Alternative Toxicological Methods - H. Salem, S. katz (CRC, 2003) WW
Transcript of Alternative Toxicological Methods - H. Salem, S. katz (CRC, 2003) WW
ALTERNATIVETOXICOLOGICAL
METHODS
CRC PR ESSBoca Raton London New York Washington, D.C.
ALTERNATIVETOXICOLOGICAL
METHODS
Edited by
Harry SalemSidney A. Katz
This edition published in the Taylor & Francis e-Library, 2005.
“To purchase your own copy of this or any of Taylor & Francis or Routledge’scollection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.”
ISBN 0-203-00879-0 Master e-book ISBN
Dedication
Preface
The Editors
Contributors
Contents
PART I
Progress in the Validation and RegulatoryAcceptance of Alternatives
2 ALTERNATIVE TOXICOLOGICAL METHODS
3
CHAPTER 1
Historical Developments in the HumaneCare and Use of Research Animals:
The First 4000 Years
CONTENTS
BIBLICAL ORIGINS
4 ALTERNATIVE TOXICOLOGICAL METHODS
COLONIAL AMERICA
VICTORIAN ENGLAND
Table 1.1 Chronology for the Enactment of Animal Welfare Legislation
Year State Year State Year State
182818351838193818421845184818511851185218541856185718581859185918601861
New YorkMassachusettsConnneticutWisconsinNew HampshireMissouriVirginiaIowaMinnesotaKentuckyVermontTexasRhode IslandTennesseeKansasWashingtonPennsylvaniaNevada
18641864186718681868186918711871187118721873187318731875187918791880
IdahoOregonNew JerseyCaliforniaWest VirginiaIllinoisDistrict of ColumbiaMichiganMontanaColoradoDelawareIndianaNebraskaGeorgiaArkansasLouisianaMississippi
18801881188118831883188418871887188918901891189318951898191319131921
OhioNorth CarolinaSouth CarolinaAlabamaMaineHawaiiNew MexicoSouth DakotaFloridaMarylandNorth DakotaOklahomaWyomingUtahAlaskaArizonaVirgin Islands
THE FIRST 4000 YEARS 5
6 ALTERNATIVE TOXICOLOGICAL METHODS
RUSSELL AND BURCH
THE THREE Rs
THE FIRST 4000 YEARS 7
REFERENCES
9
CHAPTER 2
A History of Interagency Approachesto Alternatives and Establishment of the
Interagency Regulatory Alternatives Group
CONTENTS
INTRODUCTION
10 ALTERNATIVE TOXICOLOGICAL METHODS
ESTABLISHMENT OF IRAG
A HISTORY OF INTERAGENCY APPROACHES TO ALTERNATIVES 11
IRAG WORKSHOPS
12 ALTERNATIVE TOXICOLOGICAL METHODS
EVOLUTION OF ICCVAM
A HISTORY OF INTERAGENCY APPROACHES TO ALTERNATIVES 13
REFERENCES
15
CHAPTER 3
The Interagency Coordinating Committeeon the Validation of Alternative
Methods (ICCVAM): Recent Progressin the Evaluation of Alternative
Toxicity Testing Methods
CONTENTS
16 ALTERNATIVE TOXICOLOGICAL METHODS
INTRODUCTION
BACKGROUND AND HISTORY OF THE ICCVAM
ICCVAM EVALUATION OF ALTERNATIVE METHODS 17
Table 3.1 Member Agencies
Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)Consumer Product Safety CommissionDepartment of DefenseDepartment of EnergyDepartment of Health and Human Services
Agency for Toxic Substances and Disease RegistryFood and Drug AdministrationNational Institute for Occupational Safety and HealthNational Institutes of Health, Office of the DirectorNational Cancer InstituteNational Institute of Environmental Health SciencesNational Library of Medicine
Department of the InteriorDepartment of Labor
Occupational Safety and Health AdministrationDepartment of Transportation
Research and Special Programs AdministrationDepartment of AgricultureEnvironmental Protection Agency
Table 3.2 Test Method Validation and Acceptance Criteriaa
Validation Criteria
Clear statement of proposed useBiological basis/relationship to effect of interest providedFormal detailed protocolReliability assessedRelevance assessedLimitations describedAll data available for reviewData quality: Ideally good laboratory practices (GLPs)Independent scientific peer review
Acceptance Criteria
Fits into the regulatory testing structureAdequately predicts the toxic endpoint of interestGenerates data useful for risk assessmentAdequate data available for specified usesRobust and transferableTime and cost effectiveAdequate animal welfare consideration (3 Rs)
a These are shortened versions of the adopted criteria. For the full text see: National Instituteof Environmental Health Sciences (NIEHS), Validation and Regulatory Acceptance ofToxicological Test Methods: A Report of the ad hoc Interagency Coordinating Committeeon the Validation of Alternative Methods (ICCVAM), NIH publication 97-3981, ResearchTriangle Park, NC, 1997.
18 ALTERNATIVE TOXICOLOGICAL METHODS
Establishment of the ICCVAM
ICCVAM Authorization Act of 2000
Table 3.3 The Purposes of the ICCVAMa
Increase the efficiency and effectiveness of federal agency test method reviewEliminate unnecessary duplicative efforts and share experiences between federal regulatory agencies
Optimize use of scientific expertise outside the federal governmentEnsure that new and revised test methods are validated to meet the needs of federal agencies
Reduce, refine, or replace the use of animals in testing where feasible
a ICCVAM Authorization Act (U.S. Code, 2000).
ICCVAM EVALUATION OF ALTERNATIVE METHODS 19
THE NATIONAL TOXICOLOGY PROGRAM INTERAGENCY CENTER FOR THE EVALUATION OF ALTERNATIVE
TOXICOLOGICAL METHODS (NICEATM)
The ICCVAM Scientific Advisory Committee
Table 3.4 The Duties of the ICCVAMa
Consider petitions from the public for review and evaluation of new and revised test methods for which there is evidence of scientific validity
Coordinate the technical review and evaluation of new and revised test methods of interagency interest
Submit ICCVAM test recommendations to each appropriate federal agencyFacilitate and provide guidance on validation criteria and processesFacilitate:
Interagency and international harmonization of test protocols that encourage the reduction, refinement, and replacement of animal test methodsAcceptance of scientifically valid test methods and awareness of accepted methods
Make ICCVAM final test recommendations and agency responses available to the publicPrepare reports on the progress of this act and make these available to the public
a ICCVAM Authorization Act (U.S. Code, 2000).
20 ALTERNATIVE TOXICOLOGICAL METHODS
THE ICCVAM TEST METHOD EVALUATION PROCESS
Test Method Validation
Test Method Submissions
ICCVAM EVALUATION OF ALTERNATIVE METHODS 21
ICCVAM Interagency Working Groups
Independent Scientific Peer-Review Panels
Figure 3.1 ICCVAM test method evaluation process.
NTP InteragencyNTP InteragencyCenter for the EvaluationCenter for the Evaluation
ofofAlternative ToxicologicalAlternative Toxicological
Methods (NICEATM)Methods (NICEATM)
Test SponsorTest SponsorSubmission of TestSubmission of Test
MethodMethod
Interagency CoordinatingInteragency CoordinatingCommittee on theCommittee on the
Validation of AlternativeValidation of AlternativeMethods (ICCVAM)Methods (ICCVAM)
Test RecommendationsTest Recommendationsto Agenciesto Agencies
Peer Review PanelsPeer Review PanelsExpert WorkshopsExpert Workshops
Advisory CommitteeAdvisory Committeeon Alternativeon AlternativeToxicologicalToxicological
MethodsMethods
AgencyAgencyDecisions/ActionsDecisions/Actions
ICCVAM InteragencyICCVAM InteragencyWorking GroupsWorking Groups
Report
22 ALTERNATIVE TOXICOLOGICAL METHODS
Expert Panels and Workshops
ICCVAM EVALUATION OF ALTERNATIVE METHODS 23
ICCVAM Test Recommendations
REGULATORY AGENCY CONSIDERATION OF ICCVAM RECOMMENDATIONS
ICCVAM TEST METHOD EVALUATIONS
The Local Lymph Node Assay
24 ALTERNATIVE TOXICOLOGICAL METHODS
Skin Corrosivity
Frog Embryo Teratogenesis Assay in Xenopus (FETAX)
ICCVAM EVALUATION OF ALTERNATIVE METHODS 25
Up-and-Down Procedure for Acute Oral Toxicity
In Vitro Methods for Assessing Acute Systemic Toxicity
26 ALTERNATIVE TOXICOLOGICAL METHODS
SUMMARY
ACKNOWLEDGMENTS
REFERENCES
ICCVAM EVALUATION OF ALTERNATIVE METHODS 27
28 ALTERNATIVE TOXICOLOGICAL METHODS
ICCVAM EVALUATION OF ALTERNATIVE METHODS 29
30 ALTERNATIVE TOXICOLOGICAL METHODS
31
CHAPTER 4
Validation and Regulatory Acceptanceof Alternative Test Methods: Current
Situation in the European Union
CONTENTS
INTRODUCTION
32 ALTERNATIVE TOXICOLOGICAL METHODS
VALIDATION AND REGULATORY ACCEPTANCE
ACCEPTANCE OF ALTERNATIVE TESTS IN THE EUROPEAN UNION 33
34 ALTERNATIVE TOXICOLOGICAL METHODS
SKIN CORROSION
ACCEPTANCE OF ALTERNATIVE TESTS IN THE EUROPEAN UNION 35
36 ALTERNATIVE TOXICOLOGICAL METHODS
SKIN IRRITATION
ACCEPTANCE OF ALTERNATIVE TESTS IN THE EUROPEAN UNION 37
38 ALTERNATIVE TOXICOLOGICAL METHODS
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
ACCEPTANCE OF ALTERNATIVE TESTS IN THE EUROPEAN UNION 39
40 ALTERNATIVE TOXICOLOGICAL METHODS
ACCEPTANCE OF ALTERNATIVE TESTS IN THE EUROPEAN UNION 41
43
CHAPTER 5
Integrated In Vitro Approachesfor Assessing Systemic Toxicity
CONTENTS
INTRODUCTION
44 ALTERNATIVE TOXICOLOGICAL METHODS
THE STEPWISE APPROACH FOR INTEGRATED TESTING
THE ECITTS PROJECT
INTEGRATED IN VITRO APPROACHES FOR ASSESSING SYSTEMIC TOXICITY 45
Compounds and Test Battery
Figure 5.1 Building blocks of the ECITTS scheme.
Figure 5.2 (a) Native and (b) retinoic acid-differentiated SH-SY5Y cells.
1. Experimental 2. Modeling 3. Validation(literature data)
in vitro data on kinetics kinetic modeling kinetics in vivo
in vitro data on dynamics
(e.g., CNC, EC20, EC50)
prediction of target tissue
concentrations
(e.g., NOEL, LOEL)
prediction of dynamics
prediction of systemic
toxic doses
(e.g., NOED, LOED,
LD50)
in vivo systemic
toxic doses
(e.g., NOED, LOED,
LD50)
a b
46 ALTERNATIVE TOXICOLOGICAL METHODS
Results
Table 5.1 Endpoints Studied in the in Vitro Neurotoxicity Test Battery for the ECITTS Project
Endpoint Assay Toxicity level
Cytotoxicity/Inhibition of cell growth Total cellular protein content Basal cytotoxicityNeurite degeneration (ND) Number of neurites per cell MorphologyProtein synthesis rate (PSR) [3H] leucine incorporation in
proteins during 2 hrPhysiology
Basal intracellular free Ca2+
concentration (basal Ca2+)Fura-2/Ca2+, fluorescence Physiology
Voltage operated Ca2+ channels(VOCC)
High potassium-induced Ca2+
flux, fluorescenceNeurochemistry
Phospholipase C-coupled acetylcholine receptor signal transduction (mAChR peak)
Carbachol-activated, immediate transient Ca2+ peak,fluorescence
Neurochemistry
Acetylcholine-induced capacitive Ca2+
entry (mAChR plateau)Carbachol-activated, secondary Ca2+ plateau, fluorescence
Neurochemistry
INTEGRATED IN VITRO APPROACHES FOR ASSESSING SYSTEMIC TOXICITY 47
CONCLUSIONS AND FUTURE PERSPECTIVES
Refinement of the Neurotoxicity Test Battery
Table 5.2 Effects of the Test Compounds, Determined in Differentiated Human Neuroblastoma SH-SY5Y Cells, on in Vitro Endpoints as Presented in Table 5.1
Compound In vitro effectConcentration( M or M/hr)
Acrylamide 20% cytotoxicity 920Acrylamide CNC; neurite degeneration/time 11Caffeine CNC; inhibition of VOCC 10Diazepam CNC; inhibition of VOCC 49Lindane CNC; inhibition of VOCC 3.4Lindane 20% increased basal [Ca2+]i 35Lindane 50% cytotoxicity 150Parathion/paraoxon CNC; neurite degeneration/time 0.5Phenytoin CNC; inhibition of protein synthesis 87
CNC, critical cellular neurotoxic concentration; VOCC, voltage operated calcium channels.
48 ALTERNATIVE TOXICOLOGICAL METHODS
Different Exposure Times
Figure 5.3 Estimated versus experimental doses after (a) acute and/or (s.c.) subchronicexposure. See Table 5.1 for endpoint definitions. The line represents the identity.
0.001
0.01
0.1
1
10
10.0
100.0
0.001 0.01 0.1 1 10 100 1000
Lindane (CNC: inhibition of VOCC vs. LOED: learning; s.c.)Lindane (EC20: increased basal Ca2+ vs. LOED: convulsions; s.c.)Lindane (50% cytotoxicity vs. lowest LD50; a.)Lindane (50% cytotoxicity vs. highest LD50; a.)Acrylamide (20% cytotoxicity vs. LOED: gait; a.)Acrylamide (CNC: ND vs. LOED: startle response; 10 days)Acrylamide (CNC: ND vs. LOED: startle response; 30 days)Acrylamide (CNC: ND vs. LOED: startle response; 90 days)Caffeine (CNC: inhibited VOCC vs. LOED: anti-nociception; a.)Diazepam (CNC: inhibited VOCC vs. LOED: time to emerge; a.)Phenytoin (CNC: inhibition of PSR vs. operant learning; a.)Parathion/paraoxon (CNC of paraoxon: ND vs. LOED: tail-pinch response of parathion; a.)
Experimental doses (mg/kg) or (mg/kg/day)
Est
imat
ed d
oses
(m
g/kg
) or
(m
g/kg
/day
)
INTEGRATED IN VITRO APPROACHES FOR ASSESSING SYSTEMIC TOXICITY 49
ACKNOWLEDGMENTS
REFERENCES
50 ALTERNATIVE TOXICOLOGICAL METHODS
51
CHAPTER 6
Summary of the OECD’s New GuidanceDocument on the Recognition,
Assessment, and Use of ClinicalSigns as Humane Endpoints for
Experimental Animals Usedin Safety Evaluation
CONTENTS
INTRODUCTION
52 ALTERNATIVE TOXICOLOGICAL METHODS
DEVELOPMENT OF THE GUIDANCE DOCUMENT
SUMMARY OF THE OECD’S NEW GUIDANCE DOCUMENT 53
DEFINITIONS
54 ALTERNATIVE TOXICOLOGICAL METHODS
GUIDING PRINCIPLES
SUMMARY OF THE OECD’S NEW GUIDANCE DOCUMENT 55
INITIAL CONSIDERATIONS IN THE DESIGN OFANIMAL EXPERIMENTS
56 ALTERNATIVE TOXICOLOGICAL METHODS
RECOGNITION AND ASSESSMENT OF PAIN, DISTRESS, AND SUFFERING AS AN APPROACH TO DETECTING CLINICAL
SIGNS AND ABNORMAL CONDITIONS
SUMMARY OF THE OECD’S NEW GUIDANCE DOCUMENT 57
MAKING AN INFORMED DECISION TO HUMANELY KILL ANIMALS
Table 6.1 Common Conditions and Clinical Signs
Abdominal rigidityAbortionAgalactiaAnemiaAnalgesiaAnuriaApathyAtaxia/incoordinationBleedingBlepharospasmBlood in feces or urineBlood around nose, eyesBoarded abdomenBody temperature, abnormalBody weight loss or emaciation
Breathing difficulties (Dyspnea)
CachexiaChewing, persistentChromodachryorrheaCirclingComatoseCompulsive behaviorConstipationConvulsionsCorneal ulcerationCoughing/sneezingCyanosisDehydrationDiarrhea
Discharge, abnormalDyspnea (difficult breathing)Epistaxis (nasal bleeding)ExcitableEyelid closureEyes fixed/sunkenFractured boneGaspingGrooming—failure to doHunched/stiff postureHyperreflexiaImmobile/inactiveJaundice (icterus)Joints swollenKyphosisLateral positionLimping/lamenessLocomotory behaviorLordosisLoss of condition, body muscle
Mammary gland abnormalities
MoribundMotor excitationNot eating/drinkingOedemaPale mucous membranesParalysisParesisPiloerection
Pinna reflexProstratePruritisPupillary constriction/dilation
Rales, pulmonaryRectal prolapseRecumbency, prolongedRed eye(s)/noseReflexesRetention of fecesRighting reflexSalivation, excessive or abnormal
SeizuresSelf-mutilationSkin bruising/color/crepitusSpasmStaggeringSunken flanksSuppurationSwellingsTenesmusTetanyTremorUrine retentionVaginal prolapseVocalizationVomiting
58 ALTERNATIVE TOXICOLOGICAL METHODS
SEVERE PAIN AND DISTRESS AS CRITERIA FOR HUMANE KILLING
SUMMARY OF THE OECD’S NEW GUIDANCE DOCUMENT 59
GUIDANCE ON THE HUMANE CONDUCT OF SPECIFIC TYPES OF TOXICITY TESTING
REFERENCE
61
CHAPTER 7
Pain and Distress Management in AnimalResearch and Testing: The Humane
Society of the United States Painand Distress Initiative*
CONTENTS
62 ALTERNATIVE TOXICOLOGICAL METHODS
INTRODUCTION
PUBLIC CONCERN ABOUT PAIN AND DISTRESS IN RESEARCH
PAIN AND DISTRESS MANAGEMENT IN ANIMAL RESEARCH AND TESTING 63
THE CHALLENGE
64 ALTERNATIVE TOXICOLOGICAL METHODS
EXPERIMENTAL PROCEDURES THAT CAUSE PAIN AND DISTRESS IN RESEARCH
THE HSUS PAIN AND DISTRESS INITIATIVE
Outreach to IACUCs
PAIN AND DISTRESS MANAGEMENT IN ANIMAL RESEARCH AND TESTING 65
Table 7.1 Models and Areas of Research and Specific Techniques That Cause Distress
Research Models or Areas
Non-Pain-Induced Distress
AggressionAnxiety (e.g., Vogel conflict-drinking model)Cancer (tumor burden, cachexia, carcinogenicity testing)Depression (e.g., learned helplessness, forced swimming, maternal
deprivation)DiabetesDrug addiction and withdrawalEnvironmental stress (e.g., hot, cold)FearImmunological research (e.g., vaccine potency testing)Infectious diseaseMotion sicknessNutrition research (e.g., nutrient deprivation)PanicPharmacology (some) (e.g., tumor necrosis factor, capsaicin research)Psychopathology (other than anxiety, depression, fear, etc., mentioned above)Radiation researchStress (psychological)Toxicology (induced effects)Transgenic research
Pain-Induced Distress
ArthritisBurn researchCancer research (tumor pain)Chronic pain studies1
Dental studiesInflammation studiesExperimental surgery (e.g., organ transplantation/rejection)Muricide (as a model of aggression, neophobia, etc.)Orthopedic studiesTrauma research
Specific Techniques
Non-Pain-Induced and Pain-Induced Distress
Anesthesia aftereffectsAntibody production (polyclonal and monoclonal)Aversive stimuli (e.g., electric shock)Bleeding techniques (including retro-orbital bleeding)Complete Freund’s AdjuvantControl group (animals denied experimental treatments)Deprivation (e.g., water, food, sleep, or social partners/experiences)Dosing techniques (e.g., gavage)Granuloma techniquesGut loop studies
(continued)
66 ALTERNATIVE TOXICOLOGICAL METHODS
Regulatory Aspects
Table 7.1 (continued) Models and Areas of Research and Specific Techniques That Cause Distress
Knockout technologyRestraintSurgery sequelae
1 Acute pain should not be a problem if the guidelines of the International Association for theStudy of Pain (IASP, 1979) are followed.
PAIN AND DISTRESS MANAGEMENT IN ANIMAL RESEARCH AND TESTING 67
68 ALTERNATIVE TOXICOLOGICAL METHODS
Table 7.2 The USDA’s Current Pain and Distress Categories and a Proposed Modification, as Well as Related Features of the Two Systems
A. Current Scheme
USDA Category
Pain and/or Distress
Anesthesia/Analgesia
Full ACUC Review
AlternativeLiterature Search
C (63%)1 Little or None No Yes NoD (29%) Yes or No2 Yes Yes YesE (8%) Yes No Yes Yes
B. Proposed Scheme
CategoryPain and/or
DistressAnesthesia/Analgesia
Full IACUC Review
AlternativeLiterature Search
I Minor or None No No NoII Minor or None Yes Perhaps PerhapsIII Moderate Yes or No Yes YesIV Severe Yes or No Yes Yes
1 Numbers in parentheses are USDA figures for 2000.2 Animals listed in USDA category D were given pain- or distress-relieving drugs, but
these drugs may not have been sufficient to relieve all pain and distress throughout theexperiment.
PAIN AND DISTRESS MANAGEMENT IN ANIMAL RESEARCH AND TESTING 69
Financial Support for Research on Pain and Distress
Table 7.3 State to State Variation in Reporting Animal Use in Column E (Unalleviated Pain or Distress) for States Using Greater Than 20,000 Animals
State
Percentage of Animals
in Column E State
Percentage of Animals
in Column E
Nationwide 8 Missouri 22 California 4 Nebraska 15Delaware 15 New Jersey 6Georgia 2 New York 11Illinois 3 North Carolina 6Indiana 18 Ohio 3Iowa 28 Pennsylvania 8Kansas 16 Texas 3Maryland 12 Virginia 0Massachusetts 1 Washington 20Michigan 15 Wisconsin 9Minnesota 3 Federal Agencies 7
Note: USDA data from 2000.
Table 7.4 States That Reported Less Than 1% of Animal Use in Column E between 1995 and 1997
Alaska (300) Mississippi (2,000) Tennessee (10,900)Arizona (5,000) Nevada (3,000) Utah (4,600)Hawaii (500) Oklahoma (4,300) Vermont (1,100)Kentucky (5,300) Oregon (4,700) Virginia (19,200)Louisiana (16,800) Rhode Island (2,100) West Virginia (1,700)Maine (800) S. Carolina (6,100) Wyoming (300)
Data from the USDA. Figures in parentheses indicate the average number of animalsused across all pain categories.
70 ALTERNATIVE TOXICOLOGICAL METHODS
Development of a Technical Report on Animal Pain and Distress
BEST PRACTICES AND POLICIES
PAIN AND DISTRESS MANAGEMENT IN ANIMAL RESEARCH AND TESTING 71
CONCLUSIONS
Table 7.5 Selected Elements of Institutional Policies on Monoclonal Antibody Production Available on the World Wide Web
Penn State Stanford U Iowa U Minnesota
Monitoring subj. with solid tumors
Not specified 3 /week Not specified 3 /week
Priming As low as 0.1 ml pristane
Not specified 0.2 ml max pristane
0.5 ml max pristane
Number of taps Max 3 taps, last terminal
Not specified 2 taps, last after euthanasia
Not specified
Monitoring postinoculation
Daily 3 /week for first week, then daily
Daily Daily
Replacementfluid after ascites harvest
Not specified Not specified 1–2 ml of saline subcutaneous
Not specified
Anesthesiaduring tap
Anesthesia can be used
Anesthesiaused for new personnel
Not specified Not specified
72 ALTERNATIVE TOXICOLOGICAL METHODS
ACKNOWLEDGMENTS
References
PAIN AND DISTRESS MANAGEMENT IN ANIMAL RESEARCH AND TESTING 73
PART II
Development of Predictive MethodsBased on Mechanisms of Eye Irritation
at the Ocular Surface: MeetingIndustry and Regulatory Needs
77
CHAPTER 8
Meeting Industry and Regulatory Needs forAlternative Test Methods to the Draize
Rabbit Eye Irritation Test
CONTENTS
INTRODUCTION
78 ALTERNATIVE TOXICOLOGICAL METHODS
INDUSTRY PERSPECTIVE
INDUSTRY AND REGULATORY PERSPECTIVES 79
80 ALTERNATIVE TOXICOLOGICAL METHODS
REGULATORY PERSPECTIVE
INDUSTRY AND REGULATORY PERSPECTIVES 81
82 ALTERNATIVE TOXICOLOGICAL METHODS
INDUSTRY AND REGULATORY PERSPECTIVES 83
DISCUSSION AND CONCLUSIONS
84 ALTERNATIVE TOXICOLOGICAL METHODS
INDUSTRY AND REGULATORY PERSPECTIVES 85
REFERENCES
86 ALTERNATIVE TOXICOLOGICAL METHODS
INDUSTRY AND REGULATORY PERSPECTIVES 87
89
CHAPTER 9
The Ocular Surface: Barrier Functionand Mechanisms of Injury and Repair
CONTENTS
THE OCULAR SURFACE
90 ALTERNATIVE TOXICOLOGICAL METHODS
ELECTROLYTES AND METABOLITES IN CORNEAL STROMA AND EPITHELIUM
Table 9.1 Stroma of rabbit cornea, mean ± SD, hydration = 3.2 ± 0.4
Mol/g H2O (n = 20) Mol/g net weight (n = 15)
Na 125 ± 42 Lactate 9.66 ± 1.2Cl 123 ± 25 Glucose 3.63 ± 0.8K 24 ± 4 ATP 0.20 ± 0.05S 105 ± 21 ADP 0.07 ± 0.03P 12 ± 4 ASC 4.72 ± 1.44
Note: ASC = ascorbate. Levels of some metabolites andelectrolytes in the corneal stroma. Adenosine triph-osphate (ATP) and adenosine diphosphate (ADP) arestrictly intracellular metabolites. Since the cornealstroma contains keratocytes only in about 10% of itsvolume, their levels are much lower than in the epi-thelium (see Table 9.2). Lactate is present in theextracellular as well as in the intracellular space andconsequently shows levels close to those of the epi-thelium (Reim et al., 1970, 1978; Fischern, 1996).
THE OCULAR SURFACE: BARRIER AND INJURY 91
Table 9.2 Metabolite levels of rabbit corneal epithelium Mol/g wet weight, m ± SEM
ATP 3.03 ± 0.10 (n = 14)ADP 0.26 ± 0.02 (n = 14)ATP/ADP 12.23 ± 0.89 (n = 14)AMP 0.93 ± 0.08 (n = 14)Glucose 2.02 ± 0.22 (n = 14)Lactate 9.89 ± 1.02 (n = 14)GSH 3.03 ± 0.43 (n = 21)GSSG 0.28 ± 0.05 (n = 21)ASC 11.55 ± 1.05 (n = 24)
Note: Levels of some metabolites in thecorneal epithelium, which may be sig-nificant to estimate vitality, to indicatemicrotrauma, and to show levels ofradical scavengers (Hennighausen etal., 1972; Reim et al., 1966, 1967,1970, 1976, 1982).
92 ALTERNATIVE TOXICOLOGICAL METHODS
TRAUMA TO OCULAR SURFACE
CHEMICAL AND THERMAL INJURIES TO THE OCULAR SURFACE
THE OCULAR SURFACE: BARRIER AND INJURY 93
Table 9.3 Grading of Eye Burns
I II III IV
Immediate signs
Erosion Large erosion Surface defect Epithelia destroyedHyperemia Ischemia 1/3 Ischemia >1/2 Deep ischemia >3/4
Chemosis Rose chemosis Dense corneal opacityCorneal opacity Conjunctival necroses
Sclera porcelain whiteDiscoloration and atrophy of irisFibrin exudate
Later signs
Regeneration Recirculation Persistent erosion ProliferationRegeneration Ulceration Large ulcerations
Vascularization Melting of cataractScars Glaucoma
Scarification
Note: Grading of eye burns according to clinical signs. The upper part lists immediatedamage visible, the lower one later secondary events. The classification of signswas developed from various authors and by clinical experiences (Hughes, 1946;Roper-Hall, 1965; Thoft, 1978; Reim and Kuckelkorn, 1995)
Figure 9.1 Eye with a mild alkali burn stage I (Table 9.3). The corneal epithelium was completelylost, but the stroma remained undamaged and clear. The conjunctiva showed hype-remia, but no swelling or ischemia. The damage healed within a few days.
94 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 9.2 Lime burn stage II (Table 9.3). The whole corneal and some conjunctival epitheliumwas destroyed. The corneal stroma exhibited little superficial turbidities. The lowerconjunctiva is demonstrated by upgaze. It was swollen (chemosis). Superficiallyin the conjunctiva, ischemia is recognized by the interrupted blood columns. Withthe lit lamp microscope, bloodstream could not be detected. Underneath theotherwise pale conjunctiva, intact sclera appeared with a faint rose background.
Figure 9.3 Clinical appearance of a severe chemical injury grade IV. The inner margin of theupper lid showed a white line of necrosis. The conjunctiva appeared flat and white,also from necrosis, which presumably included visible parts of the sclera. In theupper left region, some hemorrhages were deposited in necrotic conjunctiva.Ischemia was evident. The cornea was completely turbid. The outlines of iris andpupil could be hardly identified.
THE OCULAR SURFACE: BARRIER AND INJURY 95
PATHOPHYSIOLOGY OF CHEMICAL EYE INJURIES
Ischemia and Necrosis
Inflammatory Response
Figure 9.4 Melting of the anterior eye segment, nine days after most severe burn from liquidmetal. There were extended necroses of all conjunctival, subconjunctival, andscleral tissues, appearing homogeneously white and slippery. Only in the rightupper region, some hemorrhages in necrotic tissues showed red color. The corneawas opaque in the upper marginal parts. The lower and central cornea was meltedaway and the iris and lens exposed. Since at that time (1977) corneal donormaterial was not available, the eye was lost and had to be removed. In the meltingtissues of the anterior eye segment, high activities of N-acetylglucose aminidase(NAcGA, E.C.3.2.1.50) and cathepsin-D (E.C.3.4.23.5) were found (Reim, 1982a).
96 ALTERNATIVE TOXICOLOGICAL METHODS
Cytokines and Growth Factors in Cornea and Tears
Figure 9.5 Flow diagram of inflammatory cascade following chemical and thermal injuries ofthe eye. The inflammatory response is a quantitative process produced by theaffected tissues and the leucocytes involved (Ghattacherjee et al., 1979; Reim etal., 1980, 1993, 1997; Reim, 1982a; Rochels et al., 1982; Kulkarni and Srinivasan,1983; Becker et al., 1991, 1995; Reim and Leber, 1992; Reim and Becker, 1995).
Cornea and conjunctiva
PGE2α, Interleukins, LT 4, Subst-P, VIP, CGRP
Mild lesionweak response
Severe lesion, severe response
PMNs,macrophages
IL-1, IL-6IL-8, TNF
T-lymphocytesB-lymphocytes
Plasma cells
Cellular and humoral antibodies
O2
_
OH+ -radicalslysosomal enzymes
UlcerationInflammation Scars
PMNs
Restitution
THE OCULAR SURFACE: BARRIER AND INJURY 97
Table 9.4 Cytokines in Tears
EGF regeneration of epitheliumTGFbeta 2 inhibits proliferationTNFalpha, in inflammationMany others, but presumably released from damaged surface epithelia
Note: Cytokines and growth factors in tears influencing the cornealepithelium (Mishima et al., 1991; Kruse and Tseng, 1994; Soto-zono and Kinshita, 1998).
Figure 9.6 Interleukin-1 (IL-1) and Interleukin-6 (IL-6) in human corneal buttons from kerato-plasty. Total number of cases: 127. The logarithmic ordinate shows the concen-trations found in pg/mg extractable protein. The symbols represent the median,squares stand for IL-1, rhombs for IL-6. The error bars demonstrate the 75%percentiles. In the abscissa, the diagnoses of the cases were indicated corre-sponding to the position of the symbols (Becker et al., 1995). Inflamed corneasrevealed very high levels of IL-1 and IL-6. The levels in the uninflamed, quietecorneas were lower by an order of magnitude.
