The document provides an overview of genetic toxicology and genotoxicity assessment. It discusses what genetic toxicology is, why genotoxicity is evaluated, common assays and testing paradigms used, and key industry regulations and requirements. The agenda covers definitions of genetic toxicology, why we evaluate genotoxicity, common assays, and regulations from bodies like ICH, ECHA, and FDA.

1. NOT FOR U.S. AND CANADA AUDIENCE
Rohan Kulkarni, MSc, PhD, ERT
BioReliance® Services
Director, Toxicology Study Management
The Comprehensive
Guide to Genotoxicity
Assessment

2. • What is Genetic Toxicology (Genotoxicity)
• Why do we Evaluate Genotoxicity?
• Assays and Testing Paradigms
• Regulations and Industry Requirements
Genotoxicity Assessment
Agenda
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3. WHAT IS GENETIC
TOXICOLOGY
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4. … the study of chemical, physical or biological agents
that can change the sequence or structure of DNA
What is Genetic Toxicology
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DNA damage can be:
• at nucleotide level in DNA, or at the
chromosomal level
• induced by direct mechanisms (chemical or
metabolite interacts with DNA)
• induced by indirect mechanisms (chemical or
metabolite affects other cellular
macromolecules, e.g. mitotic spindle fibers)
Genotoxicity Assessment

5. What is Genetic Toxicology
5
ATC AGC
Mutation
Chromosome BreakMisrepair
or No Repair
Duplication
DNA Damage
Error Free Repair
or Apoptosis,
Cell Death
No Genotoxicity
Chromosome Aberration
Genotoxicity Assessment

6. WHY WE
EVALUATE
GENOTOXICITY
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7. Why We Evaluate Genotoxicity
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Germ Cells
spermatocytes, oocytes Somatic Cells
Heritable Damage
(genetic damage to
offspring)
Infertility Cancer Other Diseases
7
DNA damage is associated with many human diseases
Genotoxicity Assessment

8. 1. Hazard identification/Lead prioritization
 Companies want products to be safe and to be seen as safe for intended uses and
foreseeable misuses
 Predict whether a chemical is a carcinogen
 Predict whether a chemical could cause heritable germ cell damage
 Early testing “screening” allows Companies/Regulators to prioritize chemicals to spend
further resources on (e.g. EPA ToxCast™)
2. Mechanistic information
 Determine mechanism of action for carcinogens (genotoxic vs. non-genotoxic)
 Basic science: study of DNA damage/repair
Objectives of Genetic Toxicology Testing
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Genotoxicity Assessment

9. ASSAYS AND
TESTING
PARADIGMS
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10. What is in the Genetic Toxicology Toolbox?
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• Hundreds of assays developed to measure DNA damage
• Assays grouped by endpoint measured
• Types of DNA damage endpoints include:
− Mutations
− Changes in chromosome structure or number
− DNA damage
− DNA repair
− Biomarkers of DNA damage
• Endpoints can be measured in:
− bacteria, yeast, fungi, plants, invertebrates, mammalian cells in culture, animals and
humans
Genotoxicity Assessment

11. Assays throughout the Development Process
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Decision Gate Decision Gate
• in silico
− QSAR Analysis
• High Throughput
Screening
− Product
Characterization
• Non-Regulatory
− Predictive Screening
• Regulatory Battery
− GLP Assays
• Product Rescue
− Follow Up to In Vitro
Positive Assays
• Carcinogenicity
Assessment
− rasH2 Transgenic
Mouse Carcinogenicity
Assay
Genotoxicity Assessment

12. • Non-GLP assays used at early stages to select candidates for further development
Advantages
− low(er) cost
− quick turn-around time
− minimal test article requirements
− can be highly predictive
• Customize design based on available sample
• HOWEVER, design should mimic the ultimate GLP/regulatory study as closely as
possible for best predictive value
Discovery/Lead Optimization/Early Development
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Screening Assays

