04/12/23 1

Screening Tests for Toxic Chemicals: An Overview

Joseph F. Holson, Ph.D. WIL Research Laboratories

Contributions from: C. Chengelis, Ph.D., D.A.B.T. M. Nemec, B.S., D.A.B.T.

B. Varsho, B.S.

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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.

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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.

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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

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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

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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)

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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

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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)

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Evolution Process for New Testing Methods (ICCVAM)

StageStage OutcomeOutcome

Incorporate new science and technology into test methods

Further determination of reliability and relevance

Independent peer review evaluation of validation status

Optimize standardized transferable protocol

Effective use of new methods byregulators/users

Identify needs for new and/or improved testing methods

Understand toxic mechanisms

Determination of acceptability for regulatory risk assessment

Acceptance

Validation

Peer Review

Prevalidation

Development

Research

Review Risk Assessment Methods

Implementation

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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

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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

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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

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Tier IIIn VivoTier I

In Vitro

In Vivo

E TA

EDSTAC Recommendations

Invertebrates

Fish

Amphibian

Avian

Mammals

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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

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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

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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

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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

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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

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SIDS : Mammalian Toxicology

Acute Toxicology - 401, 402, 402, 420, 423, 425 Oral, Inhalation, Dermal

Repeated-Dose - 407, 410, 412 Oral preferred. Justification for other routes Combined 422 acceptable

Genetic Toxicology - 471, 476, 473, 477, 474, 475 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

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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

DART Rodent Screening StudiesA B C D E F

Premating to Conception

Conception to Implantation

Implantation to Closure of Hard Palate

Birth to Weaning Weaning to Sexual Maturity

Hard-Palate Closure to End of Pregnancy

Denotes Dosing Period

OPPTS 870.3500 Chernoff-Kavlock Assay

OECD 478OPPTS 870.5450

OECD 421/422, OPPTS 870.3550/36502W4W

Estrous Cyclicity MatingImplantation Sites Fertility

Dominant Lethal Assay Zygote/Embryolethality

Limited: Malformations Dev. Variations

Parturition Pup ViabilityGestation Length Pup WeightLitter Size Organ WeightsHistopathology

Limited: Malformations Dev. Variations

Assess recovery through multiple mating trials

OECD Reproduction Screen

EDSTAC Assays

Weaning to Sexual Maturity

Females

Males

PND 21

Uterotrophic

Hershberger

PND 21

PND 53

Pubertal Assay

EstrogenicityAnti-Estrogenicity

Pubertal Assay

PND 42Vaginal OpeningThyroid Endpoints

Preputial SeparationThyroid Endpoints

AndrogenicityAnti-Androgenicity

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WIL’s Experience with OECD 421 and 422 Screens

1) Dose range-finding, antecedent to 2-generation 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

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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

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Adult Concordance at MTD: Not All HPVs

Screen (S)

F0 F0 F1 2-G F0 vs. S F0 2-G F1 vs. S F0

Dose Extrapolated

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%

2-Generation (2-G) Concordance (%)

Endpoints

Adult

5/9 with Extrapolated Dose Levels

Reflects false positive resolution in apical study

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Other Concordance at MTD

Screen (S)

F0 F0 F1 2-G F0 vs. S F0 2-G F1 vs. S F0

Dose Extrapolated

Survival 2/8 1/8 2/8 63% 75%

Body Weight 3/8 5/8 5/8 75% 75%

Histopathology 1/1 5/8 5/8

Estrous Cyclicity 1/2 1/9 0/7

Spermatogenesis 0/8 0/7

Anogenital Distance 1/1 1/3 1/3

Balanopreputial 1/7 --

Vaginal Patency 1/7 --

Pup Organ Weights 2/7 2/7

Ovarian Follicles 0/2 2/6

Pup

Endpoints Specific to 2-Generation Study

2-Generation (2-G) Concordance (%)

Endpoints

5/9 with Extrapolated Dose Levels

Concordance percentages based on small numbers

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3 Case Studies (False Negatives in Screens)