1
10
100
1000
Keratitis DecompensationInflammat.Cone Dystrophy, Scars
Ulceration Keratoconus
Levels of IL-1 and IL-6 (n=127)pg/mg protein — human corneal buttons
98 ALTERNATIVE TOXICOLOGICAL METHODS
Chemical Alteration of Extracellular Matrix
Stem Cell Insufficiency
Enzyme Activities and Metabolites on the Ocular Surface
THE OCULAR SURFACE: BARRIER AND INJURY 99
Changes of the Contents of Na1+, K1+, Cl1–, and SO42–
Figure 9.7 Activity of N-acetylglucose aminidase (NAcGA, E.C.3.2.1.50) in human tearscollected from nine human cases with eye burns stage I and II and an atopicpatient. Please note that the ordinate is in logarithmic scale! The enzyme activity( Mol/min/ml) increased considerably in surface diseases.
N-Acetylglucose aminidase in human tears
mol/min/ml
0.1
1
10
Normal 0.24 0.09 (9)
Range of 8 samplesfrom human burns
Atopic conjunctivitis
100 ALTERNATIVE TOXICOLOGICAL METHODS
Calcification and Contamination
Scarring
Table 9.5 Stroma of Rabbit Cornea, Mol/g H2O, mean ± SD
EDXA Normal (n = 20)
Alkali Burn, Denuded, Rinsed for 16 Days, 4 daily
with 0.9% NaCl (n = 8)
Na 125 ± 42 90 ± 11Cl 123 ± 25 65 ± 15S 105 ± 21 24 ± 4P 12 ± 4 22 ± 22
Ca 3 ± 3 1 ± 3
Note: Changes of the levels of some electrolytes in the corneal stroma after alkali burn.The denuded stroma was rinsed with saline, four times daily for 16 days. Na, Cl,and especially S were decreased, P increased (Fischern et al., 1998).
Table 9.6 Stroma of Rabbit Cornea, Mol/g H2O, Mean ± SD
EDXA Normal (n = 20)
Alkali Burn, Denuded, Rinsed for 16 Days, 4 Daily with Phosphate Buffer (n = 8)
Na 125 ± 42 105 ± 22Cl 123 ± 25 88 ± 33S 105 ± 21 28 ± 4P 12 ± 4 623 ± 307
Ca 3 ± 3 435 ± 198
Note: Changes of the levels of some electrolytes in the corneal stroma after alkali burnand rinsing with isotonic phosphate buffer, four times daily for 16 days. Na, Cl, andespecially S were decreased, but P and Ca were largely increased. Clinically,calcification of the cornea was observed (Schrage, 1997; Fischern et al., 1998;Haller, 2001).
THE OCULAR SURFACE: BARRIER AND INJURY 101
Figure 9.8 Left eye of a 16-year-old boy six months after a most severe chemical injury. Inthis accident, a highly alkaline etching fluid used to work on electronic parts spilledinto both eyes of the patient. In this case, a severe inflammatory response haddeveloped and remained for years. The conjunctiva-like proliferation tissue sur-rounding the cornea was swollen and very hyperemic. The cornea was devoid ofepithelium. It showed extended ulceration especially in its marginal parts and wasgenerally thinned. The upper right cornea showed white calcification. To save theeye from melting, a keratoplasty was performed. The excised cornea was examinedwith electron dispersive x-ray analysis method (EDXA) (see Figures 9.9 and 9.10).
Figure 9.9 Scanning electron microscopy (SEM) on a cross section of the cornea seen inFigure 9.8. Magnification 200. The upper part shows calcification, the lower oneparallel corneal lamellae (Schrage et al., 1988, 1993, 1996).
102 ALTERNATIVE TOXICOLOGICAL METHODS
ASPECTS OF REPAIR
Figure 9.10 Electron dispersive x-ray analysis (EDXA) of the calcified cornea as demonstratedin Figures 9.8 and 9.9. The spectra of the x-rays backscattered at scanningelectron microscopy (SEM) showed as expected high peaks for calcium (Ca) andphosphorus (P). But the most prominent peak from this sample was emitted fromsilicon (Si). Thus, EDXA revealed an unexpected high contamination of the corneaby silicone, which might have explained the severe and longstanding inflammatoryresponse in this case (Schrage et al., 1988, 1993, 1996).
THE OCULAR SURFACE: BARRIER AND INJURY 103
Figure 9.11 Eye of a 42-year-old male 2 years after severe lime burn. Heavy scar formationcould not be prohibited. The cornea was covered with thick highly vascularizedproliferation tissue. The conjunctiva developed strong scars between the globeand the lids, reducing eye motility. The conjunctival scars also deformed the lidmargins. The hyperemic, red scar tissue showed that the inflammatory responsehad not subsided after 2 years. The eye was practically blind and had badprognoses for surgical rehabilitation.
104 ALTERNATIVE TOXICOLOGICAL METHODS
References
THE OCULAR SURFACE: BARRIER AND INJURY 105
106 ALTERNATIVE TOXICOLOGICAL METHODS
THE OCULAR SURFACE: BARRIER AND INJURY 107
108 ALTERNATIVE TOXICOLOGICAL METHODS
109
CHAPTER 10
Evaluation and Refinement of the BovineCornea Opacity and Permeability Assay
CONTENTS
INTRODUCTION
110 ALTERNATIVE TOXICOLOGICAL METHODS
METHODS
THE BCOP ASSAY 111
RESULTS
Figure 10.1 Cross-section of the new cornea holder. In contrast to the old holder, the newholder clamps onto sclera rather than cornea. Also, the shape of the chamber fitsthe normal curvature of the cornea in contrast to the flat chamber of the currentBCOP holder.
Quartz Window
Quartz Window
Stainless Steel Ring
Stainless Steel Ring
O-Ring
O - Ring
O-Ring
Bottom Holder
Top HolderEpithelialChamber
EndothelialChamber
Cornea
Vents
Sclera
112 ALTERNATIVE TOXICOLOGICAL METHODS
Table 10.1 Absorbance at 570 nm (A570) of Bovine Corneas Exposed to Various Treatments and Incubated in MEM for 3 h
TreatmentExposure
None 10 min 1 min 30 sec
Intact, 35ºC, MEM 0.05 ± 0.03 — — —w/o Epi, 35ºC, MEM 0.11 ± 0.03 — — —w/o Epi/Endo, 4ºC, H2O 0.67 ± 0.13 — — —Isopropanol — 0.59 ± 0.08 0.23 ± 0.04 0.24 ± 0.07Acetone — 1.38 ± 0.22 1.07 ± 0.21 0.87 ± 0.2530% TCA — 1.43 ± 0.08 2.28 ± 0.20 1.96 ± 0.061% NaOH — 1.69 ± 0.22 1.28 ± 0.29 0.36 ± 0.1930% SLS — 0.095 ± 0.03 0.48 ± 0.28 0.21 ± 0.11
Note: Intact is untreated cornea incubated in MEM. w/o Epi is cornea with epitheliumremoved, not exposed to chemical, and incubated in MEM for 3 h. w/o Epi/Endo iscornea with both epithelium and endothelium removed, not exposed to chemical,and incubated in H2O at 4ºC. Values are mean ±SD, n = 5–10. (Data cited fromUbels et al. [2000]. With permission of Elsevier Science.)
Figure 10.2 Corneal hydration (mg H2O/mg cornea) following exposure to test substances for30 sec, 1 min, or 10 min and incubation in MEM for 3 h. Intact is untreated corneaincubated in MEM. w/o Epi is cornea with epithelium removed, not exposed tochemical, and incubated in MEM for 3 h. w/o Epi-Endo is cornea with bothepithelium and endothelium removed, not exposed to chemical, incubated in H2Oat 4 C. Mean ±SD, n = 5. Values for intact, without Epi, and without Epi-Endowere all significantly different from each other. 30-sec values and unmarked 1-min values are not different than intact value. # Significantly different than 30-secand 1-min values within group. * Significantly different than 30-sec and 10-minvalues within group. (ANOVA and Dunnett’s test, P 0.05). (Data cited from Ubelset al. [2000]. With permission of Elsevier Science.)
Aceto
neIP
A
1% N
aOH
30%
SLS
30%
TCA
mg
H20
/ m
g c
orn
ea
0
2
4
6
8
10
12
14
16
18
1 min
10 min
30 sec
## #* *
#
Inta
ct
w/o E
pi
w/o E
pi-Endo
THE BCOP ASSAY 113
DISCUSSION
114 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 10.3 Corneal endothelial cell layers stained with Alizarin Red S and trypan blue. Twentypercent of the endothelial layer is damaged after mounting in the old cornealholder (left), and none of the endothelial layer is damaged after mounting in thenew holder (right). The streaks of damaged cells exhibited after mounting in theold holder are characteristic of wrinkling caused by the holder. Magnification 35 .(Reproduced from Ubels et al. [2002]. With permission of Elsevier Science.)
a b
THE BCOP ASSAY 115
ACKNOWLEDGMENTS
REFERENCES
117
CHAPTER 11
Corneal Organ Culture forOcular Toxicity Test of Commercial
Hair Care Products
CONTENTS
INTRODUCTION
118 ALTERNATIVE TOXICOLOGICAL METHODS
METHODS
Corneal Organ Culture
CORNEAL ORGAN CULTURE FOR OCULAR TOXICITY TEST 119
Culture Treatment
Surface Biotinylation-Tight Junction Permeability Assay
Electrophoretic Mobility Shift Assay (EMSA)
RESULTS AND DISCUSSION
120 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 11.1 Biotin surface labeling to visualize epithelial barrier. Cultured bovine cornea wasincubated with sulfo-NHS-LC-biotin for 30 min and then embedded in OCT, snap-frozen, and sectioned (6 m). Cryostat sections (8 m) were (A) stained directlywith hematoxylin to reveal corneal morphology (B) or incubated with rhodamine-avidin D to visualize the bound biotin. The rhodamine staining represents biotiny-lation of accessible surface of normal bovine cornea and linear staining at thecorneal surface indicates functional epithelial TJ barrier in cultured corneas. Ep,epithelium; BM, basement membrane; St, stroma that consists of fibroblasts. Aand B are mirror orientations of the same corneal sections.
CORNEAL ORGAN CULTURE FOR OCULAR TOXICITY TEST 121
Figure 11.2 Tight junction permeability assay of cultured bovine corneas in response to chal-lenge of three hair care products. Corneas in culture were treated with 100%,50% (not shown), and 25% chemicals, and TJ permeability of corneal epitheliumwas assessed by surface biotinylation as described in Figure 11.1. Inserts: cornealsections stained directly with hematoxylin to reveal corneal morphology. Note:extended biotinylation of the corneal surface caused by GA and GB exposure ina concentration dependent manner. However, no disruption of TJ was observedin GC treated cornea.
122 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 11.3 EMSA analysis of NF- B DNA-binding activity in bovine corneal epithelial cells inresponse to consumer product challenge. Panels showed cultured corneas weretreated with different concentrations of three hair care products for 5 min, untreatedcells were used as control (C). The corneas were then cultured for 10 min withoutthe presence of the chemicals. Cell extracts from corneal epithelial cells treatmentwere probed with 32P-labeled AP-1 (upper panel) or NF- B (lower panel) consen-sus oligonucleotide. EMSA experiments were repeated two times, and gels pre-sented in the figure are from a representative set.
CORNEAL ORGAN CULTURE FOR OCULAR TOXICITY TEST 123
REFERENCES
124 ALTERNATIVE TOXICOLOGICAL METHODS
125
CHAPTER 12
Human Corneal Equivalentsfor In Vitro Testing
CONTENTS
INTRODUCTION
126 ALTERNATIVE TOXICOLOGICAL METHODS
DEVELOPMENT OF CELL LINES AND CONSTRUCTION OF CORNEAS
CHANGES IN CORNEAL TRANSPARENCY IN RESPONSE TO CHEMICALS
HUMAN CORNEAL EQUIVALENTS FOR IN VITRO TESTING 127
CHANGES IN GENE EXPRESSION IN RESPONSE TO CHEMICAL EXPOSURE
DEVELOPMENT OF RELATED TISSUES
128 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
Figure 12.1 Confocal image of a corneal equivalent with surrounding sclera containing chickembryonic dorsal root ganglion (not shown). Neurites, labeled with nerve-specificantineurofilament antibody are seen traveling through the sclera (S) parallel to thecorneal periphery and branching into the cornea (C). White dashed line, cornealperiphery; arrows, parallel neurites; arrowheads, branching neurites.
HUMAN CORNEAL EQUIVALENTS FOR IN VITRO TESTING 129
131
CHAPTER 13
The EpiOcular Prediction Model:A Reproducible In Vitro Means
of Assessing Ocular Irritancy
CONTENTS
132 ALTERNATIVE TOXICOLOGICAL METHODS
INTRODUCTION
MATERIALS AND METHODS
EpiOcular (OCL-200) Tissue Source
THE EPIOCULAR PREDICTION MODEL 133
Histology
TISSUE VIABILITY ASSAY—MTT ET-50 METHOD
Preequilibration of Tissue
Preparation of Test Articles
134 ALTERNATIVE TOXICOLOGICAL METHODS
Application of Test Article to EpiOcular Tissue
Exposure Times
Table 13.1 Choice of Additional Exposure Times Based on the Viability of the Initial 20-Min Exposure
Viability after 20-min exposure Additional exposure times (min)
90% 60, 240<90% but > 30% 5, 60
<30% 1, 5
THE EPIOCULAR PREDICTION MODEL 135
Exposure Conditions
Removal of Test Article from EpiOcular Tissue
Tissue Viability: MTT Assay
136 ALTERNATIVE TOXICOLOGICAL METHODS
Calculation of Effective Time 50 (ET-50)
Determination of Prediction Model
Testing of Prediction Model
THE EPIOCULAR PREDICTION MODEL 137
Table 13.2 In Vitro and In Vivo Data Used to Generate the Prediction Model; In Vitro Data from EpiOcular ET-50 Determinations; In vivo Data from ECETOC Database or Commercial Sources
#Conc. tested
ET-50 (min)
Draize (MMAS)
1 Benzalkonium chloride (10%) 2.0% 1.07 108.0 2 Benzalkonium chloride (5%) 1.0% 1.0 83.8 3 Benzalkonium chloride (1%) 0.2% 5.9 45.3 4 Cetyl pyridinium bromide (10%) 2.0% 9.0 89.7 5 Cetyl pyridinium bromide (1%) 0.2% 30.1 36.0 6 Cetyl pyridinium bromide (0.1%) 0.02% 240.0 2.7 7 Glycerol 20.0% 240.0 1.7 8 Sodium hydroxide (10%) 2.0% 1.0 108.0 9 Sodium hydroxide (1%) 0.2% 2.3 25.8
10 Propylene glycol 20.0% 240.0 1.3 11 Sodium dodecyl sulfate (30%) 6.0% 2.1 60.5 12 Sodium dodecyl sulfate (15%) 3.0% 5.1 59.2 13 Sodium dodecyl sulfate (3%) 0.6% 9.0 16.0 14 Trichloro acetic acid (30%) 6.0% 1.0 106.0 15 Trichloro acetic acid (3%) 0.6% 155.1 6.7 16 Triton X-100 (10%) 2.0% 2.5 68.7 17 Triton X-100 (5%) 1.0% 5.3 33.1 18 Triton X-100 (1%) 0.2% 36.7 1.7 19 Tween 20 (100%) 20.0% 240.0 4.0 20 Body/hand wash 20% 9.1 3221 Body/hand wash 20% 14.8 3522 Eye gel/colorant 20% 240 023 Eye gel/colorant 20% 240 224 Face/body wash 20% 240 225 Face/body wash 20% 6.5 2526 Face/body wash 20% 10.2 4027 Hand/body lotion 20% 240 228 Hand/body lotion 20% 240 229 Hand/body lotion 20% 240 330 Shampoo—baby 20% 30.8 1031 Shampoo—baby 20% 25.7 1832 Conditioner 20% 240 233 Shampoo—regular 20% 6.0 3034 Shampoo—regular 20% 8.7 3535 Na2-ricinoleadmido MEA
sulfosuccinate20% 108.9 0
36 Sodium trideceth sulfate 20% 2.5 3337 Cetrimonium chloride 20% 116.9 6.6738 Stearalkonium chloride 20% 240 1439 Cocamide DEA 20% 240 040 Disodium cocoamphodipropionate 20% 11.2 15.341 Surfactant blend 20% 19.2 6.0 42 Surfactant blend 20% 40.4 2.6743 Final formulation shampoo 20% 26.1 4.044 Final formulation shampoo 20% 29.1 12.545 Final formulation shampoo 20% 4.2 32.746 Final formulation shampoo 20% 9.3 31.647 Final formulation shampoo 20% 9.0 34.4
(continued)
138 ALTERNATIVE TOXICOLOGICAL METHODS
Quality Control Testing
Interlaboratory Testing
Table 13.2 (continued) In Vitro and In Vivo Data Used to Generate the Prediction Model; In Vitro Data from EpiOcular ET-50 Determinations; In vivo Datafrom ECETOC Database or Commercial Sources
#Conc. tested
ET-50 (min)
Draize (MMAS)
48 Final formulation shampoo 20% 31.0 3.949 Final formulation shampoo 20% 63.1 3.550 Final formulation shampoo 20% 47.1 8.351 Final formulation shampoo 20% 29.4 6.5752 Final formulation shampoo 20% 42.1 4.853 Hydro-alcohol (hair spray) solution 20% 84.1 6.0 54 10% fatty alcohol ethoxylate 20% 189 3.555 Eye makeup remover (surfactant sol.) 20% 240 056 Lactic acid (3% solution) 20% 240 057 Oleic acid 20% 240 258 Skin care emulsion 20% 240 059 Body spray 20% 240 0
THE EPIOCULAR PREDICTION MODEL 139
Testing of Ultramild Materials
RESULTS
Histological Characterization of the EpiOcular Tissue Model
Figure 13.1 Hematoxylin and Eosin (H&E) stained histological cross sections of (A) EpiOculartissue model and (B) rabbit cornea epithelium and underlying stroma. Tissueswere fixed in 10% formalin, embedded in paraffin, and stained with H&E. Finalmagnification 360 .
A
B
140 ALTERNATIVE TOXICOLOGICAL METHODS
Determination of Prediction Model
Testing of Prediction Model
Figure 13.2 Graphical depiction of in vivo and in vitro data used to derive the predictionequation. All materials tested that had specific gravity >0.95 were diluted to 20%in ultrapure water. If actual ET-50 exceeded 240 min, ET-50 was set equal to 240min. If ET was less than 1 min, ET-50 was set to 1 min.
Prediction Equation:Draize (MMAS) = –4.74 + 101.7/ (ET-50)
95% Prediction Intervals:Draize (MMAS) = –30.54 + 100.4/ (ET-50)Draize (MMAS) = 21.07 + 102.9/ (ET-50)
r = 0.9059 materials
150
125
100
75
50
25
01 10 100 300
OCL-200 Prediction Equation
Dra
ize
Scor
e (M
MA
S)
ET-50 (min)
THE EPIOCULAR PREDICTION MODEL 141
Table 13.3 EpiOcular Prediction Model Testing: Comparison between In Vivo and InVitro Predicted Draize
Code ProductPredicted
DraizeActual Draize
(MMAS)
1 10599 A Body wash 14.6 16.72 10599 B Body wash 31.3 44.73 10599 C Dishwashing liquid 51.3 38.34 10599 D Hand soap liquid 28.0 24.75 10599 E Dishwashing liquid 25.2 39.36 10599 F Facial soap 15.6 9.37 10599 G Dishwashing liquid 50.8 39.08 10599 H Dishwashing liquid 60.5 50.39 10599 I Laundry detergent 37.3 37.3
10 10599 J Laundry detergent 1.8 0.711 10599 K Laundry detergent 33.4 37.712 10599 L Dishwashing liquid 96.9 37.713 10599 M Shampoo 6.5 4.014 10599 N Shampoo 32.9 41.715 10599 O Shampoo 8.4 3.316 10599 P Hand soap liquid 18.1 13.317 10599 Q Skin lotion 1.8 0.718 10599 R Shampoo 46.9 33.719 10599 S Skin lotion 1.8 0.020 10599 T Shampoo 39.3 37.721 10599 U Body wash 41.2 33.022 10599 V Laundry detergent 1.8 0.723 10599 W Laundry detergent 39.9 44.024 10599 X Skin lotion 1.8 0.7
Figure 13.3 Comparison of predicted and actual Draize scores for consumer products includ-ing: shampoos (5), face/body soap (6), dishwashing liquids (4), laundry detergents(5), and skin lotions (4). The predicted Draize scores were calculated based onthe ET-50 using the prediction equation shown in Figure 13.2.
142 ALTERNATIVE TOXICOLOGICAL METHODS
Quality Control Results: 1996–2000
Interlaboratory Reproducibility
Testing of Ultramild Materials
Table 13.4 EpiOcular QC Testing Results for Positive (0.3% Triton X-100) and Negative (ultrapure water) Controls
Calendar Year Tissue LotsTriton ET-50
(min)Neg. Control
(OD)Avg. Lot CV
(%)
2000 60 23.1 6.0 1.441 5.51999 84 22.6 5.0 1.433 5.61998 85 25.2 5.6 1.354 5.51997 81 22.9 4.7 1.343 5.41996 47 24.9 6.3 1.274 5.2
Table 13.5 Results of Interlaboratory Testing
ET-50 (mins): Intralaboratory Reproducibility Avg. CV (%)Laboratory: BAK SDS Triton
P&G: 5.75 3.12 25.40 7.75 IIVS: 6.39 3.30 26.59 7.14 MatTek: 5.97 3.72 29.75 9.56 Average: 6.04 3.38 27.24 Std. Dev.: 0.32 0.31 2.25 CV: 5.36 9.19 8.26
THE EPIOCULAR PREDICTION MODEL 143
Table 13.6 ET-50 for Ultramild Materials for Which the Draize Test Is Insensitive
In Vivo Concentration Draize (MMAS) ET-50 (min)
Benzalkonium chloride (BAK)
10.00% 108.0 1.15.00% 83.8 1.01.00% 45.3 5.90.30% 8.67 28.90.10% 0 212.70.03% 0 2053.0
Sodium dodecyl sulfate (SDS)
30.00% 60.5 2.115.00% 59.2 5.13.00% 16.0 9.01.00% 0.67 29.50.30% 0 740.10.10% 0 1938.3
Figure 13.4 Use of EpiOcular to differentiate between very mild materials that cannot bedistinguished by the in vivo Draize test (MMAS < 1.0).
�
�
144 ALTERNATIVE TOXICOLOGICAL METHODS
DISCUSSION
THE EPIOCULAR PREDICTION MODEL 145
ACKNOWLEDGMENTS
146 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
147
CHAPTER 14
Three-Dimensional Construct of the HumanCorneal Epithelium for In Vitro Toxicology
CONTENTS
INTRODUCTION
148 ALTERNATIVE TOXICOLOGICAL METHODS
MATERIALS AND METHODS
Three-Dimensional Corneal Epithelial Cell Constructs
Histology
CONSTRUCT OF THE HUMAN CORNEAL EPITHELIUM FOR TOXICOLOGY 149
IN VITRO Toxicology Assay
Western Blot Analysis
150 ALTERNATIVE TOXICOLOGICAL METHODS
RESULTS
Histologic Characteristics of the Three-Dimensional Corneal Epithelial Constructs
Figure 14.1 Light micrograph of a plastic section from a construct after 24 hr equilibration at37°C. The tissue organization reveals a stratified appearance with columnar basalcells, defined wing cells, and flattened superficial cells. Toluidine blue and basicfuchsin, original magnification 160 .
CONSTRUCT OF THE HUMAN CORNEAL EPITHELIUM FOR TOXICOLOGY 151
Figure 14.2 Transmission electron micrograph of the construct after 24 hr equilibration at 37°C.The superficial cells give rise to numerous villus processes. The nucleus is orientedparallel to the surface. Some small, spot-like junction arrangements are seenbetween cells at the surface. Desmosomes are present between adjacent cells.Original magnification 12,600 .
Figure 14.3 Transmission electron micrograph of basal cells of the three-dimensional con-struct. The nucleus of the basal cell is upright. Within the cell, there is an extensivecytoskeletal network. Mitochondria surround the nucleus and desmosomes joinadjacent cells. Original magnification 10,000 .
152 ALTERNATIVE TOXICOLOGICAL METHODS
Biochemical Characteristics of the Three-Dimensional Corneal Epithelial Constructs
Figure 14.4 High-power transmission electron micrograph of the basal membrane of a basalcell revealing numerous mature hemidesmosomes. Also visible are the compo-nents of the hemidesmosomes: the intracellular membrane placode, filamentsradiating through the membrane, and the typical band in what would be the laminalucida. The amorphous band at the bottom is the polycarbonate support membraneof the construct. Original magnification 75,000 .
CONSTRUCT OF THE HUMAN CORNEAL EPITHELIUM FOR TOXICOLOGY 153
Response to Benzalkonium Chloride
Figure 14.5 Western blots of the acidic (AE1) and the basic (AE3) keratin family in the three-dimensional construct: A. The reactivity pattern for AE1 in the three-dimensionalconstruct (Lane 1) is similar to that of immortalized human corneal epithelial cells(IHCEC) cultured for 4 weeks (Lane 2). B. The reactivity pattern for AE3 (Lane1) is similar to that of cultured IHCEC at 1 week (Lane 2) and 2 weeks (Lane 3)and human donor corneal epithelial cells obtained within 24 hr after death (Lane4). The molecular weights of several cytokeratin isoforms recognized by therespective antibodies are indicated below under AE1 and AE3, respectively.
Figure 14.6 Western blot of the 64 kDa cornea-specific keratin AE5 in the three-dimensionalconstruct. Lanes show the construct on Day 0 (immediately upon arrival), Day 1(24 hr equilibration at 37°C), and Day 2 (48 hr at 37°C), as well as the freshhuman donor corneal epithelial cell positive control (+) and the rabbit lacrimalgland negative control (–).
154 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 14.7 Western blot of laminin in the three-dimensional construct. One band is seen at220 kDa and another at 440 kDa. Lane 1, three-dimensional construct; Lane 2,fresh human corneal donor epithelial cells from cadaver eyes; Lane 3, immortal-ized human corneal epithelial cells cultured for 4 weeks.
Figure 14.8
CONSTRUCT OF THE HUMAN CORNEAL EPITHELIUM FOR TOXICOLOGY 155
Figure 14.8 (continued)
156 ALTERNATIVE TOXICOLOGICAL METHODS
DISCUSSION
Figure 14.9 Top: Western blot analysis for phosphorylated (active) p42/p44 mitogen-activatedprotein kinase (MAPK) after treatment with benzalkonium chloride. Positive control(Ctrl +), epidermal growth factor-stimulated A431 cells. Negative control (Ctrl –),unstimulated A431 cells. Bottom: The histogram shows the increase in intensitylevel (activity) of p42/p44 with increasing concentrations of benzalkonium chloride(BAK). The absence of activity with 0.1% benzalkonium chloride is likely due tocell death resulting from toxic insult.
CONSTRUCT OF THE HUMAN CORNEAL EPITHELIUM FOR TOXICOLOGY 157
158 ALTERNATIVE TOXICOLOGICAL METHODS
ACKNOWLEDGMENTS
REFERENCES
CONSTRUCT OF THE HUMAN CORNEAL EPITHELIUM FOR TOXICOLOGY 159
161
CHAPTER 15
The Human Corneal Epithelial HCE-T TEPAssay for Eye Irritation: Scientific Relevanceand Summary of Prevalidation Study Results
CONTENTS
INTRODUCTION
162 ALTERNATIVE TOXICOLOGICAL METHODS
SCIENTIFIC RELEVANCE
Background and Rationale for the Development of the HCE-T TEP Assay
THE HUMAN CORNEAL HCE-T TEP ASSAY 163
164 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 15.1 The three major steps in the execution of the HCE-T TEP assay (A–C), a typicaldose-response curve (D), and the prediction model (E).
A. B.
C.
D. Determination of In Vitro Endpoint, FR85 E. Prediction Model
THE HUMAN CORNEAL HCE-T TEP ASSAY 165
Scientific Basis for the HCE-T TEP Assay
166 ALTERNATIVE TOXICOLOGICAL METHODS
THE HUMAN CORNEAL HCE-T TEP ASSAY 167
Tab
le 1
5.1
Sim
ilari
ties
an
d D
iffe
ren
ces
in t
he
En
dp
oin
t M
easu
red
in
th
e H
CE
-T T
EP
Ass
ay a
nd
in
th
e D
raiz
e E
ye I
rrit
atio
n T
est
Test
Sp
ecie
sTe
st T
issu
eE
xpo
sure
Ro
ute
Exp
osu
reT
ime
Co
nce
ntr
atio
nTe
sted
En
dp
oin
t M
easu
red
Mec
han
ism
of T
issu
e D
amag
e
Hum
an(in
vitr
o)C
orne
al
epith
eliu
mS
uper
ficia
l to
cell
cultu
res
5 m
in5
conc
entr
atio
ns
with
hig
hest
equ
al
to th
at e
valu
ated
in
Dra
ize
test
Con
cent
ratio
n of
test
mat
eria
l ca
usin
g 15
% o
f flu
ores
cein
to
pen
etra
te t
hrou
gh t
he
corn
eal e
pith
eliu
m (
FR
85)
Cyt
otox
icity
and
junc
tiona
l di
srup
tion
in t
he c
orne
al
epith
eliu
m
Rab
bit
(in v
ivo)
Ocu
lar
surf
ace
(cor
nea,
co
njun
ctiv
a an
d iri
s)
Sup
erfic
ial t
o ey
eU
ntil
was
hed
out
by
blin
king
and
te
arin
g(<
5 m
in)
Con
cent
ratio
n de
fined
by
toxi
colo
gist
(ne
at
or d
ilute
d)
Cor
nea:
are
a of
dam
age
and
opac
ityC
onju
nctiv
a:ch
emos
is,
redn
ess
and
disc
harg
eIr
is: d
egre
e of
effe
ct
Are
a of
cor
neal
dam
age
is
rela
ted
to e
pith
elia
l cy
toto
xici
ty a
nd ju
nctio
nal
disr
uptio
n; o
paci
ty is
rel
ated
to
pen
etra
tion
of t
est
mat
eria
l int
o st
rom
a.
Con
junc
tiva
dam
age
sim
ilar
to c
orne
a—ep
ithel
ial t
oxic
ity
and
pene
trat
ion.
Ir
is—
mat
eria
l has
pe
netr
ated
to
caus
e m
ore
seve
re d
amag
e.
168 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 15.2 Cross-sectional diagram of the human corneal epithelium. A drawing illustratingthe multiple cell layers, the apical tight junctions, and the intercellular desmosomaljunctions of the corneal epithelium.
stroma
desmosomaljunction
tightjunction
tear film
THE HUMAN CORNEAL HCE-T TEP ASSAY 169
Figure 15.3 Cross sections of stratified HCE-T cultures visualized by transmission electronmicroscopy following 5-min superficial exposures to (A) cell culture medium (KGM)(5000 ); and (B) 0.01% benzalkonium chloride (BAC) (6000 ). Ap = apical surfaceof the culture; M = collagen membrane. [Photos contributed by S.D. Dimitrijevich.]
KGM 0.01% BAC
Ap
M
A B
M
Ap
170 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 15.4 Cross sections of stratified HCE-T cultures, visualized by transmission electronmicroscopy, which were fixed in a special buffer to retain the mucin layer. Thedark-stained surface material is the mucin produced by the corneal epithelial cells.(A) A continuous layer of mucin is found on the surface of a differentiated culture(maintained at the air–liquid interface in serum-free medium containing 1.15 mMcalcium) in which the TER is high (tight junctions are intact); and (B) mucin isfound between the cells in this non-differentiated culture (maintained submergedin serum-free medium containing 0.15 mM calcium) in which the TER is low (tightjunctions not intact).
A B
THE HUMAN CORNEAL HCE-T TEP ASSAY 171
Figure 15.5 Three assays were used to evaluate the dose-dependent effects of sodium dodecylsulfate (SDS) on HCE-T cultures on the day of treatment (day 1), and 48 hr later(day 3). TEP, transepithelial permeability to fluorescein; TER, transepithelial elec-trical resistance; MTT, cell viability assay using the MTT dye [3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyl tetrazolium bromide]. Each dose is represented by triplicatecultures, and the error bars are the standard deviations.
