration common)
Dosing
• Clinical signs
• Body weight
• ECGs
• Ophthalmology
• Skeletal growth
• Clinical pathology
• Immunology
• Behavioral
• TK and ADA
Recovery
• Necropsy
• Organ weights
• Gross and histopathology
• Specialized
• Immunohistochemistry
• Bone densitometry
Week -2 Day 1 Week 13 Week 26
Typical age of juvenile NHP is
10/12M to 24M

Current Trends in NHP DART
Streamlining (Consolidation) of Study Designs
– Instead of stand alone studies for M/F repro, combine
the endpoints into a chronic toxicology study
• ≥ 13 week dose duration
• Use sexually mature animals
– Instead of conducting separate EFD and PPND
studies, combine into one design known as the
enhanced PPND study (ePPND)
Each of these reduces the number of animals
required by one-half (3Rs)

Male Reproduction in Chronic Toxicity Study
Per Current ICH S6 (R1) (3, 6, 9 Months)
Dosing Recovery
Week -3 Day 1 Month 3 Month 4
As needed (for cause):
• Sperm count, motility
and morphology (at necropsy?)
• Testes volume
• Testosterone
Duration spermatogenesis ~45d
Necropsy (required)
Organ weights
Histopathology

Female Reproduction in Chronic Toxicity Study
(3, 6, 9 Months)
Observation
Cycles
1
Treatment
Cycles
As needed (ICH S6):
• Menstrual cycles (vaginal swabs)
• Hormones:
Blood samples 3x/week for 6 weeks
(Pre, EOD, EOR)
Measure E/P (LH/FSH optional)
2 3 6 74 5
Recovery
(1+ cycle)
Dosing not
synchronized to
menstrual cycle
Average NHP cycle
length 28-32 days
Necropsy
Organ weights
Histopathology

Selection Criteria for Sexual Maturity:
How successful is your strategy?
• Using males ≥5 years old and ≥5 kg has provided 97% mature
[maturity confirmed by histopathology]
Regul Toxicol Pharmacol (2012) 63:391-400
Functional Assessment of Maturity Male Cynos

NHP Enhanced PPND Study Design
(ePPND)
Cycle
checks
2-3 months
GD0 GD18-20 GD160 PP 3-12M
Treatment/
Confirm
pregnancy
(ultrasound)
Mating Infant necropsyDelivery
• Mothers: blood/milk
• Infants: blood,
physical exams,
neurobehavior
Nursing/Behavior
External Assessment/
Morphometric Eval
(Birth, 1, 3, 6 Months)
Necropsy:
External/
Visceral
Eval
Skeletal Eval
(Radiograph)
(1 Week or
1 Month)

2008-2012: Shift Towards ePPND Studies
ePPND studies have increased (with EFD’s decreased).
Chronic tox/M-F repro has also dramatically increased.

Additional Trends in NHP DART Design
ePPND
• Later dose start (GD50) if previous EFD
• Include placental transfer cohort C-sections
• Decrease group size (pregnancies + infants)
• Streamlining of endpoints to focus on infant
growth/development
Chronic Tox with M/F Repro
• Focus on ICH - organ weights/histo (fewer extras)
• Screen males and females functionally to verify
sexual maturity

Social Housing for ePPND Studies
• Can social housing reduce NHP abortion rate?
• How about infant losses?
• Is infant growth and development improved?

Use of Group (Social) Housing for NHP
• Trend is away from single
housing
• New caging allows 2 or
more NHPs to be together
• Stainless mesh and solid
pen panels to
accommodate infants on
DART studies
• Multiple perches, climbing
pole, exploratory balcony
and foraging devices

Data Interpretation Challenges with
NHP ePPND Studies

Historical Control Data (12 ePPNDs):
Fetal and Infant Losses
Category Average (%) Range (%)
Gestation length (days) 159 ± 7 130 -175
Abortions 1st trimester 8 0 - 15
2nd trimester 1 0 - 10
3rd trimester 14 0 - 29
Infant loss (BD1-28) 11 0 - 20
Data for 12 final ePPND studies (217 pregnancies, 161 births, 2008-2012)

Fetal Loss Interpretation
20
14.3
% loss
(Article)
3
0
2
4
1
2
No.
lost
27.3
0
16.7
30.8
10
18.2
% loss
(Group)
15.0
24.0
14.3
% loss
(Dose)
18.2
% loss
(Study)
11High B
9High A
12Low B
13Low A
10Control B
11Control A
No.
preg.
Group
14.3
20.0
18.2

Background Changes in Pregnancy:
B-lymphocytes
Reprod Toxicol 2009 28:443-455

Background Changes in Pregnancy:
Cholesterol

Test Article Effects in NHP ePPND’s
• Long lasting TK and PD (≥ 3M postpartum)
• GD20-50 EFD monoclonal antibody →
malformations (placental transfer)
• Placental target (CTLA-4) → decreased gestation
length + fetal/infant loss
• Metabolic target (RANK-L) → fetal/infant loss +
skeletal alterations
• Strong maternal ADA response had major impact
on maternal (and infant) exposure

Developmental Immunotoxicology

DIT in Nonhuman Primate DART Studies
Charles River – Nevada (2008-2012)
Inflammatory disease (8), immunomodulatory (5), metabolic (3), other (1)

NHP Immunologic Evaluations (B-cell modulator)
BD7 BD28 BD91 BD365
0
500
1000
1500
2000
2500
3000
3500
150 mg/kg
0 (Control)
5 mg/kg
Infant Age (Days)
CD20+/CD21+
(MatureB-lymphocytes)
Infant Spleen
Control
Lymphoid Follicle Lymphoid Follicle
Infant Spleen
High dose