( ) = Strict concordance could not be calculated (endpoint not measured in screen)

Screen (S)F0 F0 F1 2-G F0 vs. S F0 2-G F1 vs. S F0

Dose Extrapolated

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%

Sex Ratio 0/3 1/3 2/3 67% 33%

Hypospadias Not Measured 1/3 ? (0%) (0%)

Offspring

2-Generation (2-G) Concordance (%)

Endpoints

Adult

0/3 with Extrapolated Dose Levels

Selected Reproductive Endpoints Exhibiting Strong Signals from Rare Events/Low Incidence

EndpointExamples from WIL Research Historical Control in Crl:CD(SD)IGS BR

Mean Viable Litter Size

13.9 1.02 decrease of 1

Mortality PND 4Mean = 96.2% Min/Max 91-95%

91%

Total Litter LossMean = 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

Case Study: Dystocia, Extended Parturition and/or Pregnancy

2-generation with second mating phase of F1, vapor inhalation, used industrially, OTC pharmaceutically

PPM 0 70 300 500 700

F0 0 0 0 2/24 3/26

F1-1st 0 0 0 0 1/17

F1-2nd 0 0 1/21 1/18 0/12

HC then: 2/333 = 0.60% HC now: 4/1100 = 0.36%

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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.

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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.

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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)

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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

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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

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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)

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Definitive Neurotoxicity Study in Dogs with XCX-XX

Animal 5XX9 controlAnimal 5XX1 100 mg/kg

Lesion that developed only with chronic treatment

Medulla adjacent to Hypoglossal Nucleus (40X; H&E)

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Definitive Neurotoxicity Study in Dogs with XCX-XX

Selected FOB Findings (Cranial)

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Finding Control High DoseNorm. 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

Selected FOB Findings (Cranial)

Definitive Neurotoxicity Study in Dogs with XCX-XX

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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

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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 efficientEDMVS, 2002

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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.

EDMVS, 2002

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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

EDMVS, 2002

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In Vitro Tier 1 Screens

ER Binding / Reporter Gene Assay

AR Binding / Reporter Gene Assay

Steroidogenesis Assay with Minced Testis

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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 toxicityEDMVS, 2002

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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)

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Alternate Tier 1 Screens

Aromatase Inhibition

Pubertal Male

Adult 14–Day Intact Male Preferred by industry over Hershberger-

Pubertal combination A, E, PL, T, SSI, PG, PRL

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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

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

Parturition Litter Size Landmarks of Sexual DevelopmentGestation Length Pup Viability Neurobehavioral Assessment F1 Mating and Fertility Pup Weight Acoustic Startle Response

Organ Weights Motor Activity Learning & Memory

ParturitionGestation Length Pup Viability Litter SizeLandmarks of Sexual Development Pup WeightNeurobehavioral Assessment Organ Weights Acoustic Startle Response F1 Mating and Fertility Motor Activity Hormonal Analyses Learning & Memory Ovarian QuantificationHistopathology Premature Senescence

Postimplantation LossViable FetusesMalformations & VariationsFetal Weight

Postimplantation LossViable FetusesMalformationsVariationsFetal Weight

Estrous Cyclicity Mating Corpora Lutea Fertility Implantation SitesPre-Implantation Loss Spermatogenesis

Estrous CyclicityMatingFertilityCorpora LuteaImplantation SitesPre-Implantation LossSpermatogenesis

Denotes Dosing Period

Standard Designs

Single- and Multigenerational

Satellite Phase

OECD 415, OECD 416, OPPTS 870.3800, FDA Redbook I, NTP RACB

F1

F2 ????????????????

????????????????

Pre- and Postnatal Development

F1

ICH 4.1.2F0

????????????????

Prenatal DevelopmentICH 4.1.3 OECD 414

OPPTS 870.3600 870.3700

Fertility StudyICH 4.1.110W 2W4W

CMAX

AUC

CMAX

AUC