13.  Computer based (in silico) qualitative and quantitative modelling methods relating chemical structure to biological
activity leading to prediction and characterization of chemical toxicity.
QSAR – Quantitative – statistically-derived model
SAR – Qualitative – expert rule-based model
 A SAR model, also known as an expert system, establishes a qualitative association between a chemical’s substructures
and its potential toxicity.
 Uses literature, expert knowledge, toxicology, toxicokinetics and metabolism
 A QSAR is a mathematical relationship between a chemical’s quantitative molecular descriptors and its toxicological,
biological, and physicochemical activities.
 -Uses pattern recognition, rule induction, mathematical algorithms
SAR
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SAR resides at the intersection of biology, chemistry, and statistics.
James D. McKinney et al. Toxicol. Sci. 2000;56:8-17
High Throughput Screening

14.  Drugs & Candidate Selection
 Lead Optimization, Metabolites, Degradation, Impurities
(Use of (Q)SAR To Address ICH M7, Reach, and Other Compound Development)
 Leachable & extractable compounds
 Medical Devices, Drug Packaging, Food Packaging
 Consumer Products
 Pesticide impurities
 Eco toxicity
 Physico-Chemical Parameters
 Identification of Surrogates for use in risk assessment
 Read Across Risk Assessment
Areas where (Q)SAR and related methods can be applied:
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High Throughput Screening

15. Purpose: To predict if the test article has genotoxic property with clastogenic or aneugenic mode of
action (MOA), or is non-genotoxic toxicant/agent based on markers associated with DNA damage
response pathways.
Markers:
• 24hr Nucleus P53 as genotoxic marker
• 4hr gH2AX as a measure of DNA double strand breaks,
• 4hr Phosphorylation of histone H3 (p-H3) as an indicator of mitotic cells,
• 24hr Polyploidy as mitosis marker
Modeling:
• Risk assessment uses modeling to predict risk factors. It will be the future for safety assessment as well,
• Most common models are mapping and logistical regression model
CAN MultiFlow™
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High Throughput Screening

16. 1. Since prediction is based on time-related trends of biomarkers together (DNA
damage, mitosis, genotox), the prediction is more accurate than single biomarker,
e.g.GreenScreen HC assay that based on P53-activated GADD45a-GFP expression
or flow-based gH2AX analysis.
2. The CAN model can provide more information at the same time, e.g.
genotoxic/non-genotoxic, clastogenic/aneugenic MOA, and toxicant/non-toxicant.
3. CAN is a high throughput screening assay: require less test article (<10mg), test
20 doses, has short assay period (results in 2 assay days), and has automatic
data analysis.
4. CAN assay can be used to detect single biomarker, e.g. gH2AX or p53.
Why use CAN MultiFlow™
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High Throughput Screening

17. The Ames Assays are another option for High Throughput Screening
Ames II
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High Throughput Screening

18. Ames Screens – Variations
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Abbreviated standard assay
 100-mm plates
Multi-well modifications
 mini-Ames (6-well plating)
 micro-Ames (24-well plating)
Liquid micro-plate method (Ames II™)
Predictive Screening Assays

19. • In Vitro Chromosome Aberration
− Assay detects chromosome breaks/rearrangements (clastogens)
• In Vitro Micronucleus
− Assay detects whole chromosomes or fragments not segregated as normal into the
daughter nuclei at cell division
− Can distinguish clastogens (fragments) from aneugens (whole chromosomes) using
FISH or CREST analysis
− Multiple test systems (CHO, HPBL, TK6)
− Multiple evaluation types (Flow, Microscopic)
In Vitro Cytogenetics
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Predictive Screening Assays

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In silico Approach
Negative ResultsPositive Results
Predictive Screen
CAN MultiFlow™ Ames (6-well/24-well)
Ames II
In vitro MN screening (CHO/TK6)
Negative ResultsPositive Results
Discard
Modification
Impurity
Discard
Modification
Impurity
Non-GLP Assay for MoA GLP Assays
HTP
Designing the Appropriate Genotoxicity Testing Program

21. 21 Comprehensive Genotoxicity 020917
Non-GLP Assay for MoA GLP Assays
In vitro MN
assay/ CREST
In vitro Chrom
Abs
TK6, HPBL or
CHO cells
HPBL or CHO
cells
Ames Assay Ames Assay Ames Assay
In vitro MN assay In vitro MN assay with
CREST
In vivo MN assay In vivo MN assay In vivo MN + Comet
Option 1 Option 2 Option 3
Toxicology Seminar Series 06.08.2016
Designing the Appropriate Genotoxicity Testing Program