100
80
60
40
20
00.00 0.02 0.04 0.06 0.00 0.02 0.04 0.06
SDS(%) SDS(%)
% o
f Con
trol
Day 1 Day 3
TEPMTTTER
172 ALTERNATIVE TOXICOLOGICAL METHODS
The Mechanism of Action of Fluorescein TEP in the Test System Compared to the Human Eye
THE HUMAN CORNEAL HCE-T TEP ASSAY 173
Figure 15.6 Fluorescein staining data from rabbit eyes following surfactant-containing formu-lation exposure correlates well with Draize tissue score data (modified maximumaverage score, MMAS and modified maximum average corneal score, MMCS)from the same animals. The error bars are the standard deviations; n = 5–6 rabbitsevaluated per Draize test; and n = 2–4 rabbits evaluated for fluorescein stainingat 24 hr. [Data from Gettings et al., 1996.]
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
–20.000 0.000 20.000 40.000 60.000 80.000 100.000 120.000
24 Hr Fluorescein
MMAS R2 = 0.9435
MMCS R2 = 0.9711
Dra
ize
Dat
a
174 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 15.7 The relationship between the in vivo 24-hour fluorescein staining data and the invitro log FR85 values for the subset of the CTFA formulations that are representedin the HCE-T TEP assay Prediction Model. The error bars are the standarddeviations; n = 2 TEP assays; n = 5–6 rabbits per Draize test; and n = 2–4 rabbitsper fluorescein data.
-20
0
20
40
60
80
100
120
-0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6
logFR85
Dra
ize
Dat
aMMAS R2 = 0.632
MMCS R2 = 0.7057
24 Hr R2 = 0.7361Fluorescein
THE HUMAN CORNEAL HCE-T TEP ASSAY 175
PREVALIDATION STUDY RESULTS
Purpose for the HCE-T TEP Assay
Study Structure
176 ALTERNATIVE TOXICOLOGICAL METHODS
Data Analysis
THE HUMAN CORNEAL HCE-T TEP ASSAY 177
Tab
le 1
5.2
Pre
valid
atio
n S
tud
y Te
st M
ater
ials
an
d D
raiz
e D
ata
Pro
du
ct T
ype
Co
nc.
Te
sted
aS
urf
acta
nt
Ing
red
ien
ts
Per
cen
tF
orm
ula
(w
/w)
Su
rfac
tan
tC
lass
Dra
ize
S
core
sb
FH
SA
Cla
ssc
Bub
ble
bath
2.5
%
sodi
um la
uret
h su
lfate
coca
mid
opro
pyl b
etai
ne
25.0 5.0
anio
nic
amph
oter
icM
AS
4.8
CS
0.0
CO
S 0
.0
–
Hai
r co
nditi
oner
100
%st
eara
lkon
ium
chl
orid
ece
teth
-24
dim
ethy
l ste
aram
ine
glyc
eryl
ste
arat
e
1.14
1.0
0.67
0.44
catio
nic
noni
onic
catio
nic
noni
onic
MA
S 1
4.2
CS
0.0
CO
S 0
.0
–
Cle
ansi
ng g
el10
0%co
coam
phod
iace
tate
sodi
um n
onox
ynol
-6-
phos
phat
equ
ater
nium
-26
PE
G-1
20-m
ethy
l glu
cose
di
olea
te
15.0 6.0
1.5
1.5
amph
oter
ican
ioni
cca
tioni
cno
nion
ic
MA
S 2
2.0
CS
10.
0C
OS
0.5
–/+
Sha
mpo
o 1
100%
sodi
um la
uryl
sul
fate
diso
dium
laur
eth
sulfo
succ
inat
ebu
tyle
ne g
ylco
lla
uram
ide
DE
A
25.0
15.0 5.0
0.5
anio
nic
anio
nic
noni
onic
noni
onic
MA
S 3
7.8
CS
20.
0C
OS
1.0
+
Sha
mpo
o 2
100%
amm
oniu
m la
uryl
sul
fate
laur
amid
e D
EA
etho
xydi
glyc
olhy
drox
ypro
pyl m
ethy
lcel
lulo
se
12.0 2.0
0.4
0.15
anio
nic
noni
onic
— —
MA
S 5
7.4
CS
38.
0C
OS
2.0
+
aC
once
ntra
tion
used
in D
raiz
e te
st,
and
initi
al c
once
ntra
tion
for
dilu
tion
in T
EP
ass
ay.
bM
AS
is t
he m
axim
um a
vera
ge s
core
; C
S is
the
cor
neal
sco
re;
CO
S is
the
cor
neal
opa
city
sco
re.
cF
eder
al H
azar
dous
Sub
stan
ces
Act
(F
HS
A)
clas
sific
atio
ns:
–, n
egat
ive;
+,
posi
tive;
–/+
rep
eat
test
(F
HS
A,
1979
).
178 ALTERNATIVE TOXICOLOGICAL METHODS
Results
Conclusions
THE HUMAN CORNEAL HCE-T TEP ASSAY 179
Figure 15.8 Overlay of the prevalidation study test results for five test materials in the MASPrediction Model (PM). The solid line represents the nonlinear regression curveof the MAS PM, and the dashed lines are the 95% confidence intervals. (A)Overlay of the 60 log FR85 values in the MAS PM. There are 12 values for eachof the five test materials on the plot, but, due to overlap in the data, all 12 pointsmay not be distinct. (B) Fit of the average log FR85 value for each of the fivetest materials in the MAS PM. The result for each test material is the average of12 assays.
B
A
7065605550454035302520151050–0.8 –0.4 0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2
7065605550454035302520151050–0.8 –0.4 0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2
Dra
ize
MA
S
LogFR85
Dra
ize
MA
S
LogFR85
180 ALTERNATIVE TOXICOLOGICAL METHODS
SUMMARY
Table 15.3 The Nonirritant/Irritant Classification of the Five Test Materials as Determined by the Draize Test and by the HCE-T TEP Assay
Test MaterialDraize Score
Classificationa
TEP Assay MAS PMb
TEP Assay CS PMb
TEP Assay COS PMb
Bubble bath 1 1 1 1Hair conditioner 1 1 1 1Cleansing gel 2 2 2 1Shampoo 1 2 2 2 2Shampoo 2 2 2 2 2
a Draize classification was the same across the MAS, CS, and COS scores, except for thecleansing gel which was an irritant by the MAS and CS, but a nonirritant by the COS.
b Draize scores for the classifications: nonirritant MAS 15; nonirritant CS < 5; nonirritantCOS < 0.667.
Note: Draize maximum average score, MAS; corneal score, CS; corneal opacity score, COS.
Table 15.4 Average HCE-T TEP Assay Results for Five Test Materials in Three Laboratories; The Predicted MAS and Class from the TEP assay Are Compared to the Draize MAS and Class for Each Test Result
Test Material Draize MASa
Draize Classb
PredictedMASa
PredictedClassb
Bubble bath 4.8 1 4.20 1Hair conditionerc 14.2 1 1.82 1Cleansing gel 22.0 2 16.15 2Shampoo 1 37.8 3 34.62 3Shampoo 2 57.4 4 41.18 3
a MAS, maximum average score.b The four classification cutoffs for MAS are based on the scheme proposed by Kay and
Calandra (1962): MAS 0–15, minimal (class 1); MAS 15.1–25, mild (class 2); MAS25.1–55, moderate (class 3); MAS > 55, severe (class 4).
c Hair conditioner was less water soluble than other test materials, which may account forits underprediction when it was diluted.
THE HUMAN CORNEAL HCE-T TEP ASSAY 181
REFERENCES
Table 15.5 Variability in the Draize MAS Compared to the Predicted MAS for the HCE-T TEP Assay Prevalidation Study Data
Test MaterialIntralab CV (%) for
Draize MASa
Intralab CV (%) for Predicted MASb
Interlab CV (%) for Predicted MASc
Bubble bath 22.82 9.9410.06
4.94
6.03
Hair conditionerd 19.67 33.1915.8054.09
15.59
Cleansing gel 53.24 17.503.01
25.90
15.10
Shampoo 1 10.31 12.5421.2120.42
12.49
Shampoo 2 8.32 14.6014.6229.49
2.83
a Evaluated using five to six animals from one lab.b Evaluated using four replicates per lab for three labs.c Results from three testing labs.d Hair conditioner was less water soluble, which may account for its greater variability
when it was diluted.Notes: Intralab is the within lab variability (repeatability); interlab is the between lab
variability (reproducibility). Coefficient of variation, CV; maximum average score,MAS.
182 ALTERNATIVE TOXICOLOGICAL METHODS
THE HUMAN CORNEAL HCE-T TEP ASSAY 183
184 ALTERNATIVE TOXICOLOGICAL METHODS
THE HUMAN CORNEAL HCE-T TEP ASSAY 185
186 ALTERNATIVE TOXICOLOGICAL METHODS
PART III
Dermal Testing Alternatives
189
CHAPTER 16
Alternative Methodsfor Dermal Toxicity Testing
CONTENTS
INTRODUCTION
SKIN CORROSION
190 ALTERNATIVE TOXICOLOGICAL METHODS
SKIN IRRITATION
SKIN SENSITIZATION
ALTERNATIVE METHODS FOR DERMAL TOXICITY TESTING 191
SKIN PHOTOTOXICITY
PERCUTANEOUS ABSORPTION
192 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
193
CHAPTER 17
Allergic Contact Hypersensitivity:Mechanisms and Methods*
CONTENTS
194 ALTERNATIVE TOXICOLOGICAL METHODS
INTRODUCTION
PROCESS
ICCVAM and the LLNA Review
ALLERGIC CONTACT HYPERSENSITIVITY: MECHANISMS AND METHODS 195
Figure 17.1 ICCVAM review process: the local lymph node assay. (Modified from Sailstad, 2000.)
Input/ guidance/ response
AGENCYLLNA
Recommendation
AdvisoryCommittee on
AlternativeToxicological
MethodsGuidance
LLNA SPONSORSData submission/ method support
ICCVAMImmunotoxicology
Working GroupFacilitation/ recommendation
NICEATMSupport
Peer Review Panel
Evaluation/ REPORT
REGULATORY ACTION
Agency Response
196 ALTERNATIVE TOXICOLOGICAL METHODS
BACKGROUND
Contact Hypersensitivity and Allergic Contact Dermatitis Test Methods
Background: An Example of the Dilemma of GP Tests
ALLERGIC CONTACT HYPERSENSITIVITY: MECHANISMS AND METHODS 197
Mechanisms: Contact Hypersensitivity/Allergic Contact Dermatitis
Induction Phase
Figure 17.2 Illustration of contact hypersensitivity (CH) mechanisms: induction and elicitationphase. During the induction phase of CH, immature dendritic cells of the skin,called “Langerhans cells” (LC), effectively uptake and process the allergen. Simul-taneously, epidermal keratinocytes and the LCs themselves release cytokinemediators which assist the LCs in the migration to the draining lymph node andmaturation into effective antigen-presenting cells. In the lymph node, LC to Tlymphocyte interactions occur, which is followed by lymphocyte proliferation. Theproliferation results in “primed” effector lymphocytes which maintain a memory forthe specific allergen. The elicitation phase of CH appears to involve two mech-anisms of action. Initially, allergens cause direct cellular action releasing a seriesof nonspecific inflammatory mediators. These mediators are responsible for someof the cellular influx into the site of allergen challenge. Additionally, the “primed”lymphocytes are called into the area in a very specific response. These responsesand the cytokines, costimulatory factors, and adhesion molecules act to producethe end results of erythema and edema. (Modified from Selgrade et al., 2001.)
CHEMICAL ALLERGEN
LANGERHANSCELL (LC)
EDEMA AND ERYTHEMA
CYTOKINES,COSTIMULATORY,
ADHESIONMOLECULESINCREASE
INDUCTION PHASE ELICITATION PHASE
LC ANDLYMPHOCYTEINTERACTION
“PRIMED”LYMPHOCYTES
CELLULARINFLUX
INITIAL NONSPECIFICINFLAMMATORY
MEDIATORS
MIGRATION TO LOCALLYMPH NODE
LYMPHOCYTEPROLIFERATION
SPECIFIC RESPONSE
198 ALTERNATIVE TOXICOLOGICAL METHODS
Elicitation Phase
ALLERGIC CONTACT HYPERSENSITIVITY: MECHANISMS AND METHODS 199
TEST METHODS
Traditional Contact Hypersensitivity Tests—Guinea Pig
200 ALTERNATIVE TOXICOLOGICAL METHODS
The Local Lymph Node Assay (LLNA)—Murine
Figure 17.3 Guinea pig models. (Modified from Sailstad, 2002.)
Topical antigen application:closed patch
Days 0, 6–8, and 13–15
Day 27–28 topicalapplication (untreated flank
for 6 h)
21, 24, 48 h after removingpatch
Induction
Challenge
Endpoint Analysiserythema
20 animals/group
Guinea PigMaximization
Test
Topical antigen application:ID injection w/ or w/o FCA
Days 5-8
Day 20–22 topicalapplication
48, 72 h after challenge
BuehlerAssay
ALLERGIC CONTACT HYPERSENSITIVITY: MECHANISMS AND METHODS 201
Figure 17.4 The local lymph node assay. (Modified from Sailstad, 2002.)
Agent applied to ears
Isotope incorporation expressed as disintegrationper minute (dpm)
Proliferation measured:
Days 1, 2, 3
Day 6
Day 6: 5 h post IV injection Lymph nodes removed
IV tail injection of isotope
202 ALTERNATIVE TOXICOLOGICAL METHODS
Other Murine Systems
LLNA—Ex Vivo
Mouse Ear Swelling Test (MEST)
ALLERGIC CONTACT HYPERSENSITIVITY: MECHANISMS AND METHODS 203
SUMMARY OF ACD/CH ANIMAL MODELS
FUTURE
Figure 17.5 Contact hypersensitivity models and endpoint mechanisms.
Elicitation
PHASE
MEST
Induction
GPMT and BA
LLNA
204 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
ALLERGIC CONTACT HYPERSENSITIVITY: MECHANISMS AND METHODS 205
207
CHAPTER 18
Validating In Vitro Dermal AbsorptionStudies: An Introductory Case Study
CONTENTS
INTRODUCTION
208 ALTERNATIVE TOXICOLOGICAL METHODS
METHODS FOR EVALUATION OF THE REPORTS
VALIDATING IN VITRO DERMAL ABSORPTION STUDIES 209
MATERIALS AND METHODS OF THE INDIVIDUAL STUDIES
210 ALTERNATIVE TOXICOLOGICAL METHODS
VALIDATING IN VITRO DERMAL ABSORPTION STUDIES 211
212 ALTERNATIVE TOXICOLOGICAL METHODS
VALIDATING IN VITRO DERMAL ABSORPTION STUDIES 213
RESULTS OF THE STUDIES
214 ALTERNATIVE TOXICOLOGICAL METHODS
Tab
le 1
8.1
Ace
toch
lor
In V
ivo
(Mea
n d
ose
dis
trib
uti
on
as
g e
qu
ival
ents
of
acet
och
lor
per
cm
2 .M
ean
of
fou
r m
ale
rats
.)
Exp
osu
re (
hr)
Was
hC
over
Car
bo
nF
ilter
Ski
nU
rin
eF
eces
Cag
eW
ash
Car
cass
Ab
sorb
edg
/cm
2%
g/c
m2
%
3g
/cm
2
0.5
0.22
0.2
0.1
0.1
4.00
0.01
0.00
10.
001
0.1
0.1
3.79
12.
20.
20.
10.
26.
000.
010.
001
0.00
10.
10.
14.
282
2.0
0.2
0.2
0.2
7.17
0.01
0.00
10.
001
0.2
0.2
7.32
41.
80.
20.
30.
27.
930.
020.
001
0.01
0.3
0.3
9.93
101.
20.
30.
40.
39.
630.
20.
010.
010.
40.
619
.55
240.
90.
40.
70.
412
.53
0.5
0.2
0.02
0.2
0.9
31.3
7
42g
/cm
2
0.5
34.1
3.0
—1.
43.
180.
004
0.00
10.
011.
71.
73.
951
32.3
3.2
—1.
32.
990.
020.
001
0.01
2.5
2.5
5.98
234
.94.
1—
1.2
2.78
0.02
0.00
50.
31.
81.
94.
384
31.1
4.9
—1.
22.
810.
20.
003
0.01
3.6
3.9
9.24
1022
.15.
6—
1.2
2.66
2.1
0.3
0.4
7.0
9.8
23.0
624
15.1
8.1
—0.
91.
835.
52.
10.
64.
412
.629
.64
270
g/c
m2
0.5
212.
024
.2—
9.2
3.40
0.01
0.01
0.04
12.9
12.9
4.79
120
4.9
21.7
—8.
73.
210.
020.
010.
0515
.115
.25.
632
216.
923
.7—
13.3
4.91
0.02
0.01
0.05
8.1
8.2
3.03
421
8.5
21.0
—8.
73.
210.
70.
010.
0612
.313
.04.
8310
190.
428
.2—
10.8
3.99
3.5
0.1
0.56
21.1
25.2
17.5
2
2934
g/c
m2
0.5
2571
.928
5.9
—74
.22.
530.
10.
30.
440
.340
.91.
391
2175
.740
5.4
—95
.23.
240.
20.
30.
215
0.9
153.
045.
222
2322
.735
5.2
—98
.93.
370.
50.
10.
698
.599
.63.
394
2260
.150
2.7
—93
.32.
182.
30.
11.
174
.878
.32.
6710
2261
.443
5.5
—88
.33.
0115
.20.
21.
611
4.3
131.
34.
4724
1788
.962
7.2
—86
.02.
9297
.147
.98.
421
6.7
369.
912
.61
aA
bsor
bed
is s
um o
f ur
ine,
fec
es,
cage
was
h, a
nd c
arca
ss.
Sou
rce:
Lyth
goe,
199
0a.
VALIDATING IN VITRO DERMAL ABSORPTION STUDIES 215
Table 18.2 Acetochlor In Vitro (Mean dose distribution in vitro dermal absorption in rat skin. Mean of four to seven skin samples. Results presented as g/cm2.)
Exposure(hr)
AbsorbedWash
SkinDonor Loop
TotalRecoveredg/cm2 % g/cm2 %
3.02 g/cm2
0.05 0.41 13.68 1.73 0.11 3.60 0.08 0.55 2.891 0.82 27.12 1.32 0.11 3.77 0.04 0.44 2.732 1.14 37.78 0.95 0.12 3.84 0.03 0.50 2.734 1.37 45.30 0.80 0.09 2.98 0.05 0.43 2.75
10 2.15 71.19 0.17 0.08 2.52 0.01 0.59 2.9924 2.12 70.13 0.13 0.08 2.75 0.02 0.41 2.77
47.3 g/cm2
0.5 2.02 4.27 25.6 1.09 2.30 1.41 1.03 31.11 3.90 8.25 28.2 1.17 2.47 0.97 1.02 35.32 10.3 21.78 18.7 1.46 3.09 0.57 1.25 32.24 16.0 33.83 13.6 1.08 2.28 0.58 1.38 32.7
10 20.7 43.76 7.1 1.00 2.11 0.78 0.98 30.624 28.7 60.68 2.9 0.79 1.73 0.14 1.21 33.7
318 g/cm2
0.5 6.58 2.07 283 12.9 4.06 10.5 13.4 3251 21.4 6.73 383 16.6 5.22 14.3 16.7 4522 33.4 10.50 261 28.1 8.84 19.4 8.25 3504 80.9 25.44 262 16.2 5.09 4.41 11.6 375
10 182 57.23 158 22.6 7.11 5.59 11.6 37924 246 77.36 67 14.4 4.53 5.52 13.3 347
3095 g/cm2
1 17 0.55 2411 143 4.62 311 118 29992 104 3.36 3146 187 6.04 313 138 38584 137 4.43 2103 111 3.59 316 161 2828
10 264 8.53 2344 196 6.33 294 156 325324 757 24.46 1598 202 6.53 182 127 2866
Note: The 0.5-hr exposure was not performed at the high dose.
Source: Clowes and Scott, 1990a.
216 ALTERNATIVE TOXICOLOGICAL METHODS
DISCUSSION
Table 18.3 Acetochlor In Vitro (Mean dose distribution in vitro dermal absorption in human skin. Mean of six skin samples. Results expressed as g/cm2.)
Exposure(hr)
AbsorbedWash
SkinDonor Loop
TotalRecoveredg/cm2 % g/cm2 %
3.02 g/cm2
2 0.015 0.486 2.36 0.273 9.04 0.035 0.359 3.0410 0.074 2.44 2.61 0.271 9.98 0.136 0.194 3.2824 0.261 8.63 1.54 0.092 3.03 0.098 0.244 2.24
47.3 g/cm2
2 0.140 0.296 34.2 1.89 3.99 3.12 2.61 41.910 1.70 3.59 30.4 4.43 9.37 3.86 2.31 42.724 3.63 7.68 26.2 1.63 3.45 2.80 3.09 37.4
318 g/cm2
2 0.816 0.256 239 18.9 5.94 70.4 22.4 35210 5.69 1.79 221 12.1 3.82 91.8 15.8 34724 43.6 13.7 245 18.2 5.72 62.3 15.8 384
3095 g/cm2
2 4.57 0.148 2796 51.5 1.67 772 260 388410 22.4 0.722 2057 80.5 2.60 896 259 331424 32.5 1.05 1669 38.4 1.24 671 217 2628
Source: Clowes and Scott, 1990b.
VALIDATING IN VITRO DERMAL ABSORPTION STUDIES 217
Table 18.4 Comparison of the Percent Absorbed In Vitro and In Vivo in Rat Skin; Data Are from Tables 18.1 and 18.2
Dose( g/cm2) 0.5 1.0 2.0 4.0 10.0 24.0
Exposure Duration (hr)
3.0 in vivo 3.8 4.3 7.3 9.9 19.5 31.43.02 in vitro 13.7 27.1 37.8 45.3 71.2 70.142.5 in vivo 4.0 6.0 4.4 9.2 23.1 29.647.3 in vitro 4.3 8.2 21.8 33.8 43.8 60.7270 in vivo 4.8 5.6 3.0 4.8 9.3 17.5318 in vitro 2.1 6.7 10.5 25.4 57.2 77.42934 in vivo 1.4 5.2 3.4 2.7 4.5 12.63095 in vitro — 0.6 3.4 4.4 8.5 24.5
Ratio in Vitro/in Vivo
3.02/3.0 3.6 6.3 5.2 4.6 3.7 2.247.3/42.4 1.1 1.4 5.0 3.7 1.9 2.1318/270 0.4 1.2 3.5 5.3 6.2 4.43095/2934 — 0.1 1.0 1.6 1.9 1.9
Figure 18.1 The ratio of the in vitro absorption to the in vivo absorption with exposure durationin the rat. Dose ratios 1, 2, 3, and 4 are in order of low dose ratio to high doseratio. Data are from Table 18.4.
0
1
2
3
4
5
6
7
0 5 10 15 20 25
Dose 1Dose 2
Dose 3Dose 4
Exposure (h)
Rat
io v
itro/
vivo
218 ALTERNATIVE TOXICOLOGICAL METHODS
Table 18.5 Comparison of the Percent Absorbed In Vitro in Rat and Human Epidermal Membrane Preparations; Data Are from Tables 18.2 and 18.3
Dose( g/cm2) 2.0 10.0 24.0
Exposure Duration (hr)
3.03 rat 37.8 71.2 70.1human 0.49 2.44 8.63 47.3 rat 21.8 43.8 60.7human 0.30 3.59 7.68318 rat 10.5 57.2 77.4human 0.26 1.79 13.73095 rat 3.4 8.5 24.5human 0.15 0.72 1.05
Ratio Rat/Human
3.03 77.1 29.2 8.147.3 72.7 12.2 7.9318 40.4 32.0 5.63095 22.7 11.8 23.3
Figure 18.2 The ratio of the rat in vitro absorption to the human in vitro absorption withexposure duration. Dose ratios 1, 2, 3, and 4 are in order of low dose ratio to highdose ratio. Data are from Table 18.5.
0
10
20
30
40
50
60
70
80
0 5 10 15 20 25
vitro R/H 47.3 g/cm2
vitro R/H 318 g/cm2
vitro R/H 3095 g/cm2
Duration of Exposure (h)
Rat
io r
at/h
uman
VALIDATING IN VITRO DERMAL ABSORPTION STUDIES 219
REFERENCES
221
CHAPTER 19
A Molecular Diagnostic Approachto Irritant or Allergic Patch
Testing Using the DermPatch
CONTENTS
BACKGROUND
222 ALTERNATIVE TOXICOLOGICAL METHODS
Table 19.1 Skin Cytokins (adapted from Gerberick et al., 1998)
Constitutive or inducible expression inCytokines Langerhans cells Keratinocytes Fibroblasts
IL-1 – + +IL-1 + – +IL-3 – +IL-6 + + +IL-7 – + –IL-8 – + +IL-10 – + –IL-12 – +IL-15 +G-CSF – + +M-CSF – + –GM-CSF – + +TGF- – + –TGF- + + +TNF- – + –MIP-1 + – –MIP-1 + + –IP-10 – + +
IRRITANT OR ALLERGIC PATCH TESTING USING THE DERMPATCH 223
Table 19.2 mRNA Cytokine Profiles from Human Skin Biopsy or Human Cell Samples
ACD ICD
TNF- increased increasedIFN- increased increasedIL-2 increased increasedGM-CSF increased increasedIL-1 (human cells) dependent on allergen increasedIL-1 (human cells) increased increasedIL-4 increased not determinedIL-6 increased not determinedIL-10 increased no changeIL-12 p35 (human cells) no change no changeIL-12 p40 (human cells) increased no change
Source: Adapted from Wakem and Gaspari, 2000.
Table 19.3 Mechanisms of Irritant versus Allergic Contact Dermatitis
Feature Allergic Irritant
Chemical agents low molecular weight, lipid soluble
acids, alkalies, surfactants, solvents, oxidants, enzymes
Concentration of the agent less critical more criticalGenetic predisposition ++++ ++Sensitization and lag period necessary not necessaryTrigger interaction of antigen with
primed T cellsdamage to keratinocytes
Cytokine release ++++ +++T-cell activation early ++++ later ++++Mast-cell activation ++ ++Langerhans’ cells increased decreasedEicosanoid production ++ ++
Source: Adapted from Marks and DeLeo.
224 ALTERNATIVE TOXICOLOGICAL METHODS
MATERIALS AND METHODS
Tape Stripping and Extraction of Total RNA
Ribonuclease Protection Assay
Table 19.4 Clinical and Histological Aspects of Contact Dermatitis
Feature Allergic Irritant
Itch ++++ (early) +++ (late)Pain, burning ++ ++++ (early)Erythema ++++ ++++Vesicles ++++ +Pustules + +++Hyperkeratosis ++ +++Fissuring ++ ++++Sharp demarcation yes yesReaction delay after contact days minutes to hoursSpongiosis ++++ ++++Dermal edema ++++ ++++Necrotic keratinocytes ++++ ++++Ballooning degeneration + +++Lymphocytic infiltrate ++++ ++++Neurotrophilic infiltrate + +++
Source: Adapted from Marks and DeLeo.
IRRITANT OR ALLERGIC PATCH TESTING USING THE DERMPATCH 225
Induction of Erythematous Reactions on the Skin
RESULTS
226 ALTERNATIVE TOXICOLOGICAL METHODS
DISCUSSION
IRRITANT OR ALLERGIC PATCH TESTING USING THE DERMPATCH 227
REFERENCES
229
CHAPTER 20
In Vitro Skin Equivalent Modelsfor Toxicity Testing
CONTENTS
230 ALTERNATIVE TOXICOLOGICAL METHODS
INTRODUCTION
AIR–LIQUID INTERFACE TISSUE CULTURES
IN VITRO SKIN EQUIVALENT MODELS FOR TOXICITY TESTING 231
Figure 20.1 Schematic representation of the air–liquid interface (ALI) tissue culture technique.
Table 20.1 Epithelial Tissues Successfully Cultured at the ALI
Skin equivalentsa
Keratinocytes onlyKeratinocytes plus fibroblastsb
Keratinocytes plus melanocytesKeratinocytes plus Langerhans cells
Ocular corneal epitheliuma
Tracheal/bronchiala epitheliumTracheal/bronchial submucosal glandsVaginal epitheliuma,b
Gingival epitheliuma,b
a Cultured at MatTek Corp.b In development.
Tissue Culture Well
Culture Insert
ALI Tissue
Medium
Membrane
Tissue Culture Well
Culture Insert
ALI Tissue
Medium
Membrane
Tissue Culture Well
Culture Insert
ALI Tissue
Medium
Membrane
232 ALTERNATIVE TOXICOLOGICAL METHODS
(A)
(B)
Figure 20.2 (A) Histological cross section of H&E stained EpiDerm-200. Magnification = 440 .(B) Transmission electron micrograph of ruthenium tetroxide stained intercellularlamellar lipid sheets (150,000 ).
Figure 20.3 Top macroscopic view of MelanoDerm tissues containing normal human melano-cytes derived from Black donor skin (400 magnification).
IN VITRO SKIN EQUIVALENT MODELS FOR TOXICITY TESTING 233
Figure 20.4 Development of pigmentation in MelanoDerm tissue produced with melanocytesderived from various skin phototypes. The figure shows the top macroscopic viewof tissue inserts. Day 0 indicates the day of shipment of a fully differentiated tissue.Pigmentation develops during additional culture of the fully differentiated tissueby the end user.
Figure 20.5 Pigmentation of MelanoDerm tissue (Black melanocytes) induced by -MSH and-FGF as shown by top macroscopic view of tissue inserts. Tissues were cultured
in media containing the indicated concentrations of growth factors for up to 20days following shipment of commercial MelanoDerm tissue.
• Asian (A)
• Black (B)
• Caucasian (C)
10-7M MSH, 3 ng/ml FGF
5x10-8 MSH, 1.5 ng/ml FGF
10-8 MSH, 0.3 ng/ml FGF
No MSH or FGF added
10-7M MSH, 3 ng/ml FGF
5x10-8 MSH, 1.5 ng/ml FGF
10-8 MSH, 0.3 ng/ml FGF
No MSH or FGF added
234 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 20.6 Lightening effect of topical kojic acid on MelanoDerm tissue. Tissues containingBlack melanocytes were treated topically with 1% kojic acid for the indicatednumber of days (25 l applied topically every other day). The top macroscopicview of the tissues reveals the visually observable lightening effect.
Table 20.2 Lightening of MelanoDerm Tissue by Topical Treatment with 1% Kojic Acid
Melanin content ( g/tissue)Treatment Day 10 Day 14
H2O 19.9 35.31% kojic acid 11.4 18.3
Figure 20.7 EpiDerm-FT tissue contains normal human fibroblasts cultured within a collagentype I dermal matrix. A fully differentiated epidermis derived from normal humankeratinocytes is cultured on the top of the dermis.
Day: 3 7 10 14
Kojic Acid—topical
Control
IN VITRO SKIN EQUIVALENT MODELS FOR TOXICITY TESTING 235
SURVEY OF USES AND ENDPOINTS
Corrosivity
Phototoxicity
Figure 20.8 Immunohistochemical staining of HLA-DR on human Langerhans cells within (A)ImmunoDerm tissue and (B) excised human skin.
236 ALTERNATIVE TOXICOLOGICAL METHODS
Irritation
Melanogenesis
Figure 20.9 Phototoxicity of chlorpromazine. EpiDerm tissues were topically treated with the indi-cated concentrations of chlorpromazine and incubated overnight. On the followingday tissues were either irradiated with 6 J/cm2 of UVA or kept in the dark. Tissueswere then rinsed, fed with fresh medium, and incubated overnight. Finally, tissueviability was determined by the MTT assay. A decrease in tissue viability of 30% orgreater between the irradiated and nonirradiated tissues indicates a phototoxic effect.
0
20
40
60
80
100
120
0.001 0.003 0.010 0.032 0.100
[Concentration %]
MT
T (
% u
ntre
ated
con
trol
)
Chloropromazine
68%
42%
87%74%
-2%
-UVA+UVA
IN VITRO SKIN EQUIVALENT MODELS FOR TOXICITY TESTING 237
Percutaneous Absorption
Drug Metabolism
MISCELLANEOUS STUDIES
QUALITY CONTROL AND VALIDATION ISSUES
238 ALTERNATIVE TOXICOLOGICAL METHODS
HISTOLOGICAL EVALUATION OF EACH TISSUE LOT
QUANTITATIVE ASSESSMENT OF FUNCTIONAL RESPONSE TO TOPICAL TRITON X-100 TREATMENT
IN VITRO SKIN EQUIVALENT MODELS FOR TOXICITY TESTING 239
EMERGING APPLICATIONS OF ALI TISSUES
Gene Microarray Technology
Table 20.3 EpiDerm-200 Triton X-100 ET50 Database Summary
YearEPI-200
Triton ET-50EPI-200
Triton C.V. Lots Avg. C.V.
