This document provides an overview of screening tests for toxic chemicals. It defines screens as simplified tests designed to identify agents requiring further evaluation or exclude them from further testing. Key purposes of screens include economic savings, increased speed and number of chemicals evaluated while decreasing animal use. Screens must be valid, sensitive, reproducible and practical. The document discusses criteria for validation and acceptance of new testing methods and provides examples of in vitro and in vivo screens used in toxicology. It also discusses how screens fit within the regulatory testing structure and notes some limitations of screens in fully characterizing toxicity.

1. Screening Tests for Toxic
Chemicals: An Overview
Joseph F. Holson, Ph.D.
WIL Research Laboratories
Contributions from:
02/11/14
C. Chengelis, Ph.D., D.A.B.T.
M. Nemec, B.S., D.A.B.T.
B. Varsho, B.S.
1

2. Screens: Definition
Screens are simplified tests/studies or
models designed or used and conducted to
identify agents having a certain set of
attributes or characteristics that will either
exclude them from further investigation or
cause them to be assigned for further (more
rigorous) evaluations.
2

3. Traditional View of Screens
 Screens
are best described as short-term
experiments used to select and or sort a
series of molecules for a particular specific
trait.
 May be used to presage potential hazard
identification, but results are not generally
used in risk assessment.
3

4. Selected Purposes of Screens
 Economic
savings
 Increase speed
 Creation of data base for QSAR
 Reduced chemical (test article) requirements
 Decrease use of intact animals
 Increase number of chemicals evaluated
 Increase attrition of development candidates
 Evaluate potency/selectivity
Pharmaceutical Development vs. Chemical Safety Evaluation
4

5. Necessary Attributes of
Screens
 Validity

False positives vs. false negatives
 Sensitivity

Level of concern (mild, moderate, severe)
 Practicability

Economic
 Reproducibility

Intra- and inter-laboratory over time
5

6. Key Terms
 Relevance
is extent to which a test is related
to the effect of interest and the test’s utility for
a specified purpose. (ICCVAM)
 Reliability
is a measure of the degree to
which a test can be performed reproducibly
within and among laboratories over time.
(ICCVAM)
6

7. Criteria for Test Method
Validation (ICCVAM)
1. Clear statement of proposed use
2. Biological basis/relationship to effect of
interest
3. Formal detailed protocol
4. Reliability assessed
5. Relevance assessed
6. Limitations described
7. All data available for review
8. Data quality: Ideally GLPs
9. Independent scientific peer review
7

8. Criteria For Test Method
Acceptance (ICCVAM)
1. Fits into the regulatory testing structure
2. Adequately predicts the toxic endpoint of interest
3. Generates data useful for risk assessment
4. Adequate data available for specified uses
5. Robust and transferable
6. Time and cost-effective
7. Adequate animal welfare consideration (3Rs)
8

9. Evolution Process for New Testing
Methods (ICCVAM)
Stage
Review Risk Assessment Methods
Research
Development
Prevalidation
Validation
Peer Review
Acceptance
Implementation
Outcome
Identify needs for new and/or
improved testing methods
Understand toxic mechanisms
Incorporate new science and
technology into test methods
Optimize standardized transferable
protocol
Further determination of reliability and
relevance
Independent peer review evaluation of
validation status
Determination of acceptability for
regulatory risk assessment
Effective use of new methods by
regulators/users
9

10. Examples of In Vitro Screens
in Toxicology
 The
use of specific receptor binding assays
to identify estrogenic substances.
 The use of SKINTEX to identify potential
dermal irritants
 The use of the Ames Assay to identify
chemicals that cause a mutation at a specific
locus
 The use of FETAX to identify potential
ecotoxins
10

11. The “Ames Assay” As a Screen
for Potential Carcinogens
 Probably
most widely used in vitro test for the
identification of mutagens
 Rapid, economical, high through-put
 Well validated and standardized methods:

Reverse Mutations - test strains of Sal. Typh.
revert back to wild type in histidine free medium
 Examination


of the NTP Data base
positive with 45% of carcinogens
negative with 86% of non-carcinogens
11

12. The SKINTEX as a Screen for
Dermal Irritants
 Rapid,
economical, high through-put
 Well validated and standardized methods:



Keratin/collagen membrane barrier disruption
leads to dye release
Over 5,300 test samples studied in validation;
results with any one chemical very reproducible
80-89% correlation with Draize scoring
 Negatives
generally confirmed in vivo
12

13. EDSTAC Recommendations
In Vitro
Tier I
In Vivo
Invertebrates
Fish
Amphibian In Vivo
Avian
Mammals
E
A
T
13
Tier II

14. Attributes of In Vivo
Toxicology Screens
 Reduced
group size
 Reduced number of endpoints
 Reduced histopathology component
 Reduced exposure regime and total conduct
time
 Not always intended to be GLP compliant
 Will identify potent toxicants if appropriate
endpoints are included or are correlated
Power and endpoints equated to simplification
14

15. Examples of In Vivo Screens in
Toxicology
 The
mouse micronucleus test for genetic
toxicity (clastogenesis)
 The canine cardiac sensitization test
 The screening developmental toxicity study in
mice for teratogenic retinoids “class”
 The Hershberger assay in rats for androgenic
substances
 Kavlock-Chernoff assay for developmental/
reproductive effects
15

16. Examples of In vivo Screens in
Toxicology (continued)
RACB Reproductive Assessment by Continuous
breeding in mice
 The Dominant-Lethal Assay in rats for germ cell
mutation
 The Local Lymph Node Assay in mice for
delayed hypersensitivity (Type IV
Immunotoxicity)
 The Sheep Red Blood Cell Assay for
immunomodulation
 The p53 mouse assay for carcinogenicity

16

17. The “Local Lymph Node Assay” As a
Screen for Potential Sensitizers
 Well
validated in multiple laboratories
 Successful in identifying weak sensitizers
 Gained increased regulatory acceptance
 Replacement for traditional Guinea Pig
Protocols



Examines only induction (5 days vs. 6 weeks)
Mice less expensive than guinea pigs
Quantifiable endpoints
17

18. Screens in the Regulatory
Environment

SIDS -Screening Informational Data Set






Mandated by OECD
Thousands of chemicals in commerce not tested
Minimum data to set testing priorities
Physical Chemical Data (9), Environmental Fate (4),
Ecotoxicology (5), Mammalian Toxicology (6)
Far from being screens, each category requires a
series of robust studies
Considered screens only in so far as the results are
used to rank and prioritize
18

19. SIDS : Mammalian Toxicology

Acute Toxicology - 401, 402, 402, 420, 423, 425


Repeated-Dose - 407, 410, 412



Oral preferred. Justification for other routes
Combined 422 acceptable
Genetic Toxicology - 471, 476, 473, 477, 474, 475



Oral, Inhalation, Dermal
Gene Mutation (with bacteria)
Chromosomal Aberration (non-bacteria)
Toxicity to Reproduction - 414, 415, 416, 421, 422



Prenatal Development (414) + 407 acceptable
One- (415) or Two-Generation (416) studies
421 and combined 422 acceptable
19

20. SIDS: Mammalian Toxicology

Repeated-Dose 407, 410, 412
14-28 Days of Dosing
 Decreased Group Size (5 vs. 10/sex)
 Hematology and Clinical Pathology
 Smaller Organ List
 Recovery
 Functional Observation Batteries
 Multiple Endpoints
 No TK Requirement

20

21. DART Rodent Screening Studies
A
B
C
D
E
F
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
OECD Reproduction Screen
4W 2W
Estrous Cyclicity
Implantation Sites
OECD 421/422, OPPTS 870.3550/3650
Mating
Fertility
Chernoff-Kavlock Assay
Limited:
Malformations
Dev. Variations
Parturition
Gestation Length
Litter Size
Histopathology
Pup Viability
Pup Weight
Organ Weights
OPPTS 870.3500
Limited:
Malformations
Dev. Variations
Dominant Lethal Assay
Zygote/Embryolethality
OECD 478
OPPTS 870.5450
Assess recovery through
multiple mating trials
EDSTAC Assays
Weaning to Sexual Maturity
Uterotrophic
Females
Estrogenicity
Anti-Estrogenicity
Pubertal Assay
PND 21
Vaginal Opening
Thyroid Endpoints
PND 42
PND 21
PND 53
Pubertal Assay
Males
Preputial Separation
Thyroid Endpoints
Hershberger
Androgenicity
Anti-Androgenicity
Denotes Dosing Period