22. REGULATIONS
AND INDUSTRY
REQUIREMENTS
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23. 23
What are the Best Gene Tox Choices for My Substance?
Designing the Appropriate Genotoxicity Testing Program

24. Regulatory Agencies agreed (within reason) on
 a battery of assays to be performed
 the appropriate design to be used for each assay (OECD TG)
Address two main endpoints
 Mutagenicity - changes in DNA base sequence
 Clastogenicity - breaks in chromosomes/chromatids resulting in
rearrangements, gains, losses
 both are known causes of many genetic diseases
 both shown to effect oncogenes and tumour suppressor genes involved in
tumour formation in humans
Includes both In Vitro and In Vivo tests
Gene Tox Testing Guidelines
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Designing the Appropriate Genotoxicity Testing Program

25. Designing Specific Regulatory Programs
ICH
International Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use
• Provides globally accepted approaches – US, Japan, Europe
• ICHS2 guideline finalised Nov 2011
• ICH S2A: Guidance on specific aspects “How to do assays” (1995)
• ICH S2B: Guidance on standard battery “What assays to do” (1997)
• Builds on OECD guidelines - “protocols”
www.ich.org
Guidelines for Pharmaceuticals
25

26. Designing Specific Regulatory Programs
Former - ICH S2A/S2B Revised - ICHS2R1
Option 1* Option 2
Ames microbial mutagenesis & repeat Ames
(no repeat if clear positive or negative)
Ames
(no repeat if clear positive or
negative)
In vitro mammalian cell assay (using 5 mg/ml
or 10mM):
Chromosome aberrations,
OR
tk mutations in mouse lymphoma cells
In vitro mammalian cell assay:
(1 mM top concentration)
Chromosome aberrations,
OR:
tk mutations in mouse lymphoma cells
OR:
in vitro micronucleus assay
No in vitro mammalian cell assays
In vivo cytogenetic assay In vivo micronucleus or chromosome aberration
assay (rat/mouse, blood/bone marrow)**
In vivo gene tox with two tissues**:
1) Micronuclei (not chrom ab)
2) Typically DNA breakage in liver
3) Prefer both endpoints in 1 study
Pharma - ICH S2R1 Guidance
before phase 1
before phase 2
* Required for chemicals toxic to bacteria (e.g. antibiotics)
** Can be integrated into repeat dose studies.
Micronuclei analysis can be evaluated by flow cytometry

27. Designing Specific Regulatory Programs
27
European Medicines Agency

28. Designing Specific Regulatory Programs
ICH M7 addresses Mutagenic (DNA
Reactive) impurities in
pharmaceuticals
Provides some current approaches to
dealing with Ames Positive results
ICH M7 Guideline

29. Designing Specific Regulatory Programs
Compounds demonstrated to induce genetic mutations, chromosomal
breaks, and/or chromosomal rearrangements
 are considered genotoxic
 have the potential to cause cancer in humans
• Focus on DNA-reactive impurities, i.e. mutagenic impurities typically positive in
the bacterial mutagenicity assay
• Threshold of Toxicological Concern (TTC) concept applies – 1x10-5 lifetime risk
• Less than Lifetime (LTL) principle applies
• Clinical development and marketed products with shorter treatment durations
have higher acceptable levels
• Evaluate actual impurities plus risk-based subset of potential impurities
• When an impurity is also a metabolite, safety evaluation is primarily focused on
the risk assessment of the metabolite
ICH M7 Guideline on Mutagenic Impurities

30. Designing Specific Regulatory Programs
“ECHA is becoming the world’s leading regulatory
authority on the safe use of chemicals. We make sure
that information on the hazards and safe use of
chemicals is available to everyone. You and your
environment will be better protected.”
Official REACH Guideline
• REGULATION (EC) No 1907/2006 OF THE EUROPEAN PARLIAMENT
AND OF THE COUNCIL
Chemicals in Europe – ECHA/REACH