2000a 6.76 16.4 89 6.21999 6.75 18.2 146 5.71998 7.24 17.9 175 9.21997 6.78 15.9 228 9.91996 6.74 14.6 184 9.61995 6.65 77.8 112 4.9
a Through September 2000.
240 ALTERNATIVE TOXICOLOGICAL METHODS
IN VITRO SKIN EQUIVALENT MODELS FOR TOXICITY TESTING 241
Figure 20.10 Changes in cancer-related gene expression following UVB-irradiation of Epi-Derm tissue. AtlasTM human cancer cDNA expression array analysis of geneexpression changes induced in EpiDerm tissues 6 hr after irradiation with 175mJ/cm2 UVB.
Table 20.4 UVB-Irradiation of EpiDerm Tissue
WAF-1 (+)MAP Kinase p38 (+)Growth-arrest-specific protein (+)c-Myc binding protein MM-1 (+)TRAF-interacting protein (+)Caspases (+)Death-associated protein kinase (DAP kinase 1, +)p53-induced protein (+)GADD45 (+)DNA excision repair protein ERCC1 (+)DNA-repair protein XRCC1 (+)Placenta growth factors 1+2 (+)TIMP-1 (+)T-plasminogen activator (+)Rho GDP dissociation inhibitor 1 (+)Endothelin 2 (–)IL-6 (–)Leukocyte interferon-inducible peptide (–)60S ribosomal protein (–)
242 ALTERNATIVE TOXICOLOGICAL METHODS
High-Throughput ALI Tissue Formats
Figure 20.11 Induction of PlGF expression following UVB-irradiation of EpiDerm tissue. Aga-rose gel electrophoresis of products obtained by RT-PCR of total RNA isolatedfrom EpiDerm tissue 20 hours following UVB-irradiation. (–) no irradiation. (+)UVB-irradiated. The expected PlGF PCR product is 273 bp.
Figure 20.12 EpiDerm tissues cultured in 24 well (EpiDerm-224) or 96 well (EpiDerm-296)high throughput ALI formats. Each tissue has its own individual media reservoirto avoid cross-contamination of samples.
20 25 30 35
Cycles
– + – +– + – +
273 bp
Markers
IN VITRO SKIN EQUIVALENT MODELS FOR TOXICITY TESTING 243
SUMMARY
Figure 20.13 Total RNA-96 isolation kit designed for high throughput total RNA isolation fromEpiDerm-296.
Figure 20.14 Agarose gel electrophoresis of total RNA isolated from EpiDerm-296 with theTotal RNA-96 isolation kit. Average yield is approximately 10 g DNA-free totalRNA/EpiDerm-296 tissue.
244 ALTERNATIVE TOXICOLOGICAL METHODS
ACKNOWLEDGMENTS
REFERENCES
IN VITRO SKIN EQUIVALENT MODELS FOR TOXICITY TESTING 245
246 ALTERNATIVE TOXICOLOGICAL METHODS
IN VITRO SKIN EQUIVALENT MODELS FOR TOXICITY TESTING 247
PART IV
A Case Study in the Use of Alternativesto Determine the Mechanism
of Sulfur Mustard Action
250 ALTERNATIVE TOXICOLOGICAL METHODS
251
CHAPTER 21
Cellular Resistance of Tetrahymenato Sulfur Mustard
CONTENTS
INTRODUCTION
252 ALTERNATIVE TOXICOLOGICAL METHODS
MATERIALS AND METHODS
Cell Culture and SM Exposures
Coulter Counter Studies
CELLULAR RESISTANCE OF TETRAHYMENA TO SULFUR MUSTARD 253
RESULTS
Figure 21.1 Cell viability vs. [SM]. Viability of six different cell lines was analyzed over a rangeof SM concentrations. Symbols are represented within the graph.
10 -110 -210 -310 -410 -510 -610 -710 -80
20
40
60
80
100
CHO and HeLa
PBL
HEK
Tetrahymena
Yeast
SM Concentration [M]
Per
cent
age
Via
ble
254 ALTERNATIVE TOXICOLOGICAL METHODS
DISCUSSION
Figure 21.2 Effects of SM on cell number over time. The number of Tetrahymena was deter-mined after exposure to a range of SM concentrations and over a period of 72hr. Symbols are represented within the graph.
807060504030201000
10
20
30
40
50
60
8.0 mM4.0 mM2.0 mM
0.5 - 1.0 mM
0.3 mM & Positive Control
Time (h)
Cel
l Num
ber
CELLULAR RESISTANCE OF TETRAHYMENA TO SULFUR MUSTARD 255
256 ALTERNATIVE TOXICOLOGICAL METHODS
ACKNOWLEDGMENTS
REFERENCES
CELLULAR RESISTANCE OF TETRAHYMENA TO SULFUR MUSTARD 257
259
CHAPTER 22
Studies of Cellular Biochemical ChangesInduced in Human Cells by Sulfur Mustard
CONTENTS
260 ALTERNATIVE TOXICOLOGICAL METHODS
INTRODUCTION
MATERIALS AND METHODS
Cell Cultures
HEK
HeLa
BIOCHEMICAL CHANGES INDUCED IN CELLS BY SULFUR MUSTARD 261
Lymphocytes
DNA Isolation
Reagents
Phenol-Chloroform Extraction
Commercial Kits
262 ALTERNATIVE TOXICOLOGICAL METHODS
Modified Kit Usage
Pulse Field Gel Electrophoresis (PFGE)
PARP Assay
Calcium Measurement
Interleukin ELISA
BIOCHEMICAL CHANGES INDUCED IN CELLS BY SULFUR MUSTARD 263
Measurement of Fc and C1q Receptors
RESULTS
Table 22.1 Problems Seen with DNA Isolation from HEK
Procedure Date Sample
Purity Level of DNA (260/280
Ratio)
DNAYield
(mg/ml)
Purification of HEK DNA using Boehringer Mannheim Kit for mammalian cells
8/4/98 Control 1.35 608/18/98 Control 1.65 458/24/98 Control 1.35 25
Purification of lymphocyte DNA using Boehringer Mannheim Kit for mammalian blood
9/2/98 Control 1.82 1899/8/98 Control 1.81 1859/29/98 Control 1.93 194
Phenol/chloroform extraction of HEK DNA
10/1/98 Control 1.22 6010/7/98 Control 1.35 3310/13/98 Control 1.47 67
Purification of HEK DNA using Boehringer Mannheim Kit for mammalian cells (modified procedure)
10/19/98 Control 1.90 9410/27/98 Control 1.86 9311/5/98 Control 1.86 98
264 ALTERNATIVE TOXICOLOGICAL METHODS
Table 22.2 Purification of HEK DNA using Boehringer Mannheim Kit for Mammalian Cells Exposed to Sulfur Mustard (HD)
Date SamplePurity Level of DNA
(260/280 Ratio)DNAYield
11/19/98 Control 1.81 12250 mM HD 2.10 163100 mM HD 1.89 150300 mM HD 1.82 199
11/26/98 Control 1.85 19450 mM HD 1.99 213100 mM HD 1.86 198300 mM HD 1.81 172
12/8/98 Control 1.98 18350 mM HD 1.96 179100 mM HD 1.91 176300 mM HD 1.86 199
BIOCHEMICAL CHANGES INDUCED IN CELLS BY SULFUR MUSTARD 265
DISCUSSION
Figure 22.1 Time-dependent response of PARP activity following HD exposure of (top) HeLacells and (bottom) HEK cells. Two concentrations of HD were used, 10 and 100 M,and PARP activities are presented as percent maximal response.
HeLa PARP enzyme activity
Time post-HD (h)1 2 64 9 24
% o
f max
imum
res
pons
e
0
20
40
60
80
100
120
100 µM HD10 µM HD
HEK PARP enzyme activity
Time post-HD (h)1 2 4 6 24
% o
f max
imum
res
pons
e
0
20
40
60
80
100
120
140
x-axis vs. 10 µM HDx-axis vs. 100 µM HD
266 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 22.2 Increase in intracellular calcium in HEK (passage 2) exposed to 300 M HD asmeasured by 340:380 nm ratio of Fura-2 AM.
Table 22.3 Binding in HD-Exposed HEKa
Hrs post HD Dose HD ( M)C1q 0 100 200 300
8 – – – –16 – – NT ++24 – + +++ +++
CD32 0 50 100 200
8 – + + +24 – + ++ +++
a + = weak; ++ = moderate; +++ = intense; NT = not tested. Grading of stainingwas judged visually by fluorescence microscope and HEK controls not exposedto HD were negative for fluorescence.
Time (s)
0 200 400 600 800 1000 1200 1400
Rat
io (
340:
380
nm)
1.7
1.8
1.9
2.0
2.1
2.2
2.3
2.4
2.5
2.6
BIOCHEMICAL CHANGES INDUCED IN CELLS BY SULFUR MUSTARD 267
REFERENCES
Figure 22.3 Concentration-dependent increase in the secretion of IL-8 from HEK exposed to HD.
HD Concentration
-50 0 50 100 150 200 250 300 350
pg/m
l IL-
8
0
500
1000
1500
2000
2500
3000
3500
4000
HD-exposed HEK
269
CHAPTER 23
Human Keratinocyte InflammatoryTranscript Gene Activity
Following Sulfur Mustard*
CONTENTS
INTRODUCTION
270 ALTERNATIVE TOXICOLOGICAL METHODS
MATERIALS AND METHODS
Materials
HUMAN KERATINOCYTE SM INFLAMMATORY TRANSCRIPT GENE ACTIVITY 271
Cell Culture and Exposure
RNA Preparation
Probing cDNA Array Blot
272 ALTERNATIVE TOXICOLOGICAL METHODS
Table 23.1 HEK Housekeeping Gene Transcripts at 16 Hr Following Sulfur Mustard
Gene Ratioa
Gene TranscriptControl
Intensityb 25 M SM 200 M SM
14-3-3 zeta protein 317 0.7 0.4423 kDa Highly Basic Protein 1689 2.0 2.3
-Tubulin 307 0.57 1.4-Actin 236 1.2 1.2
Glyceraldehyde 3-phosphate dehydrogenase 612 0.44 1.1HLA class I histocompatibility antigen C-4 alpha chain
487 0.94 1.0
Hypoxanthine-guanine phosphoribosyltransferase
59 0.9 3.7
Ribosomal Protein S9 978 1.2 2.1Ubiquitin 2064 0.8 0.73
a Induction ratio from phosphorimage densitometeric measurements of listed gene transcriptnormalized to the sum total of the detected transcript intensity/pixel density.
b Control intensity (grayscale pixel density) adjusted from background. Background was setat the median intensity of the blank space between the six array panels. The subtractedbackground intensity ranged from 19 to 43.
Table 23.2 HEK Inflammation-Associated Transcripts at 16 Hr Following Sulfur Mustard
Gene Ratioa
Gene TranscriptControl
Intensityb 25 M SM 200 M SM
CD40 86 4.9 0.3Interleukin 1 67 0.1 1.4Interleukin 1 101 0.6 3.5Interleukin 2 receptor subunit 148 0.3 0.4Interleukin 6 494 0.7 0.6Interleukin 7 receptor subunit 131 0.4 0.7Interleukin 8 16 —c 20Interleukin 13 349 3.0 0.7Interleukin 15 215 — 0.03Macrophage inflammatory protein 2 11 — 13S19 ribosomal protein 886 0.9 2.1Tumor necrosis factor 50 — 1.4
a Induction ratio from phosphorimage densitometeric measurements of listed gene transcriptnormalized to the sum total of the detected transcript density.
b Control intensity (grayscale pixel density) adjusted from background. Background was setat the median intensity of the blank space between the six array panels. The subtractedbackground intensity ranged from 19 to 43.
c Transcript not detected above background.
HUMAN KERATINOCYTE SM INFLAMMATORY TRANSCRIPT GENE ACTIVITY 273
Subtraction Library Construction
Subtraction Library Sequence and Analysis
Figure 23.1 Atlas cDNA array nylon blot image. HEK were exposed to 200 M sulfur mustardfor 16 h and mRNA isolated and 32P labeled as cDNA. The array contains double-dot blots of cDNA from 588 transcriptionally regulated genes and 9 housekeepinggenes at the bottom of the array (see 3 housekeeping genes in rectangular box).Boxes 1 and 2 show expression of macrophage inflammatory protein 2 andinterleukin 8, respectively. These inflammatory transcripts were at very low expres-sion levels in control HEK.
1
2
274 ALTERNATIVE TOXICOLOGICAL METHODS
RESULTS
HUMAN KERATINOCYTE SM INFLAMMATORY TRANSCRIPT GENE ACTIVITY 275
DISCUSSION AND CONCLUSIONS
276 ALTERNATIVE TOXICOLOGICAL METHODS
HUMAN KERATINOCYTE SM INFLAMMATORY TRANSCRIPT GENE ACTIVITY 277
278 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
HUMAN KERATINOCYTE SM INFLAMMATORY TRANSCRIPT GENE ACTIVITY 279
281
CHAPTER 24
Evaluation of Cytotoxicity Assays of HumanEpidermal Keratinocytes Exposed
In Vitro to Sulfur Mustard
CONTENTS
INTRODUCTION
282 ALTERNATIVE TOXICOLOGICAL METHODS
EXPERIMENT
Reagents
Keratinocyte Growth
Agent Exposure
Calcein AM
Alamar Blue
CYTOTOXICITY ASSAYS OF KERATINOCYTES EXPOSED TO SULFUR MUSTARD 283
Neutral Red
MTS
Propidium Iodide
RESULTS
284 ALTERNATIVE TOXICOLOGICAL METHODS
DISCUSSION
REFERENCES
Figure 24.1 HEKs were exposed to the indicated HD concentrations and then incubated for24 hr at 37 C. Viabilities were determined by using these dyes as described inthe Experimental section.
CYTOTOXICITY ASSAYS OF KERATINOCYTES EXPOSED TO SULFUR MUSTARD 285
287
CHAPTER 25
Comparison of Spectrophotometric andFluorometric Assays of Proteolysis in
Cultured Human Cells Exposedto Sulfur Mustard
CONTENTS
288 ALTERNATIVE TOXICOLOGICAL METHODS
INTRODUCTION
MATERIALS AND METHODS
Reagents
Lymphocyte Isolation
ASSAYS OF PROTEOLYSIS INDUCED BY SULFUR MUSTARD 289
Keratinocyte Growth
HD Exposure
Chromozym® Assay (24 hr Postexposure to HD)
Keratinocytes
Lymphocytes
Intracellular Protease Activity
RESULTS
290 ALTERNATIVE TOXICOLOGICAL METHODS
Chromozym® Assay
CellProbe® Assay
Figure 25.1 Substrate descriptions.
Chromozym® SubstratesChromozym® TH Tosyl-glycyl-prolyl-arginine-4-nitranilide acetateChromozym® t-PA N-Methylsulfonyl-D-Phe-Gly-Arg-4-nitranilide acetateChromozym® TRY Carbobenzoxy- valyl-glycyl-arginine-4-nitrilanilide acetateChromozym® U Benzoyl-β-alanyl-glycyl-arginine-4-nitranilide acetate
CellProbe® Enzyme Substrates:Nonfluorescent (aa)x -Fl Fluorescent Dye +Substrate-Dye complex ➾➾➾ Nonfluorescent Substrate leaving group
Enzyme SubstrateAAPV- Elastase (AAPV) Dl-Alanyl-Alanyl-Prolyl-Rho 110D-aminopeptidase A (DAA) Asp-Asp-Rho 110K-Aminopeptidase B (KAB) Lysine-Lysine-Rho 110G-Aminopeptidase (GA) Gly-Gly-Rho 100L- Aminopeptidase (LA) Leu-Leu-RhoA- Aminopeptidase M (AAM) Alanine-Alanine-Rho 110R- Aminopeptidase B (RAB) Arginine-Arginine-Rho 110
ASSAYS OF PROTEOLYSIS INDUCED BY SULFUR MUSTARD 291
Figure 25.2 Protease activity was measured in two donors as a function of cell number.Increasing the cell concentration above 2 106/well did not increase proteaseactivity and this concentration was used in all subsequent studies.
Figure 25.3 PBL from five different donors were exposed to 250 M HD and then incubatedfor 24 hr. Protease activity using the Chromozym® TH substrate was measuredas described in Material and Methods. Significant protease activity occurredreproducibly. All assays were run in triplicate and all donors were evaluatedmultiple times on different days.
292 ALTERNATIVE TOXICOLOGICAL METHODS
SUMMARY
Figure 25.4 HEK were exposed to indicated HD concentrations and incubated for 20 hr. Chro-mogenic protease assays were performed as described in the Materials and Methods.The protease activity was only evident at the highest concentration of HD, and onlythe Chromozym® U and Chromozym® t-PA substrates were significantly hydrolyzed.
Figure 25.5 HEK were exposed to HD at the indicated concentrations, and protease activitywas measured fluorometrically 24 hr later as described in the Material and Meth-ods section. KAB and GA substrates were significantly hydrolyzed above thecontrol values while elastase (AAPV) and DAA were decreased.
ASSAYS OF PROTEOLYSIS INDUCED BY SULFUR MUSTARD 293
REFERENCES
295
CHAPTER 26
Effects of Low Dose Sulfur Mustardon Growth and DNA Damage
in Human Cells in Culture
CONTENTS
INTRODUCTION
296 ALTERNATIVE TOXICOLOGICAL METHODS
PURPOSE
MATERIALS AND METHODS
PBL Isolation
PBL Exposure
EFFECTS OF LOW DOSE SULFUR MUSTARD ON HUMAN CELLS 297
HEK Cultures
HEK Exposure
Comet Assay
Viability Assays
Growth Curve Assay
RESULTS
298 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 26.1 PBLs were exposed to buffer or to buffer plus HD. Four hours following exposure,cells were harvested and treated with 0.001% H2O2 or buffer. HD concentrationsof 5 M or greater inhibited expression of SSB in the comet assay. Points representmean ±SEM from two experiments.
Figure 26.2 HEKs were exposed to buffer or to buffer plus HD. Four hours following exposure,cells were harvested and treated with 0.002% H2O2 or buffer. Results are similarto those seen with PBLs. Points represent mean ±SEM from two experiments.
µM HD
0 10 20 30 40 50 60
Com
et M
omen
t
0
10
20
30
40
HDHD-HP
µM HD
0 10 20 30 40 50 60
Com
et M
omen
t
0
10
20
30
40
HDHD-HP
EFFECTS OF LOW DOSE SULFUR MUSTARD ON HUMAN CELLS 299
Figure 26.3 PBLs were exposed to buffer or to buffer plus CEES. Four hours following expo-sure, cells were harvested and treated with 0.001% H2O2 or buffer. CEES did notinhibit expression of H2O2-induced SSB. Points represent mean ±SEM from twoexperiments.
Figure 26.4 HEKs were exposed to buffer or to buffer plus CEES. Four hours following exposure,cells were harvested and treated with 0.002% H2O2 or buffer. Results are similar tothose seen with PBLs. Points represent mean ±SEM from two experiments.
µM CEES
0 10 20 30 40 50 60
Com
et M
omen
t
0
10
20
30
40
CEESCEES-HP
µM CEES
0 200 400 600 800 1000 1200
Com
et M
omen
t
0
10
20
30
40
CEESCEES-HP
300 ALTERNATIVE TOXICOLOGICAL METHODS
CONCLUSIONS
Figure 26.5 HEKs were exposed to buffer or to buffer plus HD. Eighteen hours followingexposure, cells were harvested and treated with 0.002% H2O2 or buffer. Pointsrepresent mean ±SEM from two experiments.
µM HD
0 10 20 30 40 50 60
Com
et M
omen
t
10
20
30
40
50
60
70
HDHD and H2O2
EFFECTS OF LOW DOSE SULFUR MUSTARD ON HUMAN CELLS 301
Figure 26.6 HEKs were exposed to buffer or to buffer plus HD. Twenty-four hours followingexposure, cells were harvested and treated with 0.002% H2O2 or buffer. Pointsrepresent mean ±SEM from two experiments.
Figure 26.7 Growth curves for HEKs following exposure to HD in culture. Lowest line is for5 M concentration.
µM HD
0 10 20 30 40 50 60
Com
et M
omen
t
0
20
40
60
80
100
HDHP and H2O2
Time (h)
20 30 40 50 60 70 80 90 100
Via
ble
Cel
ls/m
L
100x103
1x106
10x106
0.00 M HD 0.01 M HD0.10 M HD0.50 M HD1.00 M HD5.00 M HD
302 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
303
CHAPTER 27
Imaging Sulfur Mustard Lesions in BasalCells and Human Epidermal Tissues
by Confocal and MultiphotonLaser Scanning Microscopy
CONTENTS
INTRODUCTION
304 ALTERNATIVE TOXICOLOGICAL METHODS
MATERIALS AND METHODS
Figure 27.1 Basal cell adhesion complex. A microvesicle (lower left panel) showing details ofthe dermal–epidermal separations characteristic of a sulfur mustard blister.Hemidesmosomes (arrowhead), at the roof of the blister, are well displaced fromthe basement membrane (bm) and lamina densa (ld). An expanded model of theintact adhesion complex (circumscribed area) includes the intracellular keratinfilaments K5 and K14 and their facilitated attachments to the transmembrane 6 4
integrin receptors. The exodomains of 6 4 are shown linked by laminin 5 to thebasement membrane zone.
IMAGING SULFUR MUSTARD LESIONS 305
RESULTS
306 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 27.2 Keratin 5 images from control cultures of HEK recorded by multiphoton imaging(A), showed the elaborate cytoskeletal matrix and distribution of these filamentswithin the cell cytoplasm. Image intensity and K5 concentration were greatestaround the nucleus of each cell, and a lacy network of delicate filaments projectedout toward the cell extremities. Analysis of confocal images from K5 controls (B)and HD-exposed populations (C) showed a statistically significant (p < 0.01) 29.2%decrease in intensity at 1 hr postexposure to sulfur mustard.
IMAGING SULFUR MUSTARD LESIONS 307
Figure 27.3 Multiphoton keratin 14 images from control HEK cultures (A) showed elaboratecytoskeletal distribution comparable to that of keratin 5. Image intensity and K14concentration were greatest around the nucleus. Lacy networks of filaments pro-jected out to the cell extremities where they interfaced closely with those ofadjacent cells (arrow). At 1 hr postexposure to sulfur mustard, K14 images (B)indicated a disruption of organization, resulting in withdrawal of filaments from theplasma membrane margins, appearance of punctate nodules (arrows), and asubstantial loss of cytoskeletal definition.
Figure 27.4 Analysis of K14 confocal images from replicate cultures of HEK sham-treatedcontrols (A and C) and HD-exposed populations (B and D) showed a statisticallysignificant (p < 0.01) 30.14% decrease in image intensity (K14 expression) at 1hr postexposure and a nearly complete loss of expression (79% decrease in imageintensity) at 2 hr.
308 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 27.5 A multiphoton montage showing the organization of 6 integrins on an explant ofhuman epidermis. Serial slices (A and B) illustrate the receptor outline on suc-cessive cross sections through the epidermal rete pegs. The three-dimensionalreconstruction (C) and the stereo image (D) are from the same Z-series of serialslices. Together, they show the topographic complexity of ventral epidermis plusthe circular shape and extensive distribution of 6 4-integrin receptors.
Figure 27.6 A multiphoton image (slice) ofhuman epidermis showing incross section the distributionand circular shape of 4 inte-grins (arrows) on the basalcell surface.
IMAGING SULFUR MUSTARD LESIONS 309
DISCUSSION
Figure 27.7 Multiphoton images of 6 4 receptors in human epidermis exposed to sulfurmustard indicated unraveling and loss of circular shape at 1 hr postexposure (A)and an almost total loss of 6 4 expression at the basal cell surface by 2 hrpostexposure (B, C). At 2 hr postexposure, only a basolateral pattern of residualfluorescence remained to outline the constituent basal cells. Loss of 6 and 4
integrin also occurred spontaneously in epidermal tissues following dermal–epi-dermal separation; therefore, the effects may not be strictly related to sulfurmustard exposure.
310 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 27.8 Analysis of confocal images from HEK in replicate control and HD-exposed cul-tures indicated a statistically significant (p < 0.01) decrease of 27.3% and 26.3%in image intensity of 6 and 4 integrins, respectively, at 1 hr postexposure. Thedecrease was characterized by a loss of fluorescence from the surface of attachedbasal cells, resulting in a honeycomb pattern of residual, basolateral fluorescence.Postexposure image patterns from cultures were very similar to those recordedfrom intact epidermal tissues (see Figures 27.7B,C).
IMAGING SULFUR MUSTARD LESIONS 311
REFERENCES
312 ALTERNATIVE TOXICOLOGICAL METHODS
313
CHAPTER 28
Suppression of Sulfur Mustard-IncreasedIL-8 in Human Keratinocyte Cell Cultures
by Serine Protease Inhibitors: Implicationsfor Toxicity and Medical Countermeasures*
CONTENTS
INTRODUCTION
314 ALTERNATIVE TOXICOLOGICAL METHODS
METHODS
Reagents
MUSTARD-INDUCED IL-8 315
Sulfur Mustard Exposure
Protease Inhibitors
Table 28.1 Candidate Antivesicant Drug Screening: Statistically Positive Reduction of at Least 50% in Edema or Histopathology in the Mouse Ear or Hairless Mouse
Edema Histopathology
Anti-Inflammatory Drugs
Fluphenazine dihydrochloride 50Indomethacin 63 96Olvanil 53 91Hydrocortisone 65 71Olvanil (saturated) 53Retro olvanil 62 84Olvanil (urea analog) 81Octyl homovanillamide 65 100Dexamethasone 72
Scavenger Drugs
2-Mercaptopyridine-1-oxide 656-Methyl-2-Mercaptopyridine-1-oxide 564-Methyl-2-Mercaptopyridine-1-oxide 57 94Dimercaprol 43 92
Protease Inhibitor
1-(4-aminophenyl)-3-(4-chlorophenyl)urea HCl 54N-(OP)-L-Ala-L-Ala-benzy ester hydrate 631(G-T)-4-(4-methyl phenylsemithiocarbazide) 50
PADPRP Inhibitor
3-(4 -Bromophenyl)ureidobenzamide 74Benzoylene urea 54
Other
Hydrogen peroxide gel, 3% 58
Data generated by the U.S. Army Medical Research Institute of Chemical DefenseAnti-vesicant Drug Screen.
316 ALTERNATIVE TOXICOLOGICAL METHODS
DATA ANALYSIS
RESULTS
DISCUSSION
Figure 28.1 Percent IL-8 of HD-exposed HEK following treatment with TLCK.
TLCK (µM)
Control 0.0 62.5 125.0 250.0 500.0 1000.0
Per
cent
of I
L-8
0
20
40
60
80
100
120
MUSTARD-INDUCED IL-8 317
Figure 28.2 Percent IL-8 of HD-exposed HEK following treatment with 1579.
1579 (µM)
Control 0.0 62.5 125.0 250.0 500.0 1000.0
Per
cent
of I
L-8
0
20
40
60
80
100
120
31.25
318 ALTERNATIVE TOXICOLOGICAL METHODS
MUSTARD-INDUCED IL-8 319
REFERENCES
320 ALTERNATIVE TOXICOLOGICAL METHODS
MUSTARD-INDUCED IL-8 321
323
CHAPTER 29
Development of Medical Countermeasuresto Sulfur Mustard Vesication
CONTENTS
INTRODUCTION
324 ALTERNATIVE TOXICOLOGICAL METHODS
EXPERIMENTAL DESIGN
Decision Tree Network (DTN)
In Vitro Screening Modules
In Vivo Screening Modules
MUSTARD COUNTERMEASURES 325
RESULTS AND DISCUSSION
Basic Research
326 ALTERNATIVE TOXICOLOGICAL METHODS
Candidate Compound Screening
Figure 29.1 The cellular and tissue alterations induced by HD that are proposed to result inblister formation. HD can have many direct effects such as alkylation of proteinsand membrane components (Memb), as well as activation of inflammatory cells.One of the main macromolecular targets is DNA, with subsequent activation ofpoly(ADP-ribose) polymerase (PARP). Activation of PARP can initiate a series ofmetabolic changes culminating in protease activation. Within the tissue, the pen-ultimate event is the epidermal–dermal separation that occurs in the lamina lucidaof the basement membrane zone. Accompanied by a major inflammatory responseand changes in the tissue hydrodynamics (Hyd), fluid fills the cavity formed at thiscleavage plane and presents as a blister.
Table 29.1 Strategies for Pharmacologic Intervention of the HD Lesion
Biochemical Event Pharmacologic Strategy Example
DNA alkylation Intracellular scavengers N-acetyl cysteineDNA strand breaks Cell cycle inhibitors MimosinePARP activation PARP inhibitors NiacinamideDisruption of calcium Calcium modulators BAPTAa
Proteolytic activation Protease inhibitors AEBSFa
Inflammation Antiinflammatories Indomethacin; Olvanil
a BAPTA is a calcium chelator; AEBSF is a sulfonyl fluoride compound.
Proposed Mechanism of HD Action
DNA
Proteaserelease
Breakdown ofepithelial
attachment
Epidermal-dermalseparation
VESICATION
Memb
MetabolicDisruption
Strand Breaks PARP
Toxicity
HydInflam
NAD+
depletion
ProteinsInflammatory
Cells
Inhibition ofglycolysis
Membranes
InflamCa++
Ca++
CELLULAR
TISSUE
HD
MUSTARD COUNTERMEASURES 327
Future
Table 29.2 Candidate Countermeasures with Greater Than 50% Efficacy in Mouse Ear Model
Percentage Reduction in Pathology
Anti-Inflammatory Drugs
Fluphenazine dihydrochloride 50Indomethacin 96Olvanil 91Olvanil (saturated) 53Retro olvanil 84Olvanil (urea analog) 81Octyl homovanillamide 100
Scavenger Drugs
2-Mercaptopyridine-1-oxide 666-Methyl-2-mercaptopyridine-1-oxide 564-Methyl-2-mercaptopyridine-1-oxide 94Dimercaprol 78Na 3-sulfonatopropyl glutathionyl disulfide 64Hydrogen peroxide gel, 3% 58
Protease Inhibitors
1-(4-aminophenyl)-3-(4-chlorophenyl) urea 54N-(OP)-L-Ala-L-Ala-benzy ester hydrate 62Ethyl p-guanidino benzoate hydrochloride 62
PARP Inhibitors
3-(4 -Bromophenyl)ureidobenzamide 74Benzoylene urea 544-Amino-1-naphthol hydrochloride tech 80Total number of positive compounds = 19
328 ALTERNATIVE TOXICOLOGICAL METHODS
CONCLUSIONS
REFERENCES
PART V
Neurotoxicology: Molecular Biomarkers,Transgenics, and Imaging Technologies
SUMMARY
330 ALTERNATIVE TOXICOLOGICAL METHODS
GENOMICS/PROTEOMICS
NEUROTOXICOLOGY: MOLECULAR BIOMARKERS AND IMAGING TECHNOLOGIES 331
IMAGING
SECTION OUTLINE
332 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
333
CHAPTER 30
Molecular Neurotoxicologyof 6-Hydroxydopamine
and Methamphetamine: LessonsDerived from Transgenic Models
CONTENTS
INTRODUCTION
334 ALTERNATIVE TOXICOLOGICAL METHODS
SIX-OHDA-INDUCED NEURODEGENERATION
NEUROTOXICOLOGY OF 6-OHD AND METHAMPHETAMINE 335
METHAMPHETAMINE-INDUCED NEURODEGENERATION
CONCLUSIONS
336 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
Figure 30.1 Molecular neurotoxicology of methamphetamine.