7 28 91 182 273 365
0
1
2
3
4
5
6
7
8
9
10
11
12
Days After Birth
*
7 28 91 182 273 365
0
250
500
750
1000 Control
Low (5 mg/kg)
High (150 mg/kg)
Days After Birth
* *
* *
Monitoring Immunoglobulins in NHP Infants
IgMIgG
Total IgG: transient decrease BD7 to BD91  transition of maternal to infant
source
Total IgM: dose-independent decrease BD7 and BD91 vs. controls, with
recovery

ImmuneResponse
Adaptive
Memory
KLH
Immunization #1
KLH
Immunization #2

Juvenile Toxicity Study
Purpose
• Evaluate possible effects on postnatal development when
exposure occurs in some/all of the neonatal to pre-adult period
Why not just use adult clinical data for pediatric safety?
• Children are not simply miniature adults
• Differences in physiology and metabolism can alter sensitivity
to drug effects
• These factors are constantly changing during maturation, along
with unique timelines for development of each organ system
• Therefore juvenile toxicity studies need careful design
– Factor in animal age + stage of organ system development
38

Physiology/Pharmacokinetics in
Neonate/Infant Compared to Adult
39
Neonate/Infant
GI Motility/pH
Lower GI motility ( GI absorption), higher pH ( absorption
of basic molecule,  absorption of acidic molecule)
Protein Binding Typically lower, more free compound
Total Water Content Higher, affects volume of distribution
Metabolism Enzymes not fully developed, affecting pharmacokinetics
Glomerular Filtration Lower, may  half-life
Biliary Excretion Lower, may  half-life

Organ Systems of Potential Concern
(and Maturation Periods in Humans)
Behavior/cognition development up to adulthood
Immune system development up to twelve years
Reproduction development up to adulthood
Skeleton (growth) development up to adulthood
Renal development up to one year
Pulmonary development up to two years
Cardiovascular development up to five to seven years
Gastrointestinal development up to one to two years
Test what is pertinent (not everything)!

41
Non-Clinical Species/Ages to Support Pediatric Use
Designed on a case-by-
case basis, and the
dosing is designed so
that the organs of
concern would be at the
same maturational stage
in both the animals (likely
rats) and intended patient
population.

Challenges of Acquiring Juvenile NHPs
• Can purchase 9M (weaning) to 12M old from vendors
• If need <9M: purpose breed ($$) or obtain from PPND
• Testing juveniles or infants?
• Is goal to close gap below standard age for tox?
– PPN (6M-1Y old) to tox study (2-3Y old)
– Use of sexually immature NHP’s in chronic toxicity
studies may eliminate the need for an additional study
in juvenile NHP’s
– Use of sexually mature NHPs for tox widens gap
• Not practical to use NHP for testing to sexual maturity (4-6
years old)

Conclusions
• DART studies in NHP have become more
common due to the increasing number of
biopharmaceuticals in drug development.
• NHPs may be used when traditional species or
alternate models cannot be used (rodents,
rabbits, surrogate molecule).
• The ePPND design has become one of the most
frequently conducted NHP DART designs.
• Work with regulatory agency(s) & CRO to
determine what DART studies are needed and
timeline – plan ahead!

1. Chellman GJ, et al. (2009) Developmental and reproductive toxicology studies
in nonhuman primates. Birth Defects Research (Part B) 86:446-462.
2. Rocca MS and Wehner NG. (2009) The guinea pig as an animal model for
developmental and reproductive toxicology studies. Birth Defects Research
(Part B) 86:92-97.
3. Morford LL, et al. (2011) Preclinical safety evaluations supporting pediatric
drug development with biopharmaceuticals: strategy, challenges, current
practices. Birth Defects Research (Part B) 92:359-380.
4. Collinge M, et al. (2012) Developmental immunotoxicity (DIT) testing of
pharmaceuticals: current practices, state of the science, knowledge gaps, and
recommendations. J Immunotoxicol 9(2):210-230.
5. Chapman K, et al. (2012) Pharmaceutical toxicology: Designing studies to
reduce animal use, while maximizing human translation. Regulatory Toxicology
and Pharmacology 66(1):88-103.
6. Dybdal, N. (2010) Impact of monoclonal antibody administration to pregnant
cynomolgus monkeys on early fetal health and development. In: 16th Annual
Charles River Biotech Symposium - Biotechnology Derived Therapeutics,
Perspectives on Non-clinical Development, San Diego, CA
7. Wang H, et al. (2011) Assessment of placental transfer and the effect on
embryo-fetal development of a humanized monoclonal antibody targeting
lymphotoxin-α in non-human primates. The Toxicologist. Abstract No. 2772.
Publications Related to Biologics DART

ICH References Related to Biologics DART
• ICH M3(R2) Nonclinical Safety Studies for the Conduct of Human
Clinical Trials and Marketing Authorization for Pharmaceuticals (June
2009)
• ICH S3A Note for Guidance on Toxicokinetics: The Assessment of
Systemic Exposure in Toxicity Studies (Oct 1994)
• ICH S5(R2): Detection of Toxicity to Reproduction for Medicinal
Products & Toxicity to Male Fertility (Nov 2005)
• ICH S6(R1): Addendum to ICH S6: Preclinical Safety Evaluation of
Biotechnology-derived Pharmaceuticals (June 2011)
• ICH S9:Nonclinical Evaluation for Anticancer Pharmaceuticals (Oct
2009)

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