31. Designing Specific Regulatory Programs
31
Genetic Toxicology Battery of Assays - ECHA/REACH

32. Designing Specific Regulatory Programs
Subpart F—Genetic Toxicity
§798.5195 Mouse biochemical specific locus test.
§798.5200 Mouse visible specific locus test.
§798.5265 The salmonella typhimurium reverse mutation assay.
§798.5275 Sex-linked recessive lethal test in drosophila melanogaster.
§798.5300 Detection of gene mutations in somatic cells in culture.
§798.5375 In vitro mammalian cytogenetics.
§798.5385 In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.
§798.5395 In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay.
§798.5450 Rodent dominant lethal assay.
§798.5460 Rodent heritable translocation assays.
§798.5500 Differential growth inhibition of repair proficient and repair deficient bacteria:
“Bacterial DNA damage or repair tests.”
§798.5955 Heritable translocation test in drosophila melanogaster.
New TSCA Guidelines

33. Designing Specific Regulatory Programs
EFSA is the European Union risk assessment body for food
and feed safety
 independent scientific advice to risk managers
Scientific opinion on genotoxicity testing strategies
applicable to food and feed safety assessment
 EFSA Journal 2011;9(9):2379
More information in later slides
European Food Safety Authority (EFSA)

34. Designing Specific Regulatory Programs
• Bacterial reverse mutation (Ames) assay
(OECD TG 471)
And
• In vitro micronucleus test
(OECD TG 487)
“Addition of any further in vitro mammalian cell tests in the basic battery would
significantly reduce specificity with no substantial gain in sensitivity”
• Positive results in initial tests are evaluated in in vivo tests such as in vivo
micronucleus assay, in vivo Comet assay, Transgenic rodent somatic and
germ cell gene mutation (TGR) assays “Big Blue®”
Initial Tests for EFSA

35. Designing Specific Regulatory Programs
Conduct ICH battery if intake is > 1.5 µg/day (0.5 ppb in diet)
If < 1.5 µg/day then only in vitro assays (a and b) needed
a. Gene mutation in bacteria (Ames test)
b. In vitro cytogenetic test using mammalian cells
OR
In vitro mouse lymphoma gene mutation assay
c. In vivo test for chromosome damage in bone marrow (Micronucleus assay)
Regulations listed in Redbook 2000 (2007)
http://www.cfsan.fda.gov/~redbook/red-ivb2.html
Food Additives: US FDA, CFSAN

36. Designing Specific Regulatory Programs
New Tobacco Rule
Food and Drug Administration
21 CFR Parts 1100, 1140, and 1143
[Docket No. FDA–2014–N–0189] RIN 0910–AG38
Deeming Tobacco Products To Be Subject to the
Federal Food, Drug, and Cosmetic Act, as Amended
by the Family Smoking Prevention and Tobacco
Control Act; Restrictions on the Sale and Distribution
of Tobacco Products and Required Warning
Statements for Tobacco Products
• Effective August 8, 2016
Tobacco Products

37. Designing Specific Regulatory Programs
Regulated since June 22, 2009 (Tobacco Control Act)
Regulated since August 8, 2016 (New Tobacco Rule)
Cigarettes Roll-Your-Own Tobacco Smokeless Tobacco
Hookah
Tobacco
Dissolvables Pipe TobaccoE-Cigarettes
Cigars Novel and
Future Products
Tobacco Products - FDA

38. Designing Specific Regulatory Programs
FDA – New Tobacco Rule
• Substantial Equivalence
• New tobacco products may not be legally marketed in
the United States unless FDA has issued an order
permitting their marketing
• A substantially equivalent tobacco product has been
found by FDA to either have the same characteristics
as a predicate tobacco product; or has different
characteristics than the predicate tobacco product but
the information submitted demonstrates that the new
product does not raise different questions of public
health
Tobacco Products

39. Designing Specific Regulatory Programs
FDA Guidance – Nonclinical Studies
• Your nonclinical investigation should evaluate the
toxicity, abuse liability and carcinogenicity of your
new tobacco product as compared to other
tobacco products on the market
• You should generate data to evaluate these product
properties using some combination of in vitro, in
vivo, and/or ex vivo studies
Tobacco Products