METHAMPHETAMINE
Mitochondria
DNA Damage
Cell Death DNA Fragmentation
Pig3 ROS
↑ Bax, ↓ Bcl2 Caspases
O 2,Cytochrome c∑
∑ O 2 , H2O2,NO
p53
NEUROTOXICOLOGY OF 6-OHD AND METHAMPHETAMINE 337
338 ALTERNATIVE TOXICOLOGICAL METHODS
NEUROTOXICOLOGY OF 6-OHD AND METHAMPHETAMINE 339
341
CHAPTER 31
A Microassay Method Usinga Neuroblastoma Cell Line to Examine
Neurotoxicity of Organophosphate Mixtures
CONTENTS
INTRODUCTION
342 ALTERNATIVE TOXICOLOGICAL METHODS
MATERIALS AND METHODS
IN VITRO ASSESSMENT OF ORGANOPHOSPHATE NEUROTOXICITY 343
RESULTS
Figure 31.1 Determination of optimal cell number per well and reaction time for AChE activity.
-0.200
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
0 15 30 45 60
Time (min)
Abs
orba
nce
(405
nm
)
500,000
250,000
125,000
62,500
31,000
16,000
8,000
344 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 31.2 Effect of dichlorvos or paraoxon on AChE activity of SH-SY5Y neuroblastomas.*p < 0.05.
Figure 31.3 Effect of dichlorvos (D) or paraoxon (Px) alone or in combination on AChE activityof SH-SY5Y neuroblastomas. *p < 0.05.
***
*
**
*
**
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
Control 1.E-07 1.E-06 1.E-05 5.E-05 1.E-04 5.E-04 1.E-03 5.E-03 1.E-02 5.E-02 1.E-01
Concentration Paraoxon (µM)
Abs
orba
nce
(405
nm
)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
Control 1.E-02 5.E-02 1.E-01 5.E-01 1.E+00 5.E+00 1.E+01
Concentration Dichlorvos (µM)
Paraoxon Dichlorvos
*
*
*
*
**
* **
*
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
cont
rol
1.0
D
10-1
D
10-2
D
10-3
Px
10-4
Px
10-5
Px
1.0
D/1
0-3
Px
1.0
D/1
0-4
Px
1.0
D/1
0-5
Px
10-1
D/1
0-3
Px
10-1
D/1
0-4
Px
10-1
D/1
0-5
Px
10-2
D/1
0-3
Px
10-2
D/1
0-4
Px
10-2
D/1
0-5
Px
µM Dichlorvos (D) or Paraoxon (Px)
Abs
orba
nce
(405
nm
)
*
IN VITRO ASSESSMENT OF ORGANOPHOSPHATE NEUROTOXICITY 345
DISCUSSION
Table 31.1 Effect of Paraoxon (Px) or Dichlorvos (D) on AChE Activity of SH-SY5Y Neuroblastomas, Significance Level 0.05
OPconcentration
( M)
Absorbanceat 405 nm ±
SEMMeasured
inhibition (%)Predicted
inhibition (%)Significant
vs. control?
control 1.269 ± 0.031 10–2 D 1.373 ± 0.091 –8 10–1 D 1.198 ± 0.033 6 1.0 D 0.831 ± 0.023 34 x 10–5 Px 1.352 ± 0.081 –7 10–4 Px 0.811 ± 0.086 36 x 10–3 Px 0.362 ± 0.024 72 x10–2 D/10–5 Px 1.332 ± 0.042 –5 –15 10–2 D/10–4 Px 0.630 ± 0.077 50 28 x10–2 D/10–3 Px 0.418 ± 0.017 67 63 x10–1 D/10–5 Px 1.370 ± 0.167 –8 –1 10–1 D/10–4 Px 0.647 ± 0.104 49 42 x 10–1 D/10–3 Px 0.453 ± 0.027 64 77 x1.0 D/10–5 Px 0.432 ± 0.151 66 28 x1.0 D/10–4 Px 0.590 ± 0.156 54 71 x1.0 D/10–3 Px 0.339 ± 0.052 73 106 x
346 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
347
CHAPTER 32
Development of Integrin Expressionas a Molecular Biomarker for Early,
Sensitive Detection of Neurotoxicity
CONTENTS
INTRODUCTION
348 ALTERNATIVE TOXICOLOGICAL METHODS
INTEGRIN EXPRESSION AS A BIOMARKER FOR NEUROTOXICITY 349
Figure 32.1 Integrin heterodimers transduce signals both into and out of the cell via interactionswith extracellular ligands and cytoplasmic accessory or regulatory proteins, thecytoskeleton, and signal transduction proteins. Abbreviations: arginine-glycine-aspartic acid peptide (RGD), calreticulin (Cal), focal adhesion kinase (Fak), paxillin(Pax), phosphatidylinositol-3 kinase (PI3), vinculin (Vin).
350 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 32.2 Integrin-mediated cell functions.
TissueOrganization
Cell - CellCommunication
Activation of SignalTransduction Pathways
CellMigration
NeuriteElongation
CellCycle
GeneExpression
CellSurvival
Re-Organization ofCytoskeletal Proteins
INTEGRIN
ReceptorActivation/Clustering
IntracellularAccessory Protein
ExtracellularLigand
+ +
INTEGRIN EXPRESSION AS A BIOMARKER FOR NEUROTOXICITY 351
352 ALTERNATIVE TOXICOLOGICAL METHODS
APPROACH
INTEGRIN EXPRESSION AS A BIOMARKER FOR NEUROTOXICITY 353
Figure 32.3 Approach to validation of integrin subunit expression levels as molecular biomar-kers of neurotoxicity. Abbreviations: trimethyltin (TMT), methylmercury (MeHg),3,4-methylenedioxymethamphetamine (MDMA), RNAse protection assay (RPA).
Synaptopathic
Cell Lines
Tissue Culture
Function
Expression Levels
Neuropathic
Axonpathic
In Vivo
In Vitro
In Vivo
MDMA
Glial(C6)
Neuronal (PC12, N2A)
Isolated Cells(Astrocytes, Oligoden)
Primary Cultures/Co-Cultures
Proliferation/Differentiation
Cell AdhesionAssay
mRNA(Northerns, RPA)
Protein(Westerns)
Unknown Mechanisms
SuspectedNeurotoxicants
TMT, MeHg
Acrylamide
TMT, MeHgValidateKnownAgents
DevelopIn Vitro
Test Systems
IdentifyIntegrinSubunits
TestKnownAgents
TestNew
Compounds
354 ALTERNATIVE TOXICOLOGICAL METHODS
DISCUSSION
INTEGRIN EXPRESSION AS A BIOMARKER FOR NEUROTOXICITY 355
356 ALTERNATIVE TOXICOLOGICAL METHODS
ACKNOWLEDGMENTS
REFERENCES
INTEGRIN EXPRESSION AS A BIOMARKER FOR NEUROTOXICITY 357
358 ALTERNATIVE TOXICOLOGICAL METHODS
INTEGRIN EXPRESSION AS A BIOMARKER FOR NEUROTOXICITY 359
361
CHAPTER 33
Two-Photon Fluorescence Microscopy:A Review of Recent Advances
in Deep-Tissue Imaging
CONTENTS
INTRODUCTION
362 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 33.1 Jablonski diagrams for (a) one-photon and (b) two-photon excitation. One-photonexcitation occurs through the absorption of a single photon. The two-photonprocess occurs through the simultaneous absorption of two lower energy photons.After either excitation process, the fluorophore relaxes to the lowest energy levelof the first excited electronic state. The subsequent fluorescence emission processis independent of the mode of excitation.
TWO-PHOTON FLUORESCENCE MICROSCOPY: DEEP-TISSUE IMAGING 363
BASIC TWO-PHOTON MICROSCOPY INSTRUMENTATION
364 ALTERNATIVE TOXICOLOGICAL METHODS
DEEP TISSUE IMAGING BASED ON TWO-PHOTON MICROSCOPY
Figure 33.2 A schematic of two-photon fluorescence microscope design.
TWO-PHOTON FLUORESCENCE MICROSCOPY: DEEP-TISSUE IMAGING 365
RECENT ADVANCES IN TWO-PHOTON MICROSCOPY
Video Rate Two-Photon Microscopy
366 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 33.3 Two-photon autofluorescence images of human skin. Top left: stratum corneum.Top right: basal layer. Bottom: fibrous dermal layer (Zeiss Fluar 100 oil).
TWO-PHOTON FLUORESCENCE MICROSCOPY: DEEP-TISSUE IMAGING 367
Enhancing Image Resolution Based on Maximum Likelihood Deconvolution
368 ALTERNATIVE TOXICOLOGICAL METHODS
Two-Photon Spectral Characterization of Tissue Biochemistry
Figure 33.4 Image restoration of two-photon images of ex vivo human skin using maximumlikelihood approach. Autofluorescence (left) and blind deconvoluted (right) imagesof human basal layer. Top: lateral view. Bottom: axial section (Zeiss Fluar 40 oil).
TWO-PHOTON FLUORESCENCE MICROSCOPY: DEEP-TISSUE IMAGING 369
370 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 33.5 Two independent spectral components isolated in ex vivo human skin based onSMCR: (a) a spectral component corresponds to elastin fibers in the dermis and(b) a spectral component corresponds to melanin (or a fluorophore that colocalizeswith melanin) in the epidermal–dermal junction. For (a) and (b): (left) a two-dimensional image of the concentration distribution of the spectral component;(right, top) the spectrum of this component; (right, bottom) the depth distributionprofile of this component.
TWO-PHOTON FLUORESCENCE MICROSCOPY: DEEP-TISSUE IMAGING 371
CONCLUSIONS
REFERENCES
372 ALTERNATIVE TOXICOLOGICAL METHODS
TWO-PHOTON FLUORESCENCE MICROSCOPY: DEEP-TISSUE IMAGING 373
PART VI
Role of Transgenics and Toxicogenomics inthe Development of Alternative Toxicity Tests
376 ALTERNATIVE TOXICOLOGICAL METHODS
377
CHAPTER 34
The Application of Genomics andProteomics to Toxicological Sciences
CONTENTS
INTRODUCTION
378 ALTERNATIVE TOXICOLOGICAL METHODS
TOXICOGENOMICS AND PROTEOMICS 379
MICROARRAY TECHNOLOGIES
380 ALTERNATIVE TOXICOLOGICAL METHODS
TOXICOGENOMICS AND PROTEOMICS 381
PROTEOMIC TECHNOLOGIES
382 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 34.1 Schematic of genomic and proteomic technologies: The first step in toxicoge-nomics (right) is the construction of a microarray that involves the amplificationby PCR and the immobilization of known DNA sequences (either cDNA oroligonucleotides) on a solid support. The mRNA prepared from a biologicalmodel can be labeled and hybridized to the microarray and visualized usingphosphorimager scanning. Subsequent bioinformatic analyses using appropriatesoftware allows determination of the extent of hybridization of the labeled probesto the corresponding arrayed cDNA spots, and a comparison of control with testsamples permits quantitative assessment of changes in gene expression asso-ciated with treatment. Total protein content from a biological model treated witha toxicant is separated on two-dimensional gel electrophoresis according toisoelectric point (first dimension) and molecular weight (second dimension),allowing bioinformatic analysis of differences in protein expression of treatedversus untreated samples. Therefore, there is no preliminary work such as arrayconstruction for proteomic studies (left), but proteins with altered expressionhave to be identified subsequently. Individual proteins of interest are excisedfrom two-dimensional-gels, digested with trypsin, and applied to a mass spec-trometer. Identification of these proteins is obtained by searching protein data-bases with mass spectrometry data.
Biological model
Sample preparationTreated vs. untreated
SDS-PAGE (2nd D)
Mass spectrometry
Protein database search
Protein identity
Bioinformatic analysis
Protein sample mRNA sample isolation
Research lead Toxicological marker
Protein expression and posttranslational modification
cDNA labeling
Hybridization to microarray
cDNA selection
PCR amplification
Microarray construction
Iso electrofocusing (1st D)
mRNA expressed
Toxicant signature
TOXICOGENOMICS AND PROTEOMICS 383
TOXICOGENOMICS APPLICATIONS
384 ALTERNATIVE TOXICOLOGICAL METHODS
PROTEOMICS APPLICATIONS
TOXICOGENOMICS AND PROTEOMICS 385
THE CHALLENGE OF PATTERN RECOGNITION
386 ALTERNATIVE TOXICOLOGICAL METHODS
OPPORTUNITIES IN COMBINATION: A PERSPECTIVE
TOXICOGENOMICS AND PROTEOMICS 387
REFERENCES
388 ALTERNATIVE TOXICOLOGICAL METHODS
TOXICOGENOMICS AND PROTEOMICS 389
390 ALTERNATIVE TOXICOLOGICAL METHODS
391
CHAPTER 35
Use of Transgenic Animals in RegulatoryCarcinogenicity Evaluations*
CONTENTS
INTRODUCTION
392 ALTERNATIVE TOXICOLOGICAL METHODS
TRANSGENIC ANIMALS IN REGULATORY CARCINOGENICITY EVALUATIONS 393
394 ALTERNATIVE TOXICOLOGICAL METHODS
COMMERCIAL COMPLICATIONS
OVERVIEW OF INDIVIDUAL MODELS
TRANSGENIC ANIMALS IN REGULATORY CARCINOGENICITY EVALUATIONS 395
Tg.AC
Table 35.1 IARC Class I or 2A Human Carcinogen, or NTP Reasonably Anticipated Human Carcinogen
Tg.ACTopical TgrasH2 p53+/– XPA–/–/p53+/–
GenotoxicBenzene + + + nd*Benzo(a)pyrene + nd + +Cyclophosphamide – + + nd7,12-Dimethylbenzanthracene + nd + +**Melphalan – +/– + ndPhenacetin – + – –Procarbazine nd + nd nd
NongenotoxicCyclosporin A + +/– + +Diethylstilbestrol + + + +17- -estradiol (or ethinyl estradiol#) +# – +/– +Oxymetholone + nd – nd2,3,7,8-TCDD + nd nd
* nd: no adequate data available on the performance of the compound in that model.**Positive in 6 month XPA–/– and positive in 6 month p53+/–; not tested in XPA–/–/P53+/–
bitransgenic.
Table 35.2 Genotoxic Trans-Species Rodent Carcinogens
Tg.ACTopical TgrasH2 P53+/– XPA–/–/P53+/–
p-Cresidine – + + +2,4-Diaminotoluene + nd – ndDiethylnitrosamine nd + nd ndDimethylnitrosamine nd + + ndN-Ethylnitrosourea nd + + ndGlycidol – + – ndN-Methylnitrosourea nd + + ndPhenolphthalein nd – + ndThiotepa nd + nd ndUrethane nd + + nd4-Vinyl-1-cyclohexene-diepoxide* – + + nd
* Applied dermally to each model tested.
396 ALTERNATIVE TOXICOLOGICAL METHODS
Table 35.3 Nongenotoxic Rodent Carcinogens and Human Carcinogenicity Unlikely or Uncertain
Tg.ACTopical TgrasH2 P53+/– XPA–/–/P53+/–
Chlorpromazine nd – nd ndClofibrate + + – ndDieldrin nd nd – ndDiethylhexylphthalate – + +/– –Haloperidol nd – – –D-Limonene nd nd – ndMetaproterenol nd – – ndPentachlorophenol + nd – ndPhenobarbital – – – –Reserpine – – – –Sulfamethoxazole – – – –WY-14643 – + – +*
* Positive in 6 month XPA–/–, not tested in XPA–/–/p53+/– bitransgenic.
Table 35.4 Rodent Noncarcinogens
Tg.ACTopical TgrasH2 P53+/– XPA–/–/P53+/–
Genotoxicp-Anisidine – – – nd2-Chloroethanol – nd nd nd1-Chloro-2-propanol – nd – nd2,6-Diaminotoluene – nd – nd8-Hydroxy-quinoline – – – nd
NongenotoxicAmpicillin nd – nd ndBenzethonium chloride – nd nd ndD-Mannitol nd – nd –Oleic acid diethanolamine – nd – ndPhenol – nd nd ndResorcinol + – – ndRotenone – – – ndSulfisoxazole – – nd nd
TRANSGENIC ANIMALS IN REGULATORY CARCINOGENICITY EVALUATIONS 397
398 ALTERNATIVE TOXICOLOGICAL METHODS
TgrasH2
TRANSGENIC ANIMALS IN REGULATORY CARCINOGENICITY EVALUATIONS 399
400 ALTERNATIVE TOXICOLOGICAL METHODS
P53+/– MODEL
TRANSGENIC ANIMALS IN REGULATORY CARCINOGENICITY EVALUATIONS 401
402 ALTERNATIVE TOXICOLOGICAL METHODS
XPA–/–/P53+/–
TRANSGENIC ANIMALS IN REGULATORY CARCINOGENICITY EVALUATIONS 403
404 ALTERNATIVE TOXICOLOGICAL METHODS
REGULATORY EXPERIENCE
TRANSGENIC ANIMALS IN REGULATORY CARCINOGENICITY EVALUATIONS 405
SUMMARY AND CONCLUSIONS
406 ALTERNATIVE TOXICOLOGICAL METHODS
TRANSGENIC ANIMALS IN REGULATORY CARCINOGENICITY EVALUATIONS 407
REFERENCES
408 ALTERNATIVE TOXICOLOGICAL METHODS
TRANSGENIC ANIMALS IN REGULATORY CARCINOGENICITY EVALUATIONS 409
410 ALTERNATIVE TOXICOLOGICAL METHODS
TRANSGENIC ANIMALS IN REGULATORY CARCINOGENICITY EVALUATIONS 411
413
CHAPTER 36
Changes in Gene Expression after Exposureto Organophosphorus (OP) Agents
CONTENTS
414 ALTERNATIVE TOXICOLOGICAL METHODS
INTRODUCTION
CHANGES IN GENE EXPRESSION AFTER EXPOSURE TO AGENTS 415
METHODS
Dosing Regimen
Collection of Tissue
416 ALTERNATIVE TOXICOLOGICAL METHODS
Preparation of Total RNA and mRNA
Figure 36.1 Rat toxicology U34 gene array. This gene expression display was created byGeneSpring® (Silicon Genetics) gene array analysis software from our data (ratbrain RNA 1 h postexposure to CPF) read from an Affymetrix Rat Toxicology U34GeneChip®. In the original display (depicted in gray tones here), red and purpleblocks represented up-regulated genes and the blue represented down-regulatedgenes. Gray blocks represented genes whose expression is essentially the sameas in the control animal.
CHANGES IN GENE EXPRESSION AFTER EXPOSURE TO AGENTS 417
Synthesis of Biotin-Labeled cRNA and Target Preparation
Hybridization, Staining, and Washing of DNA Microarray
Probe Array Scan
Analysis of DNA Microarray Data
Measurement of Butylcholinesterase (BChE) Activity
RESULTS
Selection of Genes to Measure
418 ALTERNATIVE TOXICOLOGICAL METHODS
Analysis of Gene Expression Patterns
Quantification of Gene Expression Patterns in the Brain
Figure 36.2 Illustrations of the six expression pattern categories. The numerals under the x-axis represent hours after CPF exposure. Y-axis represents relative gene expres-sion level. (A) No alteration of gene expression; (B) initial up-regulation, thenreturn to normal by 24 h; (C) initial up-regulation, then return to normal by 4 h;(D) delayed up-regulation by 4 h, then return to normal by 24 h; (E) delayed up-regulation by 24 h; (F) rapid down-regulation, then return to normal by 4 h.
14 24
14 24
14 24
14 24
14 24
14 24
A. B.
C. D.
E. F.
CHANGES IN GENE EXPRESSION AFTER EXPOSURE TO AGENTS 419
Quantification of Gene Expression Patterns in the Liver
Figure 36.3 The relative percentages of the six patterns of gene expression detected in theCPF-exposed rats.
X12%
F4% E
7% D7%
C13%
B23%
A34%
A.
X23%
F10%
E10%
D7%
C 11%
B 5%
A34%
B.
A33%
B 9%
C 5%
D14%
E6%
F4%
X29%
C.
420 ALTERNATIVE TOXICOLOGICAL METHODS
Identification of Up-Regulated Genes Involved in Key Cellular Functions and Biochemical Pathways
Figure 36.4 Scatterplot of data from rat toxicology U34 GeneChip.
CHANGES IN GENE EXPRESSION AFTER EXPOSURE TO AGENTS 421
Identification of Down-Regulated Genes Involved in Key Cellular Functions and Biochemical Pathways
Measurement of BChE Activity in Blood of CPF-Exposed Rats
Figure 36.5 In vivo effects of CPF on BuChE activity.
Effect of CPF on Rat BuChE Activity
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Control 1 4 24
Hours Post-Exposure
Bu
Ch
e A
ctiv
ity
(O.D
. at
412
nm
)
422 ALTERNATIVE TOXICOLOGICAL METHODS
DISCUSSION
CHANGES IN GENE EXPRESSION AFTER EXPOSURE TO AGENTS 423
424 ALTERNATIVE TOXICOLOGICAL METHODS
CHANGES IN GENE EXPRESSION AFTER EXPOSURE TO AGENTS 425
426 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
CHANGES IN GENE EXPRESSION AFTER EXPOSURE TO AGENTS 427
PART VII
Recent Innovations in Alternatives
431
CHAPTER 37
Archival Data in Toxicology: MinimizingNeed for Animal Experiments
CONTENTS
432 ALTERNATIVE TOXICOLOGICAL METHODS
INTRODUCTION
Perspective
PROBLEM SOLVING
ARCHIVAL DATA IN TOXICOLOGY: MINIMIZING NEED FOR ANIMAL EXPERIMENTS 433
INHALATION TOXICOLOGY OF HYDROGEN CYANIDE
Modeling Approaches
Relevant Toxicological Features and Methodology
434 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 37.1 Comparison of toxicological features of HCN and nerves gases, such as sarin.The larger arrow represents inhaled HCN. The smaller arrow represents theeffective dosage, after detoxification and loss of the 30% of inhaled HCN that isexhaled (Moore and Gates, 1946), leaving 0.7 retained. HCN concentrations upto 30 mg/m3 are normally neutralized by human detoxification systems beforeeffects become observable (Prentiss, 1937).
ARCHIVAL DATA IN TOXICOLOGY: MINIMIZING NEED FOR ANIMAL EXPERIMENTS 435
Figure 37.2 Contrasting plots of data from the same rat exposures to HCN (after Ballantyne,1987). One plot (left) reflects use of exposures with fixed duration and varied HCNconcentrations. The steep initial slope shows that HCN must be very concentratedfor a small number of breaths to deliver a lethal dosage. The second plot presentsthe data results as customarily displayed, given a fixed toxic gas concentrationand varied exposure durations. LCt50 values tend to increase as more timebecomes available for detoxification.
Figure 37.3 Comparison of responses from different strains of rats under similar HCN exposureconditions (after Levin et al., 1985). Log–log plots of the data reveal differencesof effective HCN concentrations and probit slopes of lines but do not show whichstrain better represents the human race.
436 ALTERNATIVE TOXICOLOGICAL METHODS
Mechanisms and Rationales
Extrapolations of Human Data
Figure 37.4 Relationships of anatomical and pharmacological factors leading to lethality formammals that inhale HCN. Nonionized HCN is rapidly absorbed into blood tomove the short distance from alveoli to the sensor. The sensor response (or lackof a signal) communicates a need for oxygen to respiratory neurones that initiatehyperventilation. Increased minute volumes multiply HCN intake until respiratorycenter poisoning leads to apnea.
ARCHIVAL DATA IN TOXICOLOGY: MINIMIZING NEED FOR ANIMAL EXPERIMENTS 437
A Biological Common Denominator
Table 37.1 Hydrogen Cyanide Concentrations and Effects Associated with Various HCN Exposure Conditions in Animals and Man
Mg/m3 Effects Ref.
30,000a Rat, inhalation LC50 Levin et al., 19859,300b Men, 11/11 hyperventilate Bodansky and Helm, 19447,740b Men, “most hyperventilate” Cope and Abramovitz, 19594,000a Rat, inhalation LC50 Ballantyne, 19873,200b Men, “50% hyperventilate” Wexler et al., 19472,230 Pig, LC50 within 2 min of inhalation Stemler et al., 1994
a Lethal concentration for 50% of subject rats, 0.1 min inhalation exposure.b Equivalent HCN mg/m3 dosage calculated for intravenous sodium cyanide solution.
438 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 37.5 Observed breathing rates and blood cyanide concentrations of three miniaturepigs exposed to HCN for 2 min (after Stemler et al., 1994). Values for bloodconcentrations of cyanide, at the top and right side of Figure 37.5, are presentedwith the corresponding respiratory rate values. Although data acquired after theonset of HCN exposure for 2 min are very limited, they are consistent with otherindicators that an aortic blood HCN concentration of 4 mg/m3 at 5 min is a thresholdvalue for a lethal outcome.
Figure 37.6 Average whole blood cyanide levels in dogs after four continuous intravenousslow infusion trials (1 mg/kg/min) with NaCN solution at 4.0 mg/ml (after Vicket al., 2000). RA indicates respiratory arrest and cessation of NaCN infusion.Methemoglobin formation by 20 mg/ml hydroxylamine hydrochloride solution wasinitiated 30 sec after RA. Although 3.6 g/kg was the average value observedat RA, it appears that survival is dependent upon avoidance of blood cyanideconcentrations above 4 g/kg.
ARCHIVAL DATA IN TOXICOLOGY: MINIMIZING NEED FOR ANIMAL EXPERIMENTS 439
Acute and Peracute Exposures
440 ALTERNATIVE TOXICOLOGICAL METHODS
Lessons Learned
PROJECTION OF DELAYED EYE EFFECTS OF MUSTARD EXPOSURES
Vive la Difference!
ARCHIVAL DATA IN TOXICOLOGY: MINIMIZING NEED FOR ANIMAL EXPERIMENTS 441
Dose Responses—with Time
Dosage-Degree-Duration
442 ALTERNATIVE TOXICOLOGICAL METHODS
Applications of Figure 37.7
Figure 37.7 Triaxial depiction that was hand drawn to integrate (1) time course informationreported hourly or by the day; (2) mustard vapor dosage information; and (3) levelsof human effect severity for the given time and dosage. The dashed line representsdata from one particularly relevant experiment (Unde and Dunphy, 1944) amongmore than 100 experimental reports that were considered.
ARCHIVAL DATA IN TOXICOLOGY: MINIMIZING NEED FOR ANIMAL EXPERIMENTS 443
ESTIMATION OF PERFORMANCE DEGRADATION BY “FOOD POISONING”
Traveler’s Diarrhea
444 ALTERNATIVE TOXICOLOGICAL METHODS
From Cases to Predictions
Figure 37.8 Bar chart designed to illustrate relative severity of enteric disease effects ascorrelated with time after arrival of a mixed population in Mexico City. Each of the19 represented cases involved isolation of enterotoxigenic Escherichia coli organ-isms that accounted for 45% of traveler’s diarrhea cases observed during thisprospective study (Merson et al., 1976).
ARCHIVAL DATA IN TOXICOLOGY: MINIMIZING NEED FOR ANIMAL EXPERIMENTS 445
SUMMARY
ACKNOWLEDGMENTS
Table 37.2 Estimates of Percent of Military Personnel with Indicated Degree of Performance-Degradation per Day after Onset of Gastrointestinal Symptoms/Signs from Infection with Enterotoxigenic Escherichia coliBacteriaa
Percentage of cases with degraded military performanceDay of Onsetb
% New Casesc No.d (25%)c No.e (50%)c No.f (100%)c
Recoveredg
(%)c
1 (14.6) 9 (47.4) 7 (36.8) 3 (15.5) 0 (0)2 (19.0) 7 (36.8) 7 (36.8) 3 (15.8) 2 (10.5)3 (19.3) 4 (21.1) 10 (52.6) (0) 5 (26.3)4 (17.1) 4 (21.1) 8 (42.1) (7) (36.8)5 (9.8) 2 (10.5) 8 (42.2) (9) (47.4)6 (6.1) 2 (10.5) 6 (31.6) (11) (57.9)7 (4.1) 4 (21.1) 3 (15.8) (12) (63.2)8 (2.9) 4 (21.1) 2 (10.5) (13) (68.4)9 (2.0) 3 (15.8) 1 (5.3) (15) (78.9)
10 (1.5) 3 (15.8) (0) (16) (84.2)11 (1.3) 2 (10.5) (17) (89.5)12 (1.1) 1 (5.3) (18) (94.7)13 (0.7) 0.5 (2.6) (18.5) (97.4)14 (0.5) (0) (19) (100.0)
a Percentages based upon data from 19 proven cases of enterotoxigenic Escherichia coliinfection acquired in Mexico City (Merson et al., 1976).
b Onset day 1 is day 3 from exposure to infection.c Calculated from daily rate curve (Fischer and Mershon, 1994).d Cases (9) with uncomplicated traveler’s diarrhea, performance 25% degraded on day 1.e Cases (7) with changed activities, performance 50% degraded during onset day 1.f Cases (3) with recovery time in bed; performance 100% degraded during onset day 1.g Cases (19) all had some degree of incapacitation during onset day 1.
446 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
Figure 37.9 This cartoon is included to suggest that modeling represents a combination of artand science. The art of modeling methodology is applied when conventional testingmethods are not applicable for collection of experimental results. In each case,previously collected data were reacquired and analyzed to provide a best possibleestimate of reality.
ARCHIVAL DATA IN TOXICOLOGY: MINIMIZING NEED FOR ANIMAL EXPERIMENTS 447
449
CHAPTER 38
Information Management at the Library ofCongress: An Overview with Special
Reference to Biomedicine*
CONTENTS
450 ALTERNATIVE TOXICOLOGICAL METHODS
INTRODUCTION
A HISTORICAL SKETCH
INFORMATION MANAGEMENT AT THE LIBRARY OF CONGRESS 451
452 ALTERNATIVE TOXICOLOGICAL METHODS
CURRENT HOLDINGS
SCIENCE AND TECHNOLOGY
INFORMATION MANAGEMENT AT THE LIBRARY OF CONGRESS 453
CLIENT SERVICES STATISTICS
INFORMATION MANAGEMENT
In the Beginning…
454 ALTERNATIVE TOXICOLOGICAL METHODS
Current LC Practices
INFORMATION MANAGEMENT AT THE LIBRARY OF CONGRESS 455
A Historical Perspective
456 ALTERNATIVE TOXICOLOGICAL METHODS
SUBJECT-BASED ACCESS TO INFORMATION
Preamble
INFORMATION MANAGEMENT AT THE LIBRARY OF CONGRESS 457
Subject Headings: General Information
458 ALTERNATIVE TOXICOLOGICAL METHODS
Rationale for Subject Headings
INFORMATION MANAGEMENT AT THE LIBRARY OF CONGRESS 459
The Subject Authority Record
Subject Analysis: General Principles
Tag 010053150450450550550550
Field Data_a sh 85064594_a RC582.17_a Immunotoxicology_a Immunologic toxicology_a Immunotoxicity _w g _a Immunopathology_w g _a Toxicology_a Immunopharmacology
ExplanationsRecord NumberClass NumberSubject HeadingUF (Use For Term)UF (Use For Term)BT (Broader Term)BT (Broader Term) RT (Related Term)
Figure 38.1 LC authority record for Immunotoxicology, supplemented with explanatory notations.
460 ALTERNATIVE TOXICOLOGICAL METHODS
INFORMATION MANAGEMENT AT THE LIBRARY OF CONGRESS 461
Subject Headings: Select Biomedical Examples
462 ALTERNATIVE TOXICOLOGICAL METHODS
Select Subdivision Examples
INFORMATION MANAGEMENT AT THE LIBRARY OF CONGRESS 463
LCSH: Coda
INFORMATION RETRIEVAL
464 ALTERNATIVE TOXICOLOGICAL METHODS
The Futility Point
INFORMATION MANAGEMENT AT THE LIBRARY OF CONGRESS 465
THE MARC RECORD*
Genetic toxicology / editors, Albert P. Li, Robert H. Heflich. -- Boca Raton : CRC Press, cl991.x, 493 p. : ill. ; 24 cm.
Includes bibliographical references. Includes index. ISBN 0-8493-8815-3
1. Genetic toxicology. I. Li, A. P. II. Heflich, Robert U., 1946-
[DNLM: 1. Carcinogens. 2. Chromosome Abnormalities--chemically in-duced. 3. Mutagens--adverse effects. 4. Mutation. QH 465.C5 G328] RA1224.3.G457 1991 615.9'02--dc20 90-11279
DNLM/DLCfor Library of Congress
Figure 38.2 Traditional LC catalog card for Genetic Toxicology, Li, AP, and Heflich, RH,editors, 1991.