22. WIL’s Experience with OECD
421 and 422 Screens
Dose range-finding, antecedent to
2generation reproduction study
2)
To more economically demonstrate absence of
toxicity in innocuous classes of chemicals at limit
doses (30 studies)
3)
Use as apical regulatory study
Not to:
1)
More economically select candidates from a group
of chemicals for further definitive assessment of
reproductive toxicity
1)
22

23. Reproduction Screens at WIL
Number of Screens Conducted
with Reproductive Endpoints
42
Stand-Alone Screens
20
Screens Followed by 2-Generation
or Other Definitive Study
22*
*Nine sets not yet reported
23

24. Adult Concordance at MTD:
Not All HPVs
Screen (S)
F0
Endpoints
Dose Extrapolated
2-Generation (2-G)
F0
F1
Concordance (%)
2-G F0 vs. S F0
2-G F1 vs. S F0
5/9 with Extrapolated Dose Levels
Adult
Mortality
2/9
1/9
3/8
89%
75%
Body Weight
5/9
8/9
7/8
67%
63%
Food Consumption
5/9
8/9
6/8
67%
50%
Clin Obs
4/9
3/9
2/8
67%
63%
Fertility Index
0/9
0/9
0/8
100%
100%
Mating Index
0/9
0/9
0/8
100%
100%
Implantation Index
3/9
2/9
1/8
89%
88%
PI Loss
3/8
1/9
1/8
89%
88%
Live Birth Index
3/8
1/8
1/8
63%
63%
Organ Weights
3/6
4/8
3/8
67%
80%
Gestation Length
0/8
0/8
0/8
100%
100%
Dystocia
0/7
0/8
0/8
100%
100%
24
Reflects false positive resolution in apical study

25. Other Concordance at MTD
Screen (S)
F0
Endpoints
Dose Extrapolated
2-Generation (2-G)
F0
F1
Concordance (%)
2-G F0 vs. S F0
2-G F1 vs. S F0
5/9 with Extrapolated Dose Levels
Pup
Survival
2/8
1/8
2/8
63%
75%
Body Weight
3/8
5/8
5/8
75%
75%
Endpoints Specific to 2-Generation Study
Histopathology
1/1
5/8
5/8
Estrous Cyclicity
1/2
1/9
0/7
0/8
0/7
1/3
1/3
Balanopreputial
1/7
--
Vaginal Patency
1/7
--
Pup Organ Weights
2/7
2/7
Ovarian Follicles
0/2
2/6
Spermatogenesis
Anogenital Distance
1/1
25
Concordance percentages based on small numbers

26. 3 Case Studies (False
Negatives in Screens)
Endpoints
Screen (S)
F0
Dose Extrapolated
2-Generation (2-G)
F0
F1
Concordance (%)
2-G F0 vs. S F0 2-G F1 vs. S F0
0/3 with Extrapolated Dose Levels
Adult
Fertility Index
0/3
0/3
3/3
100%
0%
Mating Index
0/3
0/3
3/3
100%
0%
Live Birth Index
1/3
1/3
2/3
100%
67%
Organ Weights
0/1
2/3
2/2
(33%)
(0%)
Gestation Length
0/3
0/3
1/3
100%
67%
Dystocia
0/3
1/3
1/3
67%
67%
0/3
1/3
2/3
67%
33%
Not Measured
1/3
?
(0%)
(0%)
Offspring
Sex Ratio
Hypospadias
( ) = Strict concordance could not be calculated
(endpoint not measured in screen) 26

27. Selected Reproductive Endpoints Exhibiting
Strong Signals from Rare Events/Low Incidence
Endpoint
Examples from WIL Research
Historical Control in Crl:CD(SD)IGS BR
Mean Viable Litter
Size
13.9 ± 1.02
decrease of ≥ 1
Mortality ≤ PND 4
Mean = 96.2%
Min/Max 91-95%
≤ 91%
Total Litter Loss
Mean = 0.94%
(10/1061)
1 is equivocal
2 is more significant
signal
Newborn Pup
Weights
Mean = 7.0g ± 0.23
range 6.5-7.4g
n = 1100 litters
≤ 6.5g strong signal