40. Designing Specific Regulatory Programs
EU Tobacco Products Directive
The Directive lays down rules governing the
manufacture, presentation and sale of tobacco and
related products. These include cigarettes, roll your
own tobacco, pipe tobacco, cigars, cigarillos,
smokeless tobacco, electronic cigarettes and herbal
products for smoking.
Applicable 20 May 2016
Tobacco Products

41. Designing Specific Regulatory Programs
Proposed algorithm for using a toxicological battery
Tobacco Products
Michael D. Johnson et al. Cancer Epidemiol Biomarkers Prev
2009;18:3263-3304
1This algorithm only applies to in vitro toxicology testing, and it is assumed that other studies will be done
assessing the physical design and chemical analyses. 2Choice of assays may be informed by smoke chemistry
testing.

42. Designing Specific Regulatory Programs
42
Tobacco Products
Cytotoxicity
assays for
cigarette smoke,
whole smoke and
gas vapor phase
smoke
Cytotoxicity
assays used for
smokeless
tobacco extracts
Proliferation
assays for
cigarette smoke
Proliferation
assays for
smokeless
tobacco extracts
Genotoxicity
assays for
cigarette smoke
Apoptosis testing
for cigarette
smoke
Acid phosphatase
activity
Cell Survival Assay Attachment assay BrdU incorporation Sister chromatid
exchange
Annex V and PI
By FACS
ATP
bioluminescence
assay
DAPI staining BrdU incorporation
FACS analysis
Colony Formation
Assay
Chromosomal
Aberrations
Caspase 3
Activation Assay
Cell Viability assay LDH release MTT [3H] Thymidine
uptake
Micronuclei
formation
Microscopy -
Giemsa and
Acridine orange
LDH release Neutral red Survival/Proliferatio
n assay
MTS Ames test
performed with
cigarette smoke
TUNEL staining
MTT MTT WST-8 MTT PI labeling by Flow
Neutral red Trypan blue dye
exclusion
XTT assay
Trypan blue dye
exclusion

43. Designing Specific Regulatory Programs
43
 EU 7th Amendment to Cosmetics Directive
 Prohibits animal testing for cosmetics in EU
 Ban on in vivo genotoxicity assays March 2009
 Ban on repeat dose studies implemented 3/11/13
 3R’s Replacement, Reduction, Refinement
 Impacts all Global companies
 US Federal Food, Drug, Cosmetic Act (FFDCA)
enforced by FDA; Companies required to ensure
safety of cosmetics but no registration required
and no specific test guidelines provided
Regulations Impacting Specific Products, e.g., Cosmetics
Tier 1 In vitro genotoxicity assays
• Bacterial mutation – Ames
• Mammalian mutation
• Chromosomal damage
Tier 2 In vivo genotoxicity tests
• In vivo micronucleus (bone marrow)
• In vivo Comet
• Other

44. Designing Specific Regulatory Programs
44
Scientific Committee on Consumer Safety (SCCS)
Notes of Guidance for the Testing of Cosmetic Substances and Their Safety
Evaluation 8th Revision Dec 2012
Product Specific Guidelines
– Salmonella/E. coli Ames Assay AND
– In vitro mammalian mutation assay AND
– In vitro micronucleus assay OR
in vitro chromosome aberration assay
Follow-up approaches for positive results
3D Skin Micronucleus Assay- Shambhu Roy
3D Skin Comet Assay
Cell Transformation Assay
Pfuhler et al. Reg. Tox Pharm. 57:351-324, 2010
Addendum April 2014

45. SCOTT HICKMAN, MBA
HEAD OF TOXICOLOGY MARKETING
PHONE: +1 301-610-2913
SCOTT.HICKMAN@BIORELIANCE.COM
ROHAN KULKARNI, MSC, PHD, ERT
DIRECTOR, TOXICOLOGY STUDY MANAGEMENT
PHONE: +1 301 610 2140
EMAIL: ROHAN.KULKARNI@BIORELIANCE.COM
Thank you for your attention!
DIANE BRECHA
GLOBAL MANAGER TOXICOLOGY SALES
PHONE: + 240-447-4945
EMAIL: DIANE.BRECHA@SIAL.COM