466 ALTERNATIVE TOXICOLOGICAL METHODS
Tag Ind I Ind 2 Field Data 000 01037pam__2200325_a_4500 001 996520 005 9910520155750.7 008 900824sl991____njua_____b____001_0_eng_c
035 _9 (DLC) 90011279 906 _a 7 _b cbc _c orignew _d 1 _e ocip _f 19 _g y-gencatlg 955 _a CIP ver. ea10 to SL 05-13-91 010 _a 90011279 020 _z 0849388153 040 _a DNLM/DLC _c DLC _d DLC 050 0 0 _a RA1224.3 _b .G457 1991 060 _a QH 465.C5 G328 082 0 0 _a 615.9/02 _2 20 245 0 0 _a Genetic toxicology / _c editors, Albert P. Li, Robert H. Heflich. 260 _a Boca Raton : _b CRC Press, _c cl991. 300 _a x, 493 p. : _b ill. ; _c 24 cm. 504 _a Includes bibliographical references. 500 _a Includes index. 650 0 _a Genetic toxicology. 650 2 _a Carcinogens. 650 2 _a Chromosome Abnormalities _x chemically induced. 650 2 _a Mutagens _x adverse effects. 650 2 _a Mutation. 700 1 _a Li, A. P. 700 1 _a Heflich, Robert H., _d 1946- 991 _b c-GenColl _h RA1224.3 _i .G457 1991 _t Copy 1 _w
Figure 38.3 LC MARC record for Genetic Toxicology, Li, AP, and Heflich, RH, editors, 1991.
INFORMATION MANAGEMENT AT THE LIBRARY OF CONGRESS 467
468 ALTERNATIVE TOXICOLOGICAL METHODS
EPILOGUE
Genetic toxicology / editors, Albert P. Li, Robert H. Heflich
LC Control Number: 90011279Main Title: Genetic toxicology / editors, Albert P. Li, Robert H. Heflich
Published/Created: Boca Raton : CRC Press, c1991.Related Names: Li, A. P.
Heflich, Robert H., 1946-Description: x, 493 p.,: ill.; 24 cm.
ISBN: 0849388153Notes: Includes index.
Includes bibliographical references.Subjects: Genetic toxicology.
Carcinogens.Chromosome Abnormalities--chemically induced.Mutagens--adverse effects.Mutation.
LC Classification: RA1224.3 .G457 1991NLM Class No.: QH465.C5 G328
Dewey Class No.: 615.9/02 20
Figure 38.4 LC OPAC Full Record display for Genetic Toxicology, AP Li, and RH Heflich,editors, 1991.
INFORMATION MANAGEMENT AT THE LIBRARY OF CONGRESS 469
470 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
INFORMATION MANAGEMENT AT THE LIBRARY OF CONGRESS 471
472 ALTERNATIVE TOXICOLOGICAL METHODS
473
CHAPTER 39
World Wide Web Biomedical, Chemical,and Toxicological Information Resources
from the National Library of Medicine
CONTENTS
INTRODUCTION AND BACKGROUND
474 ALTERNATIVE TOXICOLOGICAL METHODS
BIBLIOGRAPHIC DATABASES
FACTUAL DATABASES
WEB RESOURCES FROM THE NATIONAL LIBRARY OF MEDICINE 475
476 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 39.1 TEHIP homepage.
WEB RESOURCES FROM THE NATIONAL LIBRARY OF MEDICINE 477
REFERENCES
Figure 39.2 TOXNET’s page search on “acrylamide.”
478 ALTERNATIVE TOXICOLOGICAL METHODS
NIH/NLM URL PAGES
479
CHAPTER 40
In Silico Approaches for PhysiologicallyBased Pharmacokinetic Modeling
CONTENTS
480 ALTERNATIVE TOXICOLOGICAL METHODS
INTRODUCTION
IN SILICO APPROACHES FOR PBPK MODELING 481
METHODOLOGICAL BASIS OF IN SILICO APPROACHES
QSARs
LFE-Type Models
Electrostatic Features in LFE-Type Models
482 ALTERNATIVE TOXICOLOGICAL METHODS
Steric Features in LFE-Type Models
Hydrophobic Features in LFE-Type Models
IN SILICO APPROACHES FOR PBPK MODELING 483
Free–Wilson Type Models
484 ALTERNATIVE TOXICOLOGICAL METHODS
Biologically Based Algorithms
IN SILICO APPROACHES FOR PBPK MODEL PARAMETERS
In Silico Approaches for Tissue:Air Partition Coefficients
IN SILICO APPROACHES FOR PBPK MODELING 485
Tab
le 4
0.1
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e T
issu
e:A
ir P
arti
tio
n C
oef
fici
ents
(P
) o
f C
hem
ical
s
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
QS
AR
s: L
FE
-typ
e eq
uat
ion
s
Ele
ctro
stat
ic d
escr
ipto
rs
log
Pad
ipos
e:ai
r=
–0.
294
– 0.
172R
2+
0.7
29 +
1.7
474
+ 0
.219
+
0.89
5 lo
g P
he:a
HIn
ert
Gas
es; L
MW
VO
Cs
Abr
aham
and
Wea
ther
sby
(199
4)
log
Pbr
ain:
air=
–1.
074
+ 0
.427
R2
+ 0
.286
+ 2
.781
+ 2
.787
+
0.60
9 lo
g P
he:a
HIn
ert
Gas
es; L
MW
VO
Cs
Abr
aham
and
Wea
ther
sby
(199
4)
log
Phe
art:a
ir=
–1.
208
+ 0
.128
R2
+ 0
.987
+ 0
.643
+ 1
.783
+
0.59
7 lo
g P
he:a
HIn
ert
Gas
es; L
MW
VO
Cs
Abr
aham
and
Wea
ther
sby
(199
4)
log
Pki
dney
:air
= –
1.08
4 +
0.4
17R
2+
0.2
26 +
3.6
24 +
2.9
26 +
0.
534
log
Phe
:a
HIn
ert
Gas
es; L
MW
VO
Cs
Abr
aham
and
Wea
ther
sby
(199
4)
log
Pliv
er:a
ir=
–1.
031
+ 0
.059
R2
+ 0
.774
+ 0
.593
+ 1
.049
+
0.65
4 lo
g P
he:a
HIn
ert
Gas
es; L
MW
VO
Cs
Abr
aham
and
Wea
ther
sby
(199
4)
log
Plu
ng:a
ir=
–1.
300
+ 0
.667
R2
+ 0
.680
+ 3
.539
+ 3
.35
+ 0
.458
lo
gP
he:a
HIn
ert
Gas
es; L
MW
VO
Cs
Abr
aham
and
Wea
ther
sby
(199
4)
log
Pm
uscl
e:ai
r=
–1.
14 +
0.5
44R
2+
0.2
16 +
3.4
714
+ 2
.924
+
0.57
8 lo
g P
he:a
HIn
ert
Gas
es; L
MW
VO
Cs
Abr
aham
and
Wea
ther
sby
(199
4)
Ste
ric
des
crip
tors
log
Pad
ipos
e:ai
r=
(0.
7341 x
v ) –
(0.
029
) –
(1.5
7(1/
1 x))
– (
0.55
9(1/
1 xv )
) –
0.09
83+
2.2
13
RH
aloa
lkan
esG
arga
s et
al.
(198
8)
(con
tinue
d)
486 ALTERNATIVE TOXICOLOGICAL METHODS
Tab
le 4
0.1
(co
nti
nu
ed)
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e T
issu
e:A
ir P
arti
tio
n C
oef
fici
ents
(P
) o
f C
hem
ical
s
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
log
Pad
ipos
e:ai
r=
0.7
341 X
v –
0.02
91 –
1.5
70/1 X
v–
0.55
91 Xv
– 0.
0984
+ 2
.213
RH
aloa
lkan
esC
sana
dy a
nd L
aib
(199
0)
log
Pad
ipos
e:ai
r=
0.5
63N
Cl+
1.0
28N
Br+
0.4
67N
C+
0.2
70Q
H–
0.19
9NF
–0.
097
RH
aloa
lkan
esG
arga
s et
al.
(198
8)
log
Pad
ipos
e:ai
r=
1.0
371 x
v –
(0.0
07(1
/))
+ 0
.022
QH
– 0.
1773
– 0.
199N
F
– 0.
0036
RH
aloa
lkan
esG
arga
s et
al.
(198
8)
log
Pliv
er:a
ir=
(1.
0721 x
v ) –
(0.
021(
1/))
+ (
0.64
7(1/
1 xv )
) –
(0.3
044
) –
1.21
2
RH
aloa
lkan
esG
arga
s et
al.
(198
8)
log
Pliv
er:a
ir=
0.3
66N
Cl–
0.58
8NB
r+
0.3
45Q
H–
0.17
9NF
– 0.
007
RH
aloa
lkan
esG
arga
s et
al.
(198
8)
log
Pliv
er:a
ir=
–0.
6851 x
v –
(0.0
20(1
/))
+ 0
.232
QH
+ (
0.29
8(1/
1 xv )
) +
0.
104N
Cl–
0.72
6
RH
aloa
lkan
esG
arga
s et
al.
(198
8)
log
Pliv
er:a
ir=
1.0
721 X
v –
0.02
1/ +
0.6
47/1 X
v–
0.30
44–
1.21
2R
Hal
oalk
anes
Csa
nady
and
Lai
b (1
990)
log
Pm
uscl
e:ai
r=
0.3
79Q
H–
0.27
8NC
l+
0.5
36N
Br–
0.19
0NF
+ 0
.169
NC
l–
0.43
9R
Hal
oalk
anes
Gar
gas
et a
l. (1
988)
log
Pm
uscl
e:ai
r=
0.3
991 x
v –(0
.007
(1/
)) +
0.2
95Q
H+
0.2
594
– 0.
194N
F
– 0.
217
RH
aloa
lkan
esG
arga
s et
al.
(198
8)
log
Pm
uscl
e:ai
r=
(0.
9951 x
v ) –
(0.
018(
1/))
– (
0.42
44)
– (0
.559
(1/1 x
v ) +
(0.6
02(1
/1 xv )
) –
1.33
4
RH
aloa
lkan
esG
arga
s et
al.
(198
8)
Hyd
rop
ho
bic
des
crip
tors
log(
Pad
ipos
e:w
ater
–V
wt)
= 0
.9P
o:w
+ 0
.31
FC
hlor
oeth
anes
; Ben
zene
Ber
tels
en e
t al
. (19
88)
log(
Pki
dney
:wat
er–
Vw
t) =
0.7
2Po:
w–
0.56
FC
hlor
oeth
anes
; Ben
zene
Ber
tels
en e
t al
. (19
88)
log(
Pliv
er:w
ater
–V
wt)
= 1
.06P
o:w
– 1.
43F
Chl
oroe
than
es; B
enze
neB
erte
lsen
et
al. (
1988
)lo
g(P
mus
cle:
wat
er–
Vw
t) =
0.6
3Po:
w–
0.60
FC
hlor
oeth
anes
; Ben
zene
Ber
tels
en e
t al
. (19
88)
lnP
adip
ose:
air=
0.0
32T
b –
5.45
6H
Hal
oalk
anes
Csa
nady
and
Lai
b (1
990)
IN SILICO APPROACHES FOR PBPK MODELING 487
lnP
liver
:air
= 0
.022
Tb
–4.
638
HH
aloa
lkan
esC
sana
dy a
nd L
aib
(199
0)lo
gP
adip
ose:
air=
0.2
09 +
0.0
628
log
Pw
:a+
0.8
868
log
Po:
aH
Iner
t ga
ses;
LM
WV
OC
sA
brah
am e
t al
. (19
85)
log
Pad
ipos
e:ai
r=
0.2
1 lo
g P
o:a
+ 0
.24
log
Pw
:aH
Hyd
roph
ilic
VO
Cs
Tic
hy (
1991
b)lo
gP
adip
ose:
air=
0.7
82 lo
g P
o:a
+ 0
.201
log
Pw
:a+
0.4
32H
Hyd
roph
obic
VO
Cs
Tic
hy (
1991
a)lo
gP
adip
ose:
air=
0.9
01 lo
g P
o:a
+ 0
.150
HLM
WV
OC
sF
iser
ova-
Ber
gero
va e
t al
. (1
984)
log
Pad
ipos
e:ai
r=
0.1
74 +
0.9
10 lo
g P
o:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
brai
n:ai
r=
–0.
16 lo
g P
o:a
+ 0
.82
log
Pw
:a+
0.4
7H
Hyd
roph
ilic
VO
Cs
Tic
hy (
1991
b)lo
gP
brai
n:ai
r=
0.2
74 +
0.5
37 lo
g P
w:a
+ 0
.444
log
Po:
aH
Iner
t ga
ses;
LM
WV
OC
sA
brah
am e
t al
. (19
85)
log
Pbr
ain:
air=
0.3
94 +
1.0
96 lo
g P
w:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
brai
n:ai
r=
0.4
71 lo
g P
o:a
+ 0
.630
log
Pw
:a–
0.30
5H
Hyd
roph
obic
VO
Cs
Tic
hy (
1991
a)lo
gP
brai
n:ai
r=
0.8
44 lo
g P
o:a
– 1.
124
HLM
WV
OC
sF
iser
ova-
Ber
gero
va e
t al
. (1
984)
log
Pbr
ain:
air=
–0.
850
+ 0
.773
log
Po:
aH
Iner
t ga
ses;
LM
WV
OC
sA
brah
am e
t al
. (19
85)
log
Pbr
ain:
air=
–3.
692
+ 1
.253
RG
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
kidn
ey:a
ir=
–0.
18 lo
g P
o:a
+ 0
.82
log
Pw
:a+
0.5
3H
Hyd
roph
ilic
VO
Cs
Tic
hy (
1991
b)lo
gP
kidn
ey:a
ir=
0.2
77 +
1.1
11 lo
g P
w:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
kidn
ey:a
ir=
0.4
66 lo
g P
o:a
+ 0
.379
log
Pw
:a–
0.33
2H
Hyd
roph
obic
VO
Cs
Tic
hy (
1991
a)lo
gP
kidn
ey:a
ir=
0.7
00 lo
g P
o:a
– 0.
877
HLM
WV
OC
sF
iser
ova-
Ber
gero
va e
t al
. (1
984)
log
Pki
dney
:air
= –
0.92
0 +
0.7
64 lo
g P
o:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
liver
:air
= –
0.38
8 +
0.5
02 lo
g P
w:a
+ 0
.497
log
Po:
aH
Iner
t ga
ses;
LM
WV
OC
sA
brah
am e
t al
. (19
85)
log
Pliv
er:a
ir=
0.4
32 +
1.0
64 lo
g P
w:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
liver
:air
= 0
.746
log
Po:
a+
0.1
78 lo
g P
w:a
– 0.
767
HH
ydro
phob
ic V
OC
sT
ichy
(19
91a)
log
Pliv
er:a
ir=
0.8
71 lo
g P
o:a
– 1.
044
HLM
WV
OC
sF
iser
ova-
Ber
gero
va e
t al
. (1
984)
log
Pliv
er:a
ir=
–0.
875
+ 0
.773
log
Po:
aH
Iner
t ga
ses;
LM
WV
OC
sA
brah
am e
t al
. (19
85)
log
Plu
ng:a
ir=
–0.
21 lo
g P
o:a
+ 0
.91
log
Pw
:a+
0.4
1H
Hyd
roph
ilic
VO
Cs
Tic
hy (
1991
b)lo
gP
lung
:air
= –
0.05
7 +
0.8
70 lo
g P
w:a
+ 0
.146
log
Po:
aH
Iner
t ga
ses;
LM
WV
OC
sA
brah
am e
t al
. (19
85)
log
Plu
ng:a
ir=
0.0
57 +
0.9
78 lo
g P
w:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
) (con
tinue
d)
488 ALTERNATIVE TOXICOLOGICAL METHODS
Tab
le 4
0.1
(co
nti
nu
ed)
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e T
issu
e:A
ir P
arti
tio
n C
oef
fici
ents
(P
) o
f C
hem
ical
s
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
log
Plu
ng:a
ir=
0.3
73 lo
g P
o:a
+ 0
.416
log
Pw
:a–
0.21
6H
Hyd
roph
obic
VO
Cs
Tic
hy (
1991
a)lo
gP
lung
:air
= 0
.644
log
Po:
a–
0.81
5H
LMW
VO
Cs
Fis
erov
a-B
erge
rova
et
al.
(198
4)lo
gP
lung
:air
= –
0.83
3 +
0.9
11 lo
g P
o:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
mus
cle:
air=
–0.
19 lo
g P
o:a
+ 0
.82
log
Pw
:a+
0.5
4H
Hyd
roph
ilic
VO
Cs
Tic
hy (
1991
b)lo
gP
mus
cle:
air=
0.4
9 lo
g P
o:a
+ 0
.39
log
Pw
:a–
0.31
HH
ydro
phob
ic V
OC
sT
ichy
(19
91b)
log
Pm
uscl
e:ai
r=
–0.
263
+ 0
.575
log
Pw
:a+
0.4
23 lo
g P
o:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
mus
cle:
air=
0.3
51 +
1.1
08 lo
g P
w:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
mus
cle:
air=
0.6
52 lo
g P
o:a
– 0.
702
HLM
WV
OC
sF
iser
ova-
Ber
gero
va e
t al
. (1
984)
log
Pm
uscl
e:ai
r=
–0.
852
+ 0
.768
log
Po:
aH
Iner
t ga
ses;
LM
WV
OC
sA
brah
am e
t al
. (19
85)
log
Pm
uscl
e:ai
r=
–3.
247
+ 0
.965
RG
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)P
adip
ose:
air=
0.4
47P
o:a
+ 0
.075
Pw
:a+
6.5
9H
LMW
VO
Cs;
CF
Cs
Meu
lenb
erg
and
Vijv
erbe
rg
(200
0)P
brai
n:ai
r=
(0.
026S
o+
0.5
1Sw)/
Sa
HLM
WV
OC
sP
ater
son
and
Mac
kay
(198
9)P
brai
n:ai
r=
0.0
20P
o:a
+ 0
.380
Pw
:a+
0.9
4H
LMW
VO
Cs;
CF
Cs
Meu
lenb
erg
and
Vijv
erbe
rg
(200
0)P
kidn
ey:a
ir=
(0.
014S
o+
0.5
1Sw)/
Sa
HLM
WV
OC
sP
ater
son
and
Mac
kay
(198
9)P
kidn
ey:a
ir=
0.0
11P
o:a
+ 0
.400
Pw
:a+
0.6
9H
LMW
VO
Cs;
CF
Cs
Meu
lenb
erg
and
Vijv
erbe
rg
(200
0)P
kidn
ey:a
ir=
–0.
391
+ 0
.550
log
Pw
:a+
0.4
40 lo
g P
o:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)P
liver
:air
= (
0.02
8So
+ 0
.51S
w)/
Sa
HLM
WV
OC
sP
ater
son
and
Mac
kay
(198
9)P
liver
:air
= 0
.028
Po:
a+
0.7
9H
LMW
VO
Cs;
CF
Cs
Meu
lenb
erg
and
Vijv
erbe
rg
(200
0)P
mus
cle:
air=
0.0
14P
o:a
+ 0
.384
Pw
:a+
0.9
4H
LMW
VO
Cs;
CF
Cs
Meu
lenb
erg
and
Vijv
erbe
rg
(200
0)ln
Pad
ipos
e:ai
r=
0.0
32T
b–
5.45
6R
LMW
VO
Cs
Csa
nady
and
Lai
b (1
990)
lnP
liver
:air
= 0
.022
Tb
– 4.
638
RLM
WV
OC
sC
sana
dy a
nd L
aib
(199
0)lo
gP
adip
ose:
air=
0.9
20 lo
g P
o:a
+ 0
.136
RLM
WV
OC
sG
arga
s et
al.
(198
9)lo
gP
adip
ose:
air=
0.9
27 lo
g P
o:a
– 0.
032
log
Pw
:a+
0.1
20R
LMW
VO
Cs
Gar
gas
et a
l. (1
989)
IN SILICO APPROACHES FOR PBPK MODELING 489
log
Pad
ipos
e:ai
r=
1.0
27 lo
g P
o:a
– 0.
046
log
Pw
:a–
0.11
9R
Hal
oalk
anes
Gar
gas
et a
l. (1
988)
log
Pliv
er:a
ir=
0.5
74 lo
g P
o:a
+ 0
.302
log
Pw
:a–
0.27
8R
Hal
oalk
anes
Gar
gas
et a
l. (1
988)
log
Pliv
er:a
ir=
0.7
30 lo
g P
o:a
+ 0
.128
log
Pw
:a–
0.55
0R
LMW
VO
Cs
Gar
gas
et a
l. (1
989)
log
Pm
uscl
e:ai
r=
0.4
77 lo
g P
o:a
+ 0
.365
log
Pw
:a–
0.37
4R
Hal
oalk
anes
Gar
gas
et a
l. (1
988)
log
Pm
uscl
e:ai
r=
0.6
44 lo
g P
o:a
+ 0
.180
log
Pw
:a–
0.72
5R
LMW
VO
Cs
Gar
gas
et a
l. (1
989)
Pad
ipos
e:ai
r=
0.5
94P
o:a
+ 0
.085
Pw
:a+
9.4
0R
LMW
VO
Cs;
CF
Cs
Meu
lenb
erg
and
Vijv
erbe
rg
(200
0)P
brai
n:ai
r=
0.0
54P
o:a
+ 0
.832
Pw
:aR
LMW
VO
Cs;
CF
Cs
Meu
lenb
erg
and
Vijv
erbe
rg
(200
0)P
kidn
ey:a
ir=
0.0
97P
o:a
+ 0
.826
Pw
:aR
LMW
VO
Cs;
CF
Cs
Meu
lenb
erg
and
Vijv
erbe
rg
(200
0)P
liver
:air
= 0
.026
Po:
a+
0.8
78P
w:a
+ 2
.36
RLM
WV
OC
s; C
FC
sM
eule
nber
g an
d V
ijver
berg
(2
000)
Pm
uscl
e:ai
r=
0.0
10P
o:a
+ 0
.772
Pw
:a+
0.2
9R
LMW
VO
Cs;
CF
Cs
Meu
lenb
erg
and
Vijv
erbe
rg
(200
0)
Bio
log
ical
ly b
ased
alg
ori
thm
s
Ptis
sue:
air=
(S
sVw
t+
SvV
nt+
0.7
SsV
pt+
0.3
SvV
pt)/
Sa
R,
HLM
WV
OC
sP
oulin
and
Kris
hnan
(19
96a)
Ptis
sue:
air=
Po:
wP
w:a(V
nt+
0.3
Vpt)
+ P
w:a(V
wt+
0.7
Vpt)
R,
HLM
WV
OC
sP
oulin
and
Kris
hnan
(19
96c)
a=
dip
olar
ity/p
olar
izab
ility
, =
ove
rall
hydr
ogen
-bon
d ac
idity
, =
ove
rall
hydr
ogen
-bon
d ba
sici
ty,
1 Xv ,
,1 X,4
,3
,4 X
vpc
= c
onne
ctiv
ityin
dice
s, N
Br=
num
ber
of b
rom
ide
atom
s in
the
mol
ecul
e, N
C=
num
ber
of c
arbo
n at
oms
in th
e m
olec
ule,
NC
l=
num
ber
of c
hlor
ide
atom
s in
the
mol
ecul
e,N
F=
num
ber
of fl
uorid
e at
oms
in t
he m
olec
ule,
Phe
:a=
hex
adec
ane:
air
part
ition
coe
ffici
ent,
Po:
a=
n-oc
tano
l:air
part
ition
coe
ffici
ent
(or
vege
tabl
e oi
l:air)
,P
o:w
= n
-oct
anol
:wat
er p
artit
ion
coef
ficie
nt (
or v
eget
able
oil:
wat
er),
Pw
:a=
wat
er:a
ir pa
rtiti
on c
oeffi
cien
t, Q
H=
var
iabl
e de
pend
ant
on t
he p
olar
ity o
f th
em
olec
ule
due
to t
he p
rese
nce
of h
ydro
gen
atom
s, R
2=
Exc
ess
mol
ar r
efra
ctio
n, R
g=
ave
rage
sol
ubili
ty in
ent
ire s
et o
f so
lven
t sy
stem
s, S
a=
sol
ubili
tyin
air,
So
= s
olub
ility
in
n-oc
tano
l (o
r ve
geta
ble
oil),
Ss
= s
olub
ility
in
salin
e, S
v=
sol
ubili
ty i
n ve
geta
ble
oil,
Sw
= s
olub
ility
in
wat
er,
Tb
= b
oilin
g po
int,
Vnt
= v
olum
e fr
actio
n of
neu
tral
lipi
ds in
tis
sues
, V
pt=
vol
ume
frac
tion
of p
hosp
holip
ids
in t
issu
es,
and
Vw
t=
vol
ume
frac
tion
of w
ater
in t
issu
es.
bF
= fi
sh,
H =
hum
an,
and
R =
rat
s.c
CF
Cs
= c
hlor
ofluo
roca
rbon
s, L
MW
VO
Cs
= lo
w m
olec
ular
wei
ght
vola
tile
orga
nic
chem
ical
s, a
nd V
OC
s =
vol
atile
org
anic
che
mic
als.
490 ALTERNATIVE TOXICOLOGICAL METHODS
In Silico Approaches for Blood:Air PCs
IN SILICO APPROACHES FOR PBPK MODELING 491
Tab
le 4
0.2
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e B
loo
d:A
ir P
arti
tio
n C
oef
fici
ents
(P
) o
f C
hem
ical
s
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
QS
AR
s: L
FE
-typ
e eq
uat
ion
s
Ele
ctro
stat
ic d
escr
ipto
rs
log
Pbl
ood:
air=
–1.
269
+ 0
.612
R2
+ 0
.916
+ 3
.614
+ 3
.381
+0.
362
log
Phe
:a
HIn
ert
Gas
es; L
MW
VO
Cs
Abr
aham
and
Wea
ther
sby
(199
4)
log
Ppl
asm
a:ai
r=
–1.
48 +
0.4
90R
2+
2.0
4 +
3.5
074
+ 3
.911
+
0.15
7 lo
g P
he:a
HIn
ert
Gas
es; L
MW
VO
Cs
Abr
aham
and
Wea
ther
sby
(199
4)
Ste
ric
des
crip
tors
log
Pbl
ood:
air=
0.0
072M
W+
0.1
97H
Trih
alom
etha
nes
Bat
term
an e
t al
. (20
02)
log
Pbl
ood:
air=
0.3
21N
Br+
1.0
6H
Trih
alom
etha
nes
Bat
term
an e
t al
. (20
02)
Pbl
ood:
air=
0.0
7MW
+ 5
.59
HA
lipha
tic h
ydro
carb
ons
Per
belli
ni e
t al
. (19
85)
log
Pbl
ood:
air=
0.4
43Q
H–
0.30
3NF
+ 0
.225
NC
l+
0.5
10N
BR
+ 0
.155
NC
– 0.
104
RH
aloa
lkan
esG
arga
s et
al.
(198
8)
Hyd
rop
ho
bic
des
crip
tors
log
(Pbl
ood:
wat
er–
Vw
b) =
0.7
Po:
w–
0.75
FC
hlor
oeth
anes
; ben
zene
Ber
tels
en e
t al
. (19
98)
lnP
bloo
d:ai
r=
0.0
38T
b–
13.3
HA
lipha
tic h
ydro
carb
ons
Csa
nady
and
Lai
b (1
990)
log
Pbl
ood:
air=
0.0
109T
b–
2.58
4H
Trih
alom
etha
nes
Bat
term
an e
t al
. (20
02)
log
Pbl
ood:
air=
–0.
14 lo
g P
o:a
+ 0
.86
log
Pw
:a+
0.4
7H
Hyd
roph
ilic
VO
Cs
Tic
hy (
1991
b)
log
Pbl
ood:
air=
0.6
85 lo
g P
o:a
– 0.
6565
HTr
ihal
omet
hane
sB
atte
rman
et
al. (
2002
)lo
gP
bloo
d:ai
r=
0.4
5 lo
g P
w:a
+ 1
.21
HV
OC
sLa
ass
(198
7)
log
Pbl
ood:
air=
–0.
003
log
Pw
:a+
1.4
7H
VO
Cs
Laas
s (1
987)
lo
gp b
lood
:air
= –
0.07
4 +
0.8
02 lo
g P
w:a
+ 0
.218
log
Po:
aH
Iner
t ga
ses;
LM
WV
OC
sA
brah
am e
t al
. (19
85)
log
Pbl
ood:
air=
–0.
07 lo
g S
w+
1.2
1H
VO
Cs
Laas
s (1
987)
(c
ontin
ued)
492 ALTERNATIVE TOXICOLOGICAL METHODS
Tab
le 4
0.2
(co
nti
nu
ed)
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e B
loo
d:A
ir P
arti
tio
n C
oef
fici
ents
(P
) o
f C
hem
ical
s
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
log
Pbl
ood:
air=
–0.
09 lo
g P
o:a
+ 2
.45
HV
OC
sLa
ass
(198
7)
log
Pbl
ood:
air=
–0.
102
+ 0
.675
log
Pw
:a+
0.3
15 lo
g P
o:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
bloo
d:ai
r=
–0.
295
+ 0
.588
log
Pw
:a+
0.4
11 lo
g P
o:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
bloo
d:ai
r=
–0.
338
log
Po:
a+
3.1
21H
Hal
ogen
ated
hyd
roca
rbon
sT
ichy
et
al. (
1984
)lo
gP
bloo
d:ai
r=
–0.
6737
+ 0
.531
9 lo
g P
o:a
log
Pw
:aH
VO
Cs
Sat
o an
d N
akaj
ima
(197
9)lo
gP
bloo
d:ai
r=
0.6
95 lo
g P
o:a
– 1.
076
HLM
WV
OC
sF
iser
ova-
Ber
gero
va e
t al
. (1
984)
log
Pbl
ood:
air=
–0.
820
+ 0
.754
log
Po:
aH
Iner
t ga
ses;
LM
WV
OC
sA
brah
am e
t al
. (19
85)
log
Pbl
ood:
air=
0.0
9 lo
g S
w+
8.2
5 lo
g V
o–
11.0
9H
VO
Cs
Laas
s (1
987)
lo
gP
bloo
d:ai
r=
0.1
1 lo
g S
w+
1.9
1H
VO
Cs
Laas
s (1
987)
lo
gP
bloo
d:ai
r=
0.1
80 lo
g P
o:a
+ 0
.889
log
Pw
:a+
0.0
54H
Hyd
roph
obic
VO
Cs
Tic
hy (
1991
a)lo
gP
bloo
d:ai
r=
0.2
0 lo
g S
w+
1.2
9H
VO
Cs
Laas
s (1
987)
lo
gP
bloo
d:ai
r=
0.2
2 lo
g P
w:a
+ 0
.67
log
Po:
a–
0.98
HV
OC
sLa
ass
(198
7)
log
Pbl
ood:
air=
0.2
2 lo
g S
w+
10.
78 lo
g V
w–
40.9
9H
VO
Cs
Laas
s (1
987)
lo
gP
bloo
d:ai
r=
0.2
62 +
0.9
96 lo
g P
w:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
bloo
d:ai
r=
0.2
7 lo
g 10
00/P
+ 5
.10
log
Vo
– 6.
67H
VO
Cs
Laas
s (1
987)
lo
gP
bloo
d:ai
r=
0.3
1 lo
g S
w+
3.9
0 lo
g V
o–
4.53
HV
OC
sLa
ass
(198
7)
log
Pbl
ood:
air=
0.3
5 lo
g 10
00/P
+ 1
.01
HV
OC
sLa
ass
(198
7)
log
Pbl
ood:
air=
0.3
5 lo
g S
w+
0.7
9 lo
g 10
00/P
+ 1
.34
log
Vo
– 2.
23H
VO
Cs
Laas
s (1
987)
lo
gP
bloo
d:ai
r=
0.3
7 lo
g S
w+
10.
09 lo
g V
w–
38.4
0H
VO
Cs
Laas
s (1
987)
lo
gP
bloo
d:ai
r=
0.3
8 lo
g S
w+
0.9
1 lo
g 10
00/P
–0.
45H
VO
Cs
Laas
s (1
987)
lo
gP
bloo
d:ai
r=
0.4
5 lo
g S
w+
0.8
1 lo
g 10
00/P
–0.
40H
VO
Cs
Laas
s (1
987)
lo
gP
bloo
d:ai
r=
0.4
8 lo
g S
w+
0.7
5 lo
g 10
00/P
+ 1
.67
log
Vo
– 2.