28. Case Study: Dystocia, Extended
Parturition and/or Pregnancy

2-generation with second mating phase of F1, vapor
inhalation, used industrially, OTC pharmaceutically
PPM
F0
300
500
700
0
0
0
2/24
3/26
0
0
0
0
1/17
F1-2nd

70
F1-1st

0
0
0
1/21
1/18
0/12
HC then: 2/333 = 0.60%
HC now: 4/1100 = 0.36%

29. Heuristic Axioms
 If
primary intent of screen is to reduce
number of animals used, we must be careful
to consider subsequent use of animals in
studies to clarify poor characterization of DR
curve (LOAEL, NOAEL, NOEL, TK).
 Also, if intent of screen is to reduce resource
consumption, an analogy to the above also
exists unless screen is applied to agents not
developed for biologic activity, with limited
human exposure and economic significance.
29

31. Screens vs. Preliminary
Studies
 Decision
to conduct a study has been made
 Preliminary studies are performed to provide
information to design a definitive study (one
used for risk assessment)
 Under most circumstances are not performed
to eliminate a test article from development,
although unexpected results can lead to that
decision.
31

32. Screens vs. Toxicity
Assessment
In the broadest sense, what is done in much of
nonclinical development and in all of hazard
identification phases of risk assessment may
be viewed a screening as the information will
be used to determine what additional work (if
any) may be required or, in fact, to determine if
the agent is commercially viable. (modified from
Zbinden et al., 1984)
32

33. Screens vs. Toxicity
Assessment
 Definitive
answers require definitive study
designs






Multiple dose groups
Large sample size
Multiple endpoints
Exposure assessment (PK)
Treatment regimen of appropriate length
Economy and speed of lesser concern
33

34. Example:
Screen vs. Definitive Study
 Case
history XCX-XX
Several short-term studies
 No evidence of neurotoxicity except with the
six-month study in dogs
 Vacuolation of the Medulla Oblongata
 FDA requested additional work

34

35. XCX-XX Definitive Neurotoxicity Study
in Dogs: Design
6 months, daily dosing, female dogs, 6/group
 standard body weight, feed consumption,
clinical observations
 FOBs pretest, Weeks 6,13,19, and 25
 At necropsy, fixation by perfusion
 Extensive neuropathology
 Recovery 2/group, 4-weeks
 (Note multiplicity of endpoints)

35

36. Definitive Neurotoxicity
Study in Dogs with XCX-XX
Lesion that developed only with chronic treatment
Animal 5XX1 100 mg/kg
Animal 5XX9 control
Medulla adjacent to Hypoglossal Nucleus (40X; H&E)
36

37. Definitive Neurotoxicity
Study in Dogs with XCX-XX
Selected FOB Findings (Cranial)
37

38. Definitive Neurotoxicity
Study in Dogs with XCX-XX
Selected FOB Findings (Cranial)
Finding
Control
High Dose
Norm. Menace React.
6/ 6
6/ 6
Norm. Vis Tracking
6/ 6
6/ 6
Norm. Ocular Position
6/ 6
6/ 6
Norm. Tongue Move.
6/ 6
6/ 6
Norm. Gag Reflex
5/ 6
6/ 6
38

39. Definitive Neurotoxicity
Study in Dogs with XCX-XX
 Summary
of Findings (Multiple Endpoints)
Caused lesion in medulla, specifically in the
hypoglossal nucleus
 Nature of lesion suggests intra-myelinic
vacuolation
 Recoverable/ Reversable
 Not accompanied by any functional deficits

39

40. EDSTAC Criteria for Screens





Detect all known modes of action for the
endocrine endpoints of concern
Include sufficient diversity among endpoints,
permitting weight-of-evidence conclusions
Maximize sensitivity to minimize false negatives
Include a sufficient range of taxonomic groups
among the test organisms to represent
differences in endocrine system and
metabolism
Relatively fast and efficient
40
EDMVS, 2002

41. Purpose of Tier 1
 To
distinguish chemical substances that
interact with the endocrine system from those
that do not.
 Upon completion of Tier 1, EPA and
stakeholders should be able to accept the
assignment that a chemical has (1) either low
or no potential for EAT activity, (2) or that it
has such potential.
41
EDMVS, 2002