77H
VO
Cs
Laas
s (1
987)
lo
gP
bloo
d:ai
r=
0.5
1 lo
g 10
00/P
+ 0
.37
HV
OC
sLa
ass
(198
7)
log
Pbl
ood:
air=
0.5
81 lo
g P
o:a
+ 0
.332
log
Pw
:a–
0.59
9H
LMW
VO
Cs
Gar
gas
et a
l. (1
989)
log
Pbl
ood:
air=
0.6
3 lo
g 10
00/P
+ 0
.38
HV
OC
sLa
ass
(198
7)
log
Pbl
ood:
air=
0.6
5 lo
g P
o:a
– 0.
84H
VO
Cs
Laas
s (1
987)
lo
gP
bloo
d:ai
r=
0.8
51 lo
g S
w+
1.7
8H
VO
Cs
Laas
s (1
987)
IN SILICO APPROACHES FOR PBPK MODELING 493
log
Pbl
ood:
air=
0.9
84 lo
g P
w:a
+ 0
.053
HK
eton
es; e
ther
s; g
ases
Tic
hy e
t al
. (19
84)
log
Pbl
ood:
air=
1.0
7 lo
g P
w:a
+ 0
.27
log
Po:
a–
0.79
HV
OC
sLa
ass
(198
7)
log
Pbl
ood:
air=
1.2
1 lo
g V
o–
0.17
HV
OC
sLa
ass
(198
7)
log
Pbl
ood:
air=
3.0
5 –
0.34
Po:
nH
Ket
ones
Cab
ala
et a
l. (1
992)
lo
gP
bloo
d:ai
r=
–3.
922
+ 1
.369
RG
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
) lo
gP
bloo
d:ai
r=
5.8
9 lo
g V
w–
21.4
3H
VO
Cs
Laas
s (1
987)
lo
gP
bloo
d:ai
r=
7.8
6 lo
g V
o–
10.4
0H
VO
Cs
Laas
s (1
987)
lo
gP
bloo
d:ai
r=
8.9
0 lo
g V
w–
33.4
0H
VO
Cs
Laas
s (1
987)
lo
gP
milk
:air
= 0
.900
log
Po:
a–
1.09
5H
Trih
alom
etha
nes
Bat
term
an e
t al
. (20
02)
log
Ppl
asm
a:ai
r=
–0.
079
+ 0
.896
log
Pw
:a+
0.1
49 lo
g P
o:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
) lo
gP
plas
ma:
air=
–0.
082
+ 0
.894
log
Pw
:a+
0.1
52 lo
g P
o:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
plas
ma:
air=
–0.
848
+ 0
.890
log
Po:
aH
Iner
t ga
ses;
LM
WV
OC
sA
brah
am e
t al
. (19
85)
log
Ppl
asm
a:ai
r=
–3.
696
+ 1
.208
RG
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)lo
gP
plas
ma:
air=
0.0
38 +
1.0
19 lo
g P
w:a
HIn
ert
gase
s; L
MW
VO
Cs
Abr
aham
et
al. (
1985
)P
bloo
d:ai
r=
0.0
072P
o:a
+ 0
.898
Pw
:a+
0.0
3H
LMW
VO
Cs;
CF
Cs
Meu
lenb
erg
and
Vijv
erbe
rg
(200
0)P
bloo
d:ai
r=
0.0
8e0.
0308
Tb
HA
lipha
tic h
ydro
carb
ons
Per
belli
ni e
t al
. (19
85)
Pbl
ood:
air=
0.0
0442
Po:
aH
Alip
hatic
hyd
roca
rbon
sP
erbe
llini
et
al. (
1985
)P
bloo
d:ai
r=
0.8
8Pw
:a+
0.0
12H
VO
Cs
Fei
ngol
d (1
976)
Pbl
ood:
air=
0.8
9Pw
:a+
0.0
11P
o:a
HLM
WV
OC
sT
ichy
et
al. (
1984
) P
bloo
d:ai
r=
0.9
0 lo
g P
w:a
– 46
1H
Est
ers;
alc
ohol
sK
anek
o et
al.
(199
4)P
bloo
d:ai
r=
Pw
:a+
(P
o:a/
100)
HA
naes
thet
ics
Ege
r an
d La
rson
(19
64)
Pbl
ood:
air=
Sw(1
+ 0
.003
5Po:
w)/
Sa
HLM
WV
OC
sP
ater
son
and
Mac
kay
(198
9)lo
gP
bloo
d:ai
r=
Pw
:a[V
lbP
o:w
0.85
+V
prb(
86.2
/Po:
w+
3.7
0)]
+ V
wb
H,
RLM
WV
OC
sC
onne
ll et
al.
(199
3)
log
Pbl
ood:
air=
0.4
26lo
gP
o:a
+ 0
.515
log
Pw
:a–
0.07
0R
Hal
oalk
anes
Gar
gas
et a
l. (1
988)
lo
gP
bloo
d:ai
r=
0.5
53 lo
g P
o:a
+ 0
.351
Pw
:a–
0.28
6R
LMW
VO
Cs
Gar
gas
et a
l. (1
989)
P
bloo
d:ai
r=
0.0
054P
o:a
+ 0
.931
Pw
:a+
1.1
6R
LMW
VO
Cs;
CF
Cs
Meu
lenb
erg
and
Vijv
erbe
rg
(200
0)(c
ontin
ued)
494 ALTERNATIVE TOXICOLOGICAL METHODS
Tab
le 4
0.2
(co
nti
nu
ed)
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e B
loo
d:A
ir P
arti
tio
n C
oef
fici
ents
(P
) o
f C
hem
ical
s
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
QS
AR
s: F
ree–
Wils
on
-typ
e eq
uat
ion
s
Pbl
ood:
wat
er=
BS
(C-C
)(2
8.4)
+ n
CL 2
(–12
.9)
+ n
CL 3
(12.
9)F
Chl
oroe
than
esF
ouch
écou
rt e
t al
. (20
00)
Pbl
ood:
air=
BS
(C-C
)(2
6.2)
+ n
H3(
–34.
9) +
nC
L(–4
.51)
+ n
CL 2
(29.
4) +
nC
L 3(1
1.5)
HC
hlor
oeth
anes
Fou
chéc
ourt
and
Kris
hnan
(2
000)
Pbl
ood:
air=
BS
(C-C
)(4
5.6)
+ n
H3(
–51.
5) +
nC
L(–8
.86)
+ n
CL 2
(36.
4) +
nC
L 3(1
1.1)
RC
hlor
oeth
anes
Fou
chéc
ourt
and
Kris
hnan
(2
000)
Bio
log
ical
ly b
ased
alg
ori
thm
s
Pbl
ood:
air=
Po:
wP
w:a(V
nb+
0.3
Vpb
) +
Pw
:a(V
wb
+ 0
.7V
pb)
R,
HLM
WV
OC
sP
oulin
and
Kris
hnan
(199
6c)
Pbl
ood:
air=
[f e
(SsV
we
+S
vVne
+ 0
.7S
sVpe
+ 0
.3S
vVpe
) +
fp(
SsV
wp
+S
vVnp
+0.
7SsV
pp+
0.3
SvV
pp)]
/Sa
R,
HLM
WV
OC
sP
oulin
and
Kris
hnan
(199
6b)
a=
dip
olar
ity/p
olar
izab
ility
, =
ove
rall
hydr
ogen
-bon
d ac
idity
, =
ove
rall
hydr
ogen
-bon
d ba
sici
ty, B
S =
Bas
ic s
truc
ture
, fe
= fr
actio
n of
ery
thro
cyte
sin
blo
od,
f p=
fra
ctio
n of
pla
sma
in b
lood
, M
W =
mol
ecul
ar w
eigh
t, N
Br
= n
umbe
r of
bro
mid
e at
oms
in t
he m
olec
ule,
NC
= n
umbe
r of
car
bon
atom
s in
the
mol
ecul
e, N
Cl=
num
ber
of c
hlor
ide
atom
s in
the
mol
ecul
e, n
CL
= n
umbe
r of
CL
frag
men
ts,
nCL 2
= n
umbe
r of
CL 2
frag
men
ts,
nCL 3
= n
umbe
r of
CL 3
frag
men
ts,
NF
= n
umbe
r of
fluo
ride
atom
s in
the
mol
ecul
e, n
H3
= n
umbe
r of
H3
frag
men
ts,
P=
vap
or p
ress
ure,
Phe
:a=
hex
adec
ane:
air
part
ition
coef
ficie
nt,
Po:
a=
n-o
ctan
ol:a
ir pa
rtiti
on c
oeffi
cien
t (o
r ve
geta
ble
oil:a
ir),
Po:
n=
veg
etab
le o
il:ni
trog
en p
artit
ion
coef
ficie
nt,
Po:
w=
n-o
ctan
ol:w
ater
par
titio
nco
effic
ient
(or
veg
etab
le o
il:w
ater
), P
w:a
= w
ater
:air
part
ition
coe
ffici
ent,
QH
= v
aria
ble
depe
ndan
t on
the
pol
arity
of
the
mol
ecul
e du
e to
the
pre
senc
eof
hyd
roge
n at
oms,
R2
= e
xces
s m
olar
ref
ract
ion,
Rg
= p
aram
eter
s re
lativ
e to
the
sol
vent
, S
a=
sol
ubili
ty i
n ai
r, S
s=
sol
ubili
ty i
n sa
line,
Sv
= s
olub
ility
in v
eget
able
oil,
Sw
= s
olub
ility
in w
ater
, Tb
= b
oilin
g po
int,
Vlb
= v
olum
e fr
actio
n of
lipi
ds in
blo
od,
Vnb
= v
olum
e fr
actio
n of
neu
tral
lipi
ds in
blo
od,
Vne
=vo
lum
e fr
actio
n of
neu
tral
lipi
ds in
ery
thro
cyte
s, V
np=
vol
ume
frac
tion
of n
eutr
al li
pids
in p
lasm
a, V
o=
sur
face
tens
ion,
Vpb
= v
olum
e fr
actio
n of
pho
spho
lipid
sin
blo
od,
Vpe
= v
olum
e fr
actio
n of
pho
spho
lipid
s in
ery
thro
cyte
s, V
pp=
vol
ume
frac
tion
of p
hosp
holip
ids
in p
lasm
a, V
prb
= v
olum
e fr
actio
n of
pro
tein
s in
bloo
d,V
w=
hea
t re
leas
ed d
ue t
o ev
apor
atio
n of
the
sub
stan
ce a
t bo
iling
tem
pera
ture
, V
wb
= v
olum
e fr
actio
n of
wat
er in
blo
od, V
we
= v
olum
e fr
actio
n of
wat
er in
ery
thro
cyte
s, a
nd V
wp
= v
olum
e fr
actio
n of
wat
er in
pla
sma.
bF
= fi
sh,
H =
hum
an,
and
R =
rat
s.c
CF
Cs
= c
hlor
ofluo
roca
rbon
s, L
MW
VO
Cs
= lo
w m
olec
ular
wei
ght
vola
tile
orga
nic
chem
ical
s, a
nd V
OC
s =
vol
atile
org
anic
che
mic
als.
IN SILICO APPROACHES FOR PBPK MODELING 495
496 ALTERNATIVE TOXICOLOGICAL METHODS
In Silico Approaches for Tissue:Blood PCs
IN SILICO APPROACHES FOR PBPK MODELING 497
Tab
le 4
0.3
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e T
issu
e:B
loo
d P
arti
tio
n C
oef
fici
ents
(P
) o
f C
hem
ical
s
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
QS
AR
s: L
FE
-typ
e eq
uat
ion
s
Ste
ric
des
crip
tors
log
Pad
ipos
e:bl
ood
= 0
.168
+ 0
.198
R2
+ 0
.130
– 1
.211
–
3.26
7 +
2.2
75V
x
HIn
ert
gase
s; L
MW
VO
Cs;
CF
Cs
Abr
aham
and
Wea
ther
sby
(199
4)
log
Pbr
ain:
bloo
d=
–0.
166
+ 0
.239
R2–
0.62
6 –
0.3
68 –
0.6
15
+ 1
.072
Vx
HIn
ert
gase
s; L
MW
VO
Cs;
CF
Cs
Abr
aham
and
Wea
ther
sby
(199
4)
log
Pbr
ain:
bloo
d=
–0.
0148
PS
A +
0.1
52 lo
g P
o:w
+ 0
.139
HIn
ert
gase
s; H
MW
OC
s; L
MW
VO
Cs
Cla
rk (
1999
) lo
gP
brai
n:bl
ood
= 1
.359
+ 0
.338
log
Pcy
h–
0.00
618V
mH
H2-
R a
ntag
onis
tsK
aliz
an a
nd M
arku
szew
ski
(199
6)
log
Phe
art:b
lood
= –
0.34
6 +
0.2
04 –
2.1
50 –
0.8
53 +
0.9
31V
xH
Iner
t ga
ses;
LM
WV
OC
s; C
FC
sA
brah
am a
nd W
eath
ersb
y (1
994)
log
Pki
dney
:blo
od=
–0.
188
+ 0
.226
R2
– 0.
559
– 0
.433
+ 0
.832
Vx
HIn
ert
gase
s; L
MW
VO
Cs;
CF
Cs
Abr
aham
and
Wea
ther
sby
(199
4)
log
Pliv
er:b
lood
= –
0.27
0 +
0.2
33R
2–
0.37
5 –
1.0
04 –
1.1
18
+ 0
.832
Vx
HIn
ert
gase
s; L
MW
VO
Cs;
CF
Cs
Abr
aham
and
Wea
ther
sby
(199
4)
log
Plu
ng:b
lood
= –
0.15
0 –
0.19
5 +
0.3
89V
xH
Iner
t ga
ses;
LM
WV
OC
s; C
FC
sA
brah
am a
nd W
eath
ersb
y (1
994)
log
Pm
uscl
e:bl
ood
= –
0.22
2 –
0.47
9 –
0.5
17 +
0.9
99V
xH
Iner
t ga
ses;
LM
WV
OC
s; C
FC
sA
brah
am a
nd W
eath
ersb
y (1
994)
Pad
ipos
e:pl
asm
a=
1.9
988
– 0.
5004
UN
S +
0.1
793N
PL
+ 0
.059
31D
IFF
2H
PC
Bs
(48)
log
Pbr
ain:
bloo
d=
0.0
88 +
0.2
64R
2–
0.96
6 –
0.7
05–
0.75
6+
1.1
89V
x
RH
2-R
ant
agon
ists
Nor
inde
r an
d H
aebe
rlein
(2
002)
(con
tinue
d)
498 ALTERNATIVE TOXICOLOGICAL METHODS
Tab
le 4
0.3
(co
nti
nu
ed)
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e T
issu
e:B
loo
d P
arti
tio
n C
oef
fici
ents
(P
) o
f C
hem
ical
s
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
log
Pbr
ain:
bloo
d=
–0.
088
+ 0
.272
log
Po:
w–
0.00
116M
WR
H2-
R a
ntag
onis
tsK
aliz
an a
nd M
arku
szew
ski
(199
6)lo
gP
brai
n:bl
ood
= 0
.001
16M
W +
0.2
72 lo
g P
o:w
– 0.
088
RIn
ert
gase
s; v
olat
ile h
ydro
carb
ons
Nor
inde
r an
d H
aebe
rlein
(2
002)
log
Pbr
ain:
bloo
d=
–0.
01V
m+
0.3
5 lo
g P
o:w
+ 0
.99I
3+
1.2
5R
Dru
g-lik
e m
olec
ules
Nor
inde
r an
d H
aebe
rlein
(2
002)
log
Pbr
ain:
bloo
d=
–0.
021P
SA
– 0
.003
MV
+ 1
.643
RIn
ert
gase
s; H
MW
OC
s; L
MW
VO
Cs
Cla
rk (
1999
) lo
gP
brai
n:bl
ood
= –
0.03
22D
PS
A +
1.3
3R
HM
WO
Cs
Nor
inde
r an
d H
aebe
rlein
(2
002)
log
Pbr
ain:
bloo
d=
–0.
038
+ 0
.198
R2
– 0.
687
– 0
.715
–
0.69
8+
0.9
95V
x
RH
2-R
ant
agon
ists
; Ine
rt g
ases
; SO
Ms
Nor
inde
r an
d H
aebe
rlein
(2
002)
log
Pbr
ain:
bloo
d=
–0.
218(
NN
+N
O)
+ 0
.235
log
Po:
w–
0.02
7R
HM
WO
Cs
Nor
inde
r an
d H
aebe
rlein
(2
002)
log
Pbr
ain:
bloo
d=
0.4
76 +
0.5
41 lo
g P
o:w
– 0.
0079
4MW
RH
2-R
ant
agon
ists
Kal
izan
and
Mar
kusz
ewsk
i (1
996)
log
Pbr
ain:
bloo
d=
1.2
96 +
0.3
09 lo
g P
cyh
– 0.
0057
0MW
RH
2-R
ant
agon
ists
Kal
izan
and
Mar
kusz
ewsk
i (1
996)
Hyd
rop
ho
bic
des
crip
tors
log
Pbr
ain:
bloo
d=
0.3
9 lo
g P
o:w
+ 0
.68
HD
rugs
, ho
rmon
esS
eyde
l and
Sch
aper
(19
82)
log
Pbr
ain:
bloo
d=
0.0
54G
o+
0.4
3H
H2-
R a
ntag
onis
ts; L
MW
VO
Cs
Lom
bard
o et
al.
(199
6)
Pad
ipos
e:bl
ood
=(V
lt+
Vw
t)/(V
lb+
Vw
b)+
BH
, R
LMW
VO
Cs
DeJ
ongh
et
al. (
1997
)
Pbr
ain:
bloo
d=
(Vlt
+V
wt)/
(Vlb
+V
wb)
+B
H,
RLM
WV
OC
sD
eJon
gh e
t al
. (19
97)
Pki
dney
:blo
od=
(Vlt
+V
wt)/
(Vlb
+V
wb)
+B
H,
RLM
WV
OC
sD
eJon
gh e
t al
. (19
97)
Pliv
er:b
lood
=(V
lt +
Vw
t)/(V
lb+
Vw
b)+
BH
, R
LMW
VO
Cs
DeJ
ongh
et
al. (
1997
)
IN SILICO APPROACHES FOR PBPK MODELING 499
Pm
uscl
e:bl
ood
=(V
lt+
Vw
t)/(V
lb+
Vw
b)+
BH
, R
LMW
VO
Cs
DeJ
ongh
et
al. (
1997
)
LnP
kidn
ey:b
lood
= 0
.006
5o
RH
MW
OC
sYa
mag
uchi
et
al. (
1996
) Ln
Pliv
er:b
lood
= 0
.025
iR
HM
WO
Cs
Yam
aguc
hi e
t al
. (19
96)
LnP
mus
cle:
bloo
d=
0.0
069
iR
HM
WO
Cs
Yam
aguc
hi e
t al
. (19
96)
log
Pbr
ain:
bloo
d=
0.0
35G
solv
+ 0
.259
RH
2-R
ant
agon
ists
; LM
WV
OC
sN
orin
der
and
Hae
berle
in
(200
2)lo
gP
brai
n:bl
ood
= 0
.427
5 –
0.38
73n a
cc,s
olv+
0.1
092
log
Po:
w–
0.00
17A
pol
RD
rugs
; LM
WV
OC
s; a
naes
thet
ics
Feh
er e
t al
. (20
00)
log
Pbr
ain:
bloo
d=
1.9
79 +
0.3
73 lo
g P
cyh
– 0.
0027
5Vw
avR
H2-
R a
ntag
onis
tsK
aliz
an a
nd M
arku
szew
ski
(199
6)lo
gP
brai
n:pl
asm
a=
–0.
48
log
Poc
t-cy
c+
0.8
9R
H2-
R a
ntag
onis
tsTe
sta
et a
l. (2
000)
ln
Pad
ipos
e:bl
ood
= 0
.05
i + 0
.021
RH
MW
OC
sYa
mag
uchi
et
al. (
1996
)
Pad
ipos
e:bl
ood
= 0
.915
Rb
Bas
ic d
rugs
Yoko
gaw
a et
al.
(199
0)
Pad
ipos
e:pl
asm
a=
0.0
16R
bB
asic
dru
gsYo
koga
wa
et a
l. (2
002)
Pbo
ne m
arro
w:b
lood
= 1
.975
Rb
Bas
ic d
rugs
Yoko
gaw
a et
al.
(199
0)
Pbo
ne:p
lasm
a=
0.0
36R
bB
asic
dru
gsYo
koga
wa
et a
l. (2
002)
Pbr
ain:
bloo
d=
3.1
57R
bB
asic
dru
gsYo
koga
wa
et a
l. (1
990)
Pbr
ain:
plas
ma
= 0
.062
Rb
Bas
ic d
rugs
Yoko
gaw
a et
al.
(200
2)
Pgu
t:blo
od=
3.0
02R
bB
asic
dru
gsYo
koga
wa
et a
l. (1
990)
Pgu
t:pla
sma
= 0
.058
Rb
Bas
ic d
rugs
Yoko
gaw
a et
al.
(200
2)
Phe
art:b
lood
= 1
.678
Rb
Bas
ic d
rugs
Yoko
gaw
a et
al.
(199
0)
Phe
art:p
lasm
a=
0.0
32R
bB
asic
dru
gsYo
koga
wa
et a
l. (2
002)
Pki
dney
:pla
sma
= 0
.075
Rb
Bas
ic d
rugs
Yoko
gaw
a et
al.
(200
2)
(con
tinue
d)
500 ALTERNATIVE TOXICOLOGICAL METHODS
Tab
le 4
0.3
(co
nti
nu
ed)
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e T
issu
e:B
loo
d P
arti
tio
n C
oef
fici
ents
(P
) o
f C
hem
ical
s
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
Pliv
er:p
lasm
a=
0.0
64R
bB
asic
dru
gsYo
koga
wa
et a
l. (2
002)
Plu
ng:b
lood
= 1
.158
Rb
Bas
ic d
rugs
Yoko
gaw
a et
al.
(199
0)
Plu
ng:p
lasm
a=
0.0
31R
bB
asic
dru
gsYo
koga
wa
et a
l. (2
002)
Pm
uscl
e:bl
ood
= 4
.928
Rb
Bas
ic d
rugs
Yoko
gaw
a et
al.
(199
0)
Pm
uscl
e:pl
asm
a=
0.0
99R
bB
asic
dru
gsYo
koga
wa
et a
l. (2
002)
Psk
in:b
lood
= 2
.997
Rb
Bas
ic d
rugs
Yoko
gaw
a et
al.
(199
0)
Psk
in:p
lasm
a=
0.0
58R
bB
asic
dru
gsYo
koga
wa
et a
l. (2
002)
Psp
leen
:blo
od=
3.0
02R
bB
asic
dru
gsYo
koga
wa
et a
l. (1
990)
QS
AR
s: F
ree–
Wils
on
-typ
e eq
uat
ion
s
Pad
ipos
e:bl
ood
= B
S(C
-C)(9
4.5)
+ n
CL 2
(–29
.2)
+ n
CL 3
(29.
2)F
Chl
oroe
than
esF
ouch
écou
rt e
t al
. (20
00)
Pliv
er:b
lood
= B
S(C
-C)(2
.93)
+ n
CL 2
(–0.
238)
+ n
CL 3
(0.2
38)
FC
hlor
oeth
anes
Fou
chéc
ourt
et
al. (
2000
)P
mus
cle:
bloo
d=
BS
(C-C
)(3.0
2) +
nC
L 2(–
0.17
5) +
nC
L 3(0
.175
)F
Chl
oroe
than
esF
ouch
écou
rt e
t al
. (20
00)
Pad
ipos
e:bl
ood
= B
S(C
-C)(4
9.2)
+ n
H3(
–0.4
40)
+ n
CL(
–14.
54)
+
nCL 2
(–6.
65)
+ n
CL 3
(26.
5)H
Chl
oroe
than
esF
ouch
écou
rt a
nd K
rishn
an
(200
0)P
liver
:blo
od=
BS
(C-C
)(2.6
4) +
nH
3(–0
.61)
+ n
CL(
–0.6
6) +
nC
L 2(–
0.18
)+
nCL 3
(1.6
8)H
Chl
oroe
than
esF
ouch
écou
rt a
nd K
rishn
an
(200
0)P
mus
cle:
bloo
d=
BS
(C-C
)(1.1
1) +
nH
3(0.
08)
+ n
CL(
–0.0
2) +
nC
L 2(–
0.21
)+
nCL 3
(0.1
5)H
Chl
oroe
than
esF
ouch
écou
rt a
nd K
rishn
an
(200
0)P
adip
ose:
bloo
d=
BS
(C-C
)(30.
1) +
nH
3(–9
.88)
+ n
CL(
–6.0
2) +
nC
L 2(–
3.90
)+
nCL 3
(17.
3)R
Chl
oroe
than
esF
ouch
écou
rt a
nd K
rishn
an
(200
0)P
liver
:blo
od=
BS
(C-C
)(1.7
9) +
nH
3(–0
.9)
+ n
CL(
–0.3
8) +
nC
L 2(–
0.21
) +
nC
L 3(1
.27)
RC
hlor
oeth
anes
Fou
chéc
ourt
and
Kris
hnan
(2
000)
IN SILICO APPROACHES FOR PBPK MODELING 501
Pm
uscl
e:bl
ood
= B
S(C
-C)(0
.69)
+ n
H3(
–0.1
2) +
nC
L(0.
04)
+ n
CL 2
(–0.
12)
+nC
L 3(0
.17)
RC
hlor
oeth
anes
Fou
chéc
ourt
and
Kris
hnan
(2
000)
Bio
log
ical
ly b
ased
alg
ori
thm
s
Ptis
sue:
bloo
d=
(S
oVnt
+S
w0.
7Vpt
+S
o0.3
Vpt
+S
wV
wt)/
(SoV
nb+
Sw0.
7Vpb
+S
o0.3
Vpb
+S
wV
wb)
HLM
WV
OC
sP
oulin
and
Kris
hnan
(19
95a)
Ptis
sue:
bloo
d=
(P
o:wV
nt+
Vw
t+
Po:
w0.
3Vpt
+ 0
.7V
pt)/
[f e(P
o:wV
ne+
Vw
e+
Po:
w0.
3Vpe
+ 0
.7V
pe)
+ f
p(P
o:wV
np+
Vw
p+
Po:
w0.
3Vpp
+ 0
.7V
pp)]
RK
eton
es; A
lcoh
ols;
Est
ers
Pou
lin a
nd K
rishn
an (
1995
b)
Ptis
sue:
bloo
d=
[P
o:w(V
nt+
0.3
Vpt)
+ (
Vw
t+
0.7
Vpt)]
/[Po:
w(V
nb+
0.3
Vpb
) +
(V
wb
+ 0
.7V
pe)]
R,
HLM
WV
OC
sP
oulin
and
Kris
hnan
(19
96b)
a =
dip
olar
ity/p
olar
izab
ility
, =
ove
rall
hydr
ogen
-bon
d ac
idity
, =
ove
rall
hydr
ogen
-bon
d ba
sici
ty,
Gso
lv =
free
ene
rgy
of s
olva
tion
in h
exad
ecan
e,i =
mol
ecul
ar s
truc
ture
Fuj
ita v
alue
, o
= m
olec
ular
str
uctu
re F
ujita
val
ue,
A1,
A2
= C
olla
nder
-typ
e co
effic
ient
, A
pol=
pol
ar s
urfa
ce a
rea,
B=
cor
rect
ion
fact
or,
BS
= b
asic
str
uctu
re,
DIF
F =
var
iabl
e de
pend
ant
on t
he n
umbe
r of
chl
orid
e at
oms
in t
he a
rom
atic
cyc
le,
DP
SA
= d
ynam
ic p
olar
sur
face
are
a,f e
= f
ract
ion
of e
ryth
rocy
tes
in b
lood
, f p
= f
ract
ion
of p
lasm
a in
blo
od,
I 3=
var
iabl
e de
pend
ant
on t
he p
rese
nce
of a
n am
ino
nitr
ogen
or
carb
oxyl
gro
up,
MV
= m
olec
ular
vol
ume,
MW
= m
olec
ular
wei
ght,
n acc
,sol
v=
num
ber
of s
olva
ted
hydr
ogen
-bon
d ac
cept
ors,
nC
L =
num
ber
of C
L fr
agm
ents
, nC
L 2 =
num
ber
of C
L 2fr
agm
ents
, nC
L 3=
num
ber
of C
L 3fr
agm
ents
, nH
3=
num
ber
of H
3fr
agm
ents
, N
N=
num
ber
of n
itrog
ens,
NO
= n
umbe
r of
oxy
gens
, N
PL
= v
aria
ble
depe
ndan
t on
the
num
ber
of c
hlor
ide
atom
s in
the
mol
ecul
e in
ort
ho p
ositi
on,
o G =
Gib
bs fr
ee e
nerg
y re
late
d to
the
solv
atio
n of
the
subs
tanc
ein
wat
er,
Pcy
h=
cyc
lohe
xane
:wat
er p
artit
ion
coef
ficie
nt,
Po:
w=
n-o
ctan
ol:w
ater
par
titio
n co
effic
ient
(or
veg
etab
le o
il:w
ater
), P
oct-
cyc
= o
ctan
ol-c
yclo
hexa
ne,
PS
A =
pol
ar s
urfa
ce a
rea,
R2
= E
xces
s m
olar
ref
ract
ion,
So
= s
olub
ility
in n
-oct
anol
(or
veg
etab
le o
il),
Sw
= s
olub
ility
in w
ater
, UN
S =
var
iabl
e de
pend
ant
on t
he n
umbe
r of
ato
ms
in t
he m
olec
ule
that
are
not
chl
orid
es, V
lb=
vol
ume
frac
tion
of li
pids
in b
lood
, Vlt
= v
olum
e fr
actio
n of
lipi
ds in
tis
sue,
Vm
= m
olar
volu
me,
Vnb
= v
olum
e fr
actio
n of
neu
tral
lip
ids
in b
lood
, V
ne=
vol
ume
frac
tion
of n
eutr
al l
ipid
s in
ery
thro
cyte
s, V
np=
vol
ume
frac
tion
of n
eutr
al l
ipid
s in
plas
ma,
Vnt
= v
olum
e fr
actio
n of
neu
tral
lip
ids
in t
issu
es,
Vpb
= v
olum
e fr
actio
n of
pho
spho
lipid
s in
blo
od,
Vpe
= v
olum
e fr
actio
n of
pho
spho
lipid
s in
eryt
hroc
ytes
, Vpp
= v
olum
e fr
actio
n of
pho
spho
lipid
s in
pla
sma,
Vpt
= v
olum
e fr
actio
n of
pho
spho
lipid
s in
tis
sues
, Vw
av=
vol
ume
of w
ater
nee
ded
in o
rder
to s
olub
ilize
the
subs
tanc
e, V
wb
= v
olum
e fr
actio
n of
wat
er in
blo
od, V
wb
= v
olum
e fr
actio
n of
wat
er in
blo
od, V
we
= v
olum
e fr
actio
n of
wat
er in
ery
thro
cyte
s,V
wp
= v
olum
e fr
actio
n of
wat
er i
n pl
asm
a, V
wt
= v
olum
e fr
actio
n of
wat
er i
n tis
sue,
Vw
t=
vol
ume
frac
tion
of w
ater
in
tissu
es,
and
Vx
= M
cGow
anch
arac
teris
tic v
olum
e.b
F =
fish
, H
= h
uman
, an
d R
= r
ats.
cC
FC
s =
chl
orofl
uoro
carb
ons,
HM
WO
Cs
= h
igh
mol
ecul
ar w
eigh
t org
anic
che
mic
als,
LM
WV
OC
s =
low
mol
ecul
ar w
eigh
t vol
atile
org
anic
che
mic
als,
PC
Bs
= p
olyc
hlor
obip
heny
ls,
and
VO
Cs
= v
olat
ile o
rgan
ic c
hem
ical
s.
502 ALTERNATIVE TOXICOLOGICAL METHODS
In Silico Approaches for Protein Binding
IN SILICO APPROACHES FOR PBPK MODELING 503
Tab
le 4
0.4
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
Pro
tein
Bin
din
g o
f C
hem
ical
sa
Ap
pro
ach
bS
pec
iesc
Ch
emic
al C
lass
Ref
eren
ce
QS
AR
s: L
FE
-typ
e eq
uat
ion
s
Hyd
rop
ho
bic
des
crip
tors
log
(1/f u
(pla
sma)
– 1)
= 0
.994
log
Po:
w–
1.10
HA
rom
atic
aci
dsTe
sta
et a
l. (2
000)
lo
g (1
/f u(p
lasm
a)–
1) =
0.9
94 lo
g P
o:w
– 1.