42. Advantages of In Vitro Assays








Sensitivity to low concentrations
High specificity of response
Low cost
Small amount of chemical required
Assays can be automated for high throughput
Results can be used in conjunction with QSAR
models
Can be used for complex mixtures
Reduces or replaces animal use
42
EDMVS, 2002

43. In Vitro Tier 1 Screens
 ER
Binding / Reporter Gene Assay
 AR
Binding / Reporter Gene Assay
 Steroidogenesis
Assay with Minced Testis
43

44. Advantages of In Vivo Assays






Account for absorption, distribution, metabolism
and excretion
Evaluate a broad range of mechanisms
Provide a comprehensive evaluation of the whole
endocrine system as a unit
Generally well-accepted methods in toxicity
testing
Some endpoints are toxicologically relevant and
have been used in hazard assessment
Give comparative perspective to other endpoints
of toxicity
44
EDMVS, 2002

45. In Vivo Tier 1 Screens

Rodent 3-Day Uterotrophic Assay
 Longstanding
assay, international validation complete

Rodent 20-Day Pubertal Female Assay with Thyroid

Rodent 5- to 7-Day Hershberger Assay
 Longstanding

assay, international validation underway
Frog Metamorphosis Assay
 Use
as a general vertebrate model called vague &
unsubstantiated, rat already sensitive species for thyroid
(Mihaich, 2002)

Fish Reproduction Screening Assay
 CLA
claims too long & too many apical endpoints to be a
screen, but not robust enough to be a test (2002)
45

46. Alternate Tier 1 Screens
 Aromatase
 Pubertal
 Adult
Inhibition
Male
14–Day Intact Male
 Preferred
by industry over HershbergerPubertal combination
 A, E, PL, T, SSI, PG, PRL
46

47. EDSTAC Tier 2 Tests

Avian Reproduction (with bobwhite quail and mallard)


Variable reproduction parameters; CLA suggests Japanese quail
Limited laboratory capacity (CLA, 2002)

Fish Life Cycle (fathead minnow)

Mysid Life Cycle (Americamysis)


Extrapolation of ecdysteroid to EAT activity unjustified (Mihaich,
Verslycke, 2002)
Debate over need for a 2-gen over a 1-gen (Mihaich, 2002)

Amphibian Development and Reproduction (Xenopus)

Two-Generation Mammalian Reproductive Toxicity Study

Under prevalidation now, early results from PTU demonstration raise
questions about interlaboratory methodology
47

48. Standard Designs
A
B
C
D
E
F
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
Fertility Study
10W
4W
2W
Estrous Cyclicity
Mating
Fertility
Implantation Sites
Pre-Implantation Loss Spermatogenesis
ICH 4.1.1
Corpora Lutea
ƒ
Prenatal Development
CMAX
AUC
ICH 4.1.3 OECD 414
OPPTS 870.3600
870.3700
Postimplantation Loss
Viable Fetuses
Malformations & Variations
Fetal Weight
F0
ƒ
Pre- and Postnatal Development
CMAX
ICH 4.1.2
AUC
F1
Parturition
Gestation Length
F1 Mating and Fertility
????????????????
Litter Size
Pup Viability
Pup Weight
Organ Weights
Landmarks of Sexual Development
Neurobehavioral Assessment
Acoustic Startle Response
Motor Activity
Learning & Memory
Single- and Multigenerational
OECD 415, OECD 416, OPPTS 870.3800, FDA Redbook I, NTP RACB
Estrous Cyclicity
Mating
Fertility
Corpora Lutea
Implantation Sites
Pre-Implantation Loss
Spermatogenesis
Satellite Phase
Postimplantation Loss
Viable Fetuses
Malformations
Variations
Fetal Weight
F1
????????????????
Parturition
F2
????????????????
Gestation Length
Pup Viability
Litter Size
Landmarks of Sexual Development
Pup Weight
Neurobehavioral Assessment
Organ Weights
Acoustic Startle Response
F1 Mating and Fertility
Motor Activity
Hormonal Analyses
Learning & Memory
Ovarian Quantification
Histopathology
Premature Senescence
Denotes Dosing Period