10H
Org
anic
aci
dsLa
znic
ek e
t al
. (19
87)
log
(1/K
a(pl
asm
a))
= –
3.91
log
Po:
w2+
13
log
Po:
w–
13.7
HC
epha
losp
orin
sTe
sta
et a
l. (2
000)
lo
g (1
– f
u(br
ain))
= 0
.36
log
Po:
w–
1.07
HB
arbi
tura
tes
Sey
del a
nd S
chap
er (
1982
) lo
g (1
– f
u(pl
asm
a))
= 0
.276
log
Po:
w+
1.2
HP
enic
illin
sS
eyde
l and
Sch
aper
(19
82)
log
(1 –
fu(
plas
ma))
= 0
.30
log
Po:
w–
1.03
HB
arbi
tura
tes
Sey
del a
nd S
chap
er (
1982
)lo
g (1
– f
u(pl
asm
a))
= 0
.33
log
Po:
w+
1.9
4H
Tetr
acyc
lines
Sey
del a
nd S
chap
er (
1982
)lo
g 1/
Ka(
albu
min
bin
ding
)=
–0.
85 lo
g P
o:w
+ 2
.73
HS
ulfa
pyrim
idin
es; s
ulfa
pyrid
ines
Sey
del a
nd S
chap
er (
1982
)lo
g 1/
Ka(
albu
min
bin
ding
)=
–0.
97 lo
g P
o:w
+ 3
.24
HS
ulfa
pyrid
ines
Sey
del a
nd S
chap
er (
1982
)lo
g 1/
Ka(
albu
min
bin
ding
)=
–0.
97 lo
g P
o:w
– 0.
70I
+ 3
.24
HS
ulfa
pyrim
idin
es; s
ulfa
pyrid
ines
Sey
del a
nd S
chap
er (
1982
)lo
g 1/
Ka(
albu
min
bin
ding
)=
–0.
99 lo
g P
o:w
+ 2
.49
HS
ulfa
pyrim
idin
esS
eyde
l and
Sch
aper
(19
82)
log
Kal
bum
in b
indi
ng=
0.8
9 lo
g P
o:w
+ 1
.47
HS
ulfa
pyrim
idin
esS
eyde
l and
Sch
aper
(19
82)
log
Kal
bum
in b
indi
ng=
1.1
5 lo
g P
o:w
+ 1
.23
HS
ulfo
nam
ides
Sey
del a
nd S
chap
er (
1982
)lo
gK
albu
min
bin
ding
= 1
.23
log
Po:
w–
0.05
6H
Ste
roid
bis
guan
ylhy
draz
ones
Sey
del a
nd S
chap
er (
1982
)lo
gK
albu
min
bin
ding
= 1
.32
log
Po:
w+
0.3
7H
Pen
icill
ins
Sey
del a
nd S
chap
er (
1982
)lo
gK
albu
min
bin
ding
= 1
.39
log
Po:
w–
1.19
HC
arde
nolid
esS
eyde
l and
Sch
aper
(19
82)
log
Kal
bum
in b
indi
ng=
1.6
5 lo
g P
o:w
– 2.
57H
Ste
roid
hor
mon
esS
eyde
l and
Sch
aper
(19
82)
log
Kpl
asm
a pr
otei
n bi
ndin
g=
0.7
3R
mui
+ 1
.46
HS
ulfa
pyrid
ines
Sey
del a
nd S
chap
er (
1982
)lo
gK
a(bl
ood
prot
ein
bind
ing)
= 0
.504
– 0.
665
HP
enic
illin
sB
ird a
nd M
arsh
all (
1967
)lo
g (1
/f u(p
lasm
a)–
1) =
1.0
11 lo
g P
o:w
– 1.
745
RO
rgan
ic a
cids
Lazn
icek
et
al. (
1987
) lo
g (1
– f
u)/f u
(adi
pose
)=
log
0.75
0 +
0.9
36 lo
g P
o:w
RB
arbi
turic
aci
ds
Nes
tero
v et
al.
(199
8)lo
g (1
– f
u)/f u
(bra
in)=
log
0.07
3 +
0.8
60 lo
g P
o:w
RB
arbi
turic
aci
ds
Nes
tero
v et
al.
(199
8)(c
ontin
ued)
504 ALTERNATIVE TOXICOLOGICAL METHODS
Tab
le 4
0.4
(co
nti
nu
ed)
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
Pro
tein
Bin
din
g o
f C
hem
ical
sa
Ap
pro
ach
bS
pec
iesc
Ch
emic
al C
lass
Ref
eren
ce
log
(1 –
fu)
/f u(g
ut)=
log
0.09
9 +
0.8
24 lo
g P
o:w
RB
arbi
turic
aci
ds
Nes
tero
v et
al.
(199
8)lo
g (1
– f
u)/f u
(hea
rt)=
log
0.13
5 +
0.7
80 lo
g P
o:w
RB
arbi
turic
aci
ds
Nes
tero
v et
al.
(199
8)lo
g (1
– f
u)/f u
(kid
ney)
= lo
g 0.
676
+ 0
.619
log
Po:
wR
Bar
bitu
ric a
cids
N
este
rov
et a
l. (1
998)
log
(1 –
fu)
/f u(li
ver)
= lo
g 1.
775
+ 0
.504
log
Po:
wR
Bar
bitu
ric a
cids
N
este
rov
et a
l. (1
998)
log
(1 –
fu)
/f u(lu
ng)=
log
0.16
4 +
0.8
41 lo
g P
o:w
RB
arbi
turic
aci
ds
Nes
tero
v et
al.
(199
8)
log
(1 –
fu)
/f u(m
uscl
e)=
log
0.08
0 +
0.8
35 lo
g P
o:w
RB
arbi
turic
aci
ds
Nes
tero
v et
al.
(199
8)lo
g (1
– f
u)/f u
(pan
crea
s)=
log
0.02
2 +
1.0
95 lo
g P
o:w
RB
arbi
turic
aci
ds
Nes
tero
v et
al.
(199
8)
log
(1 –
fu)
/f u(p
lasm
a)=
log
0.01
6 +
0.9
75 lo
g P
o:w
RB
arbi
turic
aci
ds
Nes
tero
v et
al.
(199
8)lo
g (1
– f
u)/f u
(red
blo
od c
ell)
= lo
g 0.
178
+ 0
.677
log
Po:
wR
Bar
bitu
ric a
cids
N
este
rov
et a
l. (1
998)
log
(1 –
fu)
/f u(s
kin)
= lo
g 0.
271
+ 0
.736
log
Po:
wR
Bar
bitu
ric a
cids
N
este
rov
et a
l. (1
998)
log
(1 –
fu)
/f u(s
plee
n)=
log
0.12
6 +
0.8
41 lo
g P
o:w
RB
arbi
turic
aci
ds
Nes
tero
v et
al.
(199
8)lo
g (1
– f
u)/f u
(sto
mac
h)=
log
0.05
8 +
0.9
39 lo
g P
o:w
RB
arbi
turic
aci
ds
Nes
tero
v et
al.
(199
8)lo
g (1
– f
u)/f u
(tes
tis)=
log
0.12
0 +
0.7
47 lo
g P
o:w
RB
arbi
turic
aci
ds
Nes
tero
v et
al.
(199
8)lo
gK
plas
ma
prot
ein
bind
ing
= 0
.33
Rm
ui–
0.53
I +
4.0
8R
Sul
fapy
ridin
esS
eyde
l and
Sch
aper
(19
82)
log
(1/f u
(pla
sma)
– 1)
= 1
.016
log
Po:
w–
1.27
5R
bO
rgan
ic a
cids
Lazn
icek
et
al. (
1987
)
aK
= p
rote
in a
ffini
ty c
onst
ant
(Fre
undl
ich
isot
herm
), K
a=
pro
tein
affi
nity
con
stan
t (S
catc
hard
isot
herm
) an
d f u
= u
nbou
nd f
ract
ion.
bP
o:w
= o
ctan
ol:w
ater
par
titio
n co
effic
ient
, =
mol
ecul
ar h
ydro
phob
icity
con
stan
t, I
= fa
mily
indi
cato
r va
riabl
e,
Rm
ui=
var
iabl
e de
pend
ant
on t
here
sist
ance
con
stan
t du
e to
diff
usio
n of
the
non
ioni
zed
form
in t
he li
pid
mem
bran
e.c
H =
hum
ans,
Rb
= r
abbi
t, an
d R
= r
at.
IN SILICO APPROACHES FOR PBPK MODELING 505
506 ALTERNATIVE TOXICOLOGICAL METHODS
In Silico Approaches for Clearance Constants
IN SILICO APPROACHES FOR PBPK MODELING 507
Tab
le 4
0.5
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
Cle
aran
ces
(CL
) o
f C
hem
ical
s
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
QS
AR
s: L
FE
-typ
e eq
uat
ion
s
Ele
ctro
stat
ic d
escr
ipto
rs
log
CL (
hepa
tic)=
0.6
4 lo
g P
o:w
– 0.
98IP
+ 9
.33
HB
enzo
diaz
epin
esLe
wis
(20
00)
log
CL (
hepa
tic)=
0.0
55E
nerg
y –
0.95
IP–
0.53
HB
D +
10.
63H
Ben
zodi
azep
ines
Lew
is (
2000
)lo
g C
L (he
patic
)=
0.0
67E
nerg
y –
1.01
IP–
0.34
HB
D –
0.4
3E
+ 1
4.66
HB
enzo
diaz
epin
esLe
wis
(20
00)
log
CL (
hepa
tic)=
0.0
94E
nerg
y –
1.18
IP–
0.74
E+
18.
65H
Ben
zodi
azep
ines
Lew
is (
2000
)lo
g C
L (he
patic
)=
0.6
5 lo
g P
o:w
– 0.
40IP
– 0.
37H
BD
+ 0
.002
5Hf
+ 3
.63
HB
enzo
diaz
epin
esLe
wis
(20
00)
Met
abol
ic r
atio
= 2
.72Q
6+
1.9
6EH
+ 0
.014
SN
+ 6
.43
RD
ichl
orob
iphe
nyls
Lew
is a
nd D
icki
ns (
2002
)
Ste
ric
des
crip
tors
1/lo
g C
L (in
trin
sic;
hep
atic
)=
3.5
8 –
0.05
8S_s
Cl –
0.5
7S_a
aO –
0.4
7Sha
dow
Z le
ngth
–
0.75
CIC
HC
omm
erci
ally
ava
ilabl
e dr
ugs
Eki
ns a
nd O
bach
(20
00)
1/lo
g C
L (in
trin
sic;
hep
atic
)=
–3.
11 –
0.1
0Dip
ole
– m
ag +
13.
25Ju
rs –
RP
CG
+ 0
.57J
urs
– R
PC
S +
0.0
0013
Apo
l
HC
omm
erci
ally
ava
ilabl
e dr
ugs
Eki
ns a
nd O
bach
(20
00)
CL (
intr
insi
c; h
epat
ic)=
25S
teric
+ 4
4Ele
ctro
stat
ic +
20L
UM
O +
11H
INT
RH
aloa
lkan
esW
alle
r et
al.
(199
6)
Hyd
rop
ho
bic
des
crip
tors
CL
(intr
insi
c; h
epat
ic)=
0.0
555
Po:
w1.
05H
Bas
ic d
rugs
Yoko
gaw
a et
al.
(200
2)
log
CL (
rena
l)=
–0.
24(lo
g P
o:w)2
– 0.
04 lo
g P
o:w
+ 0
.58
HP
robe
neci
d an
alog
sS
eyde
l and
Sch
aper
(19
82)
log
CL (
rena
l)=
–lo
g(0.
35 +
0.0
13)
HP
robe
neci
d an
alog
sS
eyde
l and
Sch
aper
(19
82)
log
CL (
rena
l)=
–0.
5 lo
g P
o:w
+ 3
HN
SA
IDS
mith
et
al. (
1996
)lo
g C
L (re
nal)
= –
0.5
log
Po:
w+
13
Hß
-blo
cker
sS
mith
et
al. (
1996
)lo
g C
L (he
patic
)=
–0.
54R
mui
– 0.
51R
Sul
fona
mid
esS
eyde
l and
Sch
aper
(19
82)
(con
tinue
d)
508 ALTERNATIVE TOXICOLOGICAL METHODS
Tab
le 4
0.5
(co
nti
nu
ed)
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
Cle
aran
ces
(CL
) o
f C
hem
ical
s
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
log
CL (
rena
l)=
–0.
41R
mui
– 0.
80R
Sul
fona
mid
esS
eyde
l and
Sch
aper
(19
82)
log
CL (
rena
l)=
–0.
51 lo
g P
o:w
– 0.
33R
Sul
fapy
ridin
esYa
mag
uchi
et
al. (
1996
) lo
g C
L (re
nal)
= –
log
[0.0
48 +
6.9
8 10
–4(1
0)1.
394 ]
RX
ylid
ines
Sey
del a
nd S
chap
er (
1982
)lo
g C
L (to
tal)
= –
0.74
Rm
ui+
0.2
2pK
a–
1.73
RS
ulfo
nam
ides
Sey
del a
nd S
chap
er (
1982
)lo
g E
(hep
atic
)=
0.0
45 lo
g P
o:w
– 0.
32R
HM
WO
Cs
Yam
aguc
hi e
t al
. (19
96)
CL (
intr
insi
c; h
epat
ic)=
3.8
28R
bB
asic
dru
gsIs
hiza
ki e
t al
. (19
97)
CL (
intr
insi
c; h
epat
ic)=
0.0
875
Rb
Bas
ic d
rugs
Ishi
zaki
et
al. (
1997
)
CL (
intr
insi
c; h
epat
ic)=
0.2
48R
bB
asic
dru
gsIs
hiza
ki e
t al
. (19
97)
QS
AR
S:
Fre
e–W
ilso
n-t
ype
equ
atio
ns
log
CL (
rena
l)=
0.4
17R
2(C
H3)
– 0
.744
R1(
OC
3H7)
+ 1
.33
RX
ylid
ines
Sey
del a
nd S
chap
er (
1982
) lo
g C
L (to
tal)
= 0
.49R
2(C
H3)
+ 0
.57
R2(
C2H
5) +
0.2
5R1(
OC
3H7)
+ 1
.76
RX
ylid
ines
Sey
del a
nd S
chap
er (
1982
)
aE
= v
aria
ble
rela
ted
to m
olec
ular
orb
itals
, R
mui
= v
aria
ble
depe
ndan
t on
the
res
ista
nce
cons
tant
due
to
diffu
sion
of
the
noni
oniz
ed f
orm
in t
he li
pid
mem
bran
e,
= m
olec
ular
hyd
roph
obic
ity c
onst
ant,
Apo
l=
pol
ar s
urfa
ce a
rea,
CIC
= c
ompl
emen
tary
info
rmat
ion
cont
ent,
Dip
ole-
mag
= d
ipol
e m
omen
t,E
H=
HO
MO
ene
rgy,
Ele
ctro
stat
ic =
Cou
lom
bic
inte
ract
ion
ener
gy,
Ene
rgy
= m
inim
um in
tern
al e
nerg
y, H
BD
= p
oten
tial h
ydro
gen
bond
don
or a
tom
s in
the
mol
ecul
e, H
f =
ent
halp
y of
form
atio
n, H
INT
= h
ydro
phob
ic fi
eld
ener
gy,
IP =
ioni
zatio
n po
tent
ial,
Jurs
-RP
CG
= r
elat
ive
posi
tive
char
ge,
Jurs
-RP
CS
= r
elat
ive
posi
tive
char
ge s
urfa
ce a
rea,
LU
MO
= l
owes
t un
occu
pied
mol
ecul
ar o
rbita
l en
ergy
, P
o:w
= n
-oct
anol
:wat
er p
artit
ion
coef
ficie
nt (
or v
eget
able
oil:w
ater
), P
o:w
, ap
p=
app
aren
t oc
tano
l:wat
er p
artit
ion
coef
ficie
nt,
Q6
= n
et a
tom
ic c
harg
e on
car
bon
atom
at
biph
enyl
rin
g po
sitio
n, R
2(C
H3)
= m
ethy
lfr
agm
ent
at R
2po
sitio
n,R
2(C
2H5)
= e
thyl
fra
gmen
t at
R2
posi
tion,
R1(
OC
3H7)
= p
ropy
l eth
er f
ragm
ent
at R
1po
sitio
n, S
_aaO
= E
-sta
te in
dice
s fo
r ox
ygen
atom
s w
ith tw
o ar
omat
ic b
onds
, S_s
Cl =
E-s
tate
indi
ce fo
r ch
lorin
e at
oms
with
a s
ingl
e bo
nd, S
hado
w Z
leng
th =
leng
th o
f the
mol
ecul
e in
Zdi
men
sion
,S
N=
tot
al n
ucle
ophi
llic
supe
rdel
ocal
izab
ility
, an
d S
teric
= V
an d
er W
aals
inte
ract
ion
ener
gy.
bF
= fi
sh,
H =
hum
an,
and
R =
rat
s.c
CF
Cs
= c
hlor
ofluo
roca
rbon
s, H
MW
OC
s =
hig
h m
olec
ular
wei
ght
orga
nic
chem
ical
s, L
MW
VO
Cs
= lo
w m
olec
ular
wei
ght
vola
tile
orga
nic
chem
ical
s, a
ndV
OC
s =
vol
atile
org
anic
che
mic
als.
IN SILICO APPROACHES FOR PBPK MODELING 509
Tab
le 4
0.6
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
Rea
ctio
n R
ates
of
Ch
emic
alsa
Ap
pro
ach
bS
pec
iesc
Ch
emic
al C
lass
dR
efer
ence
QS
AR
s: L
FE
-typ
e eq
uat
ion
s
Ele
ctro
stat
ic d
escr
ipto
rs
log
V(o
xida
tion)
= 0
.894
– 0
.111
diam
eter
– 0
.007
EH
Nitr
iles
Lew
is a
nd D
icki
ns (
2002
) lo
gk c
at (
oxid
atio
n)=
19.
97 –
0.0
24H
– 0.
95IP
HTo
luen
esLe
wis
and
Dic
kins
(20
02)
log
V(o
xida
tion)
= 2
6.90
– 2
.58I
PH
Hal
otha
nes
Lew
is a
nd D
icki
ns (
2002
) lo
gk c
at (
oxid
atio
n)=
0.0
24V
ol –
0.2
3–
1.14
HB
arbi
tura
tes
Lew
is a
nd D
icki
ns (
2002
) lo
gk c
at (
oxid
atio
n)=
1.3
3 –
0.15
HA
nilin
esLe
wis
and
Dic
kins
(20
02)
log
k cat
(de
met
hyla
tion)
= –
0.68
–
+ 1
.06
RX
-C6H
4N(C
H3)
2H
ansc
h an
d Le
o (1
995)
Ste
ric
des
crip
tors
log
k cat/K
m(O
xida
tion)
= 0
.034
7SA
– 2
.29
E+
1.9
2H
Tolu
enes
Lew
is a
nd D
icki
ns (
2002
) lo
gV
max
(n-
dem
ethy
latio
n)=
0.1
8 Le
ngth
– 1
.94
HE
thyl
amin
esLe
wis
(20
01)
log
Vm
ax (
n-de
met
hyla
tion)
= 3
.50
Leng
th –
0.1
3 Le
ngth
2–
23.9
HE
thyl
amin
esLe
wis
(20
01)
log
V(o
xida
tion)
= 2
.486
1 –
0.13
64N
PL
* N
SID
E +
0.5
694U
NS
– 0
.243
3NO
M *
NM
C
+ 0
.001
227M
W *
NU
NS
TOT
+ 0
.824
2IN
D –
1.1
493M
OD
RP
CB
sP
arha
m a
nd P
ortie
r (1
998)
log
Vm
ax (
oxid
atio
n)=
–1.
6764
+ 0
.424
3–
0.13
4 +
1.6
22R
Hal
oalk
anes
Gar
gas
et a
l. (1
988)
V(n
-dem
ethy
latio
n)=
0.0
05S
A–
0.52
RA
min
esLe
wis
and
Dic
kins
(20
02)
V(n
-dem
ethy
latio
n)=
0.0
38S
A–
0.00
001S
A2
– 25
.64
RA
min
esLe
wis
and
Dic
kins
(20
02)
Hyd
rop
ho
bic
des
crip
tors
log
k cat/K
m(d
emet
hyla
tion)
= 0
.53
log
Po:
w+
3.4
7R
X-C
6H4N
(CH
3)2
Han
sch
and
Leo
(199
5)
log
V=
0.5
5 lo
g P
o:w
RB
arbi
tura
tes
Han
sch
and
Leo
(199
5)(c
ontin
ued)
510 ALTERNATIVE TOXICOLOGICAL METHODS
Tab
le 4
0.6
(co
nti
nu
ed)
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
Rea
ctio
n R
ates
of
Ch
emic
alsa
Ap
pro
ach
bS
pec
iesc
Ch
emic
al C
lass
dR
efer
ence
QS
AR
S:
Fre
e–W
ilso
n-t
ype
equ
atio
ns
Vm
axc
= B
S(C
-C)(5
1.6)
+ n
H3(
14.6
) +
nC
L(–4
.84)
+ n
CL 2
(10.
2) +
nC
L 3(–
16.9
)R
, H
Chl
oroe
than
esF
ouch
écou
rt a
nd K
rishn
an
(200
0)
ak c
at=
cat
alyt
ic r
ate,
Km
= e
nzym
e af
finity
con
stan
t, V
= m
etab
olic
rat
e, V
max
= m
axim
al v
eloc
ity o
f m
etab
olis
m,
and
Vm
axc
= b
ody
wei
ght
norm
aliz
edm
axim
al v
eloc
ity o
f m
etab
olis
m.
bE
= L
UM
O e
nerg
y –
HO
MO
ene
rgy,
H
= h
ydro
gen
abst
ract
ion
ener
gy,
= d
ipol
ar m
omen
t of
the
mol
ecul
e, 4
,3
, =
con
nect
ivity
indi
ces,
BS
= b
asic
str
uctu
re,
diam
eter
= d
iam
eter
of
the
mol
ecul
e, I
ND
= v
aria
ble
depe
ndan
t on
exp
erim
enta
l da
ta u
sed,
IP
= i
oniz
atio
n po
tent
ial,
Leng
th =
leng
th o
f the
mol
ecul
e, M
OD
= v
aria
ble
depe
ndan
t on
expe
rimen
tal d
ata
used
, MW
= m
olec
ular
wei
ght,
nCL
= n
umbe
r of
CL
frag
men
ts,
nCL 2
= n
umbe
rof
CL 2
frag
men
ts,
nCL 3
= n
umbe
r of
CL 3
frag
men
ts,
nH3
= n
umbe
r of
H3
frag
men
ts,
NM
C =
num
ber
of m
eta
chlo
rines
, N
OM
= n
umbe
r of
adj
acen
tun
subs
titut
ed o
rtho
-met
a ca
rbon
pai
rs, N
PL
= v
aria
ble
depe
ndan
t on
the
num
ber
of c
hlor
ide
atom
s in
the
mol
ecul
e in
ort
ho p
ositi
on, N
SID
E =
var
iabl
ede
pend
ant
on t
he n
umbe
r of
chl
orid
e at
oms
in t
he m
olec
ule
in m
eta
posi
tion,
NU
NS
TOT
= v
aria
ble
depe
ndan
t on
the
num
ber
of c
hlor
ide
atom
s in
the
mol
ecul
e, P
o:w
= n
-oct
anol
:wat
er p
artit
ion
coef
ficie
nt (
or v
eget
able
oil:
wat
er),
SA
= s
urfa
ce a
rea,
UN
S =
var
iabl
e de
pend
ant o
n th
e nu
mbe
r of
ato
ms
in t
he m
olec
ule
that
are
not
chl
orid
e, V
ol =
vol
ume
of t
he m
olec
ule,
and
–
= H
amm
et c
onst
ant.
cF
= fi
sh,
H =
hum
an,
and
R =
rat
s.d
PC
Bs
= p
olyc
hlor
obip
heny
ls.
IN SILICO APPROACHES FOR PBPK MODELING 511
Tab
le 4
0.7
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e M
ich
aelis
–Men
ten
Affi
nit
y C
on
stan
t (K
m)
of
Ch
emic
als
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
QS
AR
s: L
FE
-typ
e eq
uat
ion
s
Ele
ctro
stat
ic d
escr
ipto
rs
log
1/K
m(d
emet
hyla
tion)
= 0
.46
log
Po:
w+
0.6
3–
+ 2
.62
HX
-C6H
4N(C
H3)
2H
ansc
h an
d Le
o (1
995)
lo
g 1/
Km
(sul
fatio
n)=
0.9
2 lo
g P
o:w
– 1.
48M
R4
– 0.
64M
R3
+ 1
.04M
R2
+ 0
.67
–+
4.0
1H
Phe
nols
Han
sch
and
Leo
(199
5)lo
gK
m(a
cety
latio
n)=
–0.
42 lo
g P
ui+
0.1
4pK
a–
2.89
HS
ulfo
nam
ides
Sey
del a
nd S
chap
er (
1982
) K
m(o
xida
tion)
= [
(ia
d() i)
/|IP
i–
b|]
+ c
RA
lken
esC
sana
dy e
t al
. (19
95)
log
Km
(ace
tyla
tion)
= 0
.17p
Ka
– 0.
69R
Sul
fona
mid
esS
eyde
l and
Sch
aper
(19
82)
log
Km
(ace
tyla
tion)
= –
0.42
Rm
u:i+
0.1
5pK
a–
1.39
RS
ulfo
nam
ides
Sey
del a
nd S
chap
er (
1982
)lo
gK
m(a
cety
latio
n)=
0.0
7pK
a+
0.3
1 lo
g P
o:w
– 0.
33R
bS
ulfo
nam
ides
Sey
del a
nd S
chap
er (
1982
)
Hyd
rop
ho
bic
des
crip
tors
log
1/K
m(o
xida
tion)
= 1
.39
log
Po:
w–
0.22
log
Po:
w2
– 0.
50H
Bar
bitu
rate
sLe
wis
and
Dic
kins
(20
02)
log
1/K
m(s
ulfa
tion)
= 2
.93F
2+
1.1
62
+ 0
.91
3+
0.8
2MR
2–
0.59
IO
H+
1.2
9IE
T+
2.5
9H
Phe
nols
Han
sch
and
Leo
(199
5)
–log
Km
(oxi
datio
n)=
43.
27 –
4.0
3E
– 0.
60 lo
g P
o:w
HTo
luen
esLe
wis
and
Dic
kins
(20
02)
log
1/K
m(g
luco
roni
datio
n)=
0.8
3 lo
g P
o:w
+ 1
.37
RP
heno
lsH
ansc
h an
d Le
o (1
995)
log
1/K
m(h
ydro
lysi
s)=
0.0
56Z
1 H2O
+ 0
.051
Z2 H
2O+
0.0
26Z
3 H2O
+ 0
.04Z
4 H2O
+ 4
.616
RP
heny
lhip
pura
tes
Kim
(19
93)
log
1/K
m(h
ydro
lysi
s)=
0.0
66Z
1 H2O
+ 4
.259
RP
heny
lhip
pura
tes
Kim
(19
93)
log
1/K
m(h
ydro
lysi
s)=
0.4
4+
4.0
8R
Phe
nylh
ippu
rate
sK
im (
1993
)lo
g 1/
Km
(hyd
roly
sis)
= 0
.40
+ 4
.40
RP
heny
lhip
pura
tes
Kim
(19
93)
log
1/K
m(N
AD
P-o
xida
tion)
= 0
.69
log
Po:
w+
2.9
0R
Dru
gsS
eyde
l and
Sch
aper
(19
82)
log
Km
(n-d
emet
hyla
tion)
= –
0.55
log
Po:
w+
2.6
7R
Mor
phin
esH
ansc
h an
d Le
o (1
995)
log
Km
(oxi
datio
n)=
0.6
1 lo
g P
o:w
+ 2
.23
RC
arba
mat
esH
ansc
h an
d Le
o (1
995)
log
Km
(oxi
datio
n)=
1.0
2 lo
g P
o:w
+ 2
.98
RP
yraz
oles
Han
sch
and
Leo
(199
5)(c
ontin
ued)
512 ALTERNATIVE TOXICOLOGICAL METHODS
Tab
le 4
0.7
(co
nti
nu
ed)
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e M
ich
aelis
–Men
ten
Affi
nit
y C
on
stan
t (K
m)
of
Ch
emic
als
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
log
Km
(oxi
datio
n)=
1.0
5 lo
g P
o:w
+ 1
.22
R4-
nitr
ophe
nyl a
lkyl
et
hers
Han
sch
and
Leo
(199
5)
log
Km
(oxi
datio
n)=
0.7
9 lo
g P
o:w
+ 1
.46
RA
lkyl
benz
enes
Han
sch
and
Leo
(199
5)lo
gK
m(o
xida
tion)
= 1
.04
log
Po:
w+
1.1
0R
bTo
luen
esH
ansc
h an
d Le
o (1
995)
QS
AR
s: F
ree–
Wils
on
-typ
e eq
uat
ion
s
Km
= B
S(C
-C)(3
.8)
+ n
H3(
–2.5
9) +
nC
L(–0
.37)
+ n
CL 2
(0.7
9) +
nC
L 3(0
.19)
R,
HC
hlor
oeth
anes
Fou
chéc
ourt
and
Kris
hnan
(2
000)
aE
= L
UM
O e
nerg
y –
HO
MO
ene
rgy,
–
= H
amm
et c
onst
ant,
= d
ipol
ar m
omen
t of
the
mol
ecul
e,
,2,
3=
mol
ecul
ar h
ydro
phob
icity
con
stan
ts,
Rm
ui
= v
aria
ble
depe
ndan
t on
the
resi
stan
ce c
onst
ant d
ue to
diff
usio
n of
the
noni
oniz
ed fo
rm in
the
lipid
mem
bran
e, a
= o
rbita
l ava
ilabi
lity,
b=
LU
MO
ene
rgy,
BS
= b
asic
str
uctu
re,
c=
var
iabl
e de
pend
ant
on t
he m
olec
ular
siz
e, d
()
= n
orm
aliz
ed e
lect
ron
dens
ity,
F2
= v
aria
ble
depe
ndan
t on
the
ele
ctric
al fi
eld
indu
ced
by o
rtho
pos
ition
ed a
tom
s, l
OH
= v
aria
ble
depe
ndan
t on
the
num
ber
of
OH
gro
ups
in t
he m
olec
ule,
lE
T=
var
iabl
e de
pend
ant
on t
he f
amily
of t
he s
ubst
ance
, IP
= io
niza
tion
pote
ntia
l, M
R2,
3,4
= m
olar
ref
ract
ivity
indi
ces,
nC
L =
num
ber
of C
L fr
agm
ents
, nC
L 2=
num
ber
of C
L 2fr
agm
ents
, nC
L 3=
num
ber
of C
L 3fr
agm
ents
, nH
3=
num
ber
of H
3fr
agm
ents
, pK
a=
log
diss
ocia
tion
cons
tant
of a
n ac
id in
wat
er, P
o:w
= n
-oct
anol
:wat
er p
artit
ion
coef
ficie
nt(o
r ve
geta
ble
oil:w
ater
), P
ui=
n-o
ctan
ol:w
ater
par
titio
n co
effic
ient
for
the
non
ioni
zed
form
, an
d Z
1, 2
, 3,
4H
2O=
var
iabl
es c
orre
spon
ding
to
the
pote
ntia
len
ergy
for
the
inte
ract
ion
betw
een
the
mol
ecul
e an
d w
ater
.b
F =
fish
, H
= h
uman
, an
d R
= r
ats.
cP
CB
s =
pol
ychl
orob
iphe
nyls
.
IN SILICO APPROACHES FOR PBPK MODELING 513
In Silico Approaches for Skin Permeability Constants
514 ALTERNATIVE TOXICOLOGICAL METHODS
Tab
le 4
0.8
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e S
kin
Per
mea
bili
ty C
oef
fici
ent
(Kp)
of
Ch
emic
als
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
cR
efer
ence
QS
AR
s: L
FE
-typ
e eq
uat
ion
s
Ele
ctro
stat
ic d
escr
ipto
rs
log
Kp
= –
0.62
6C
a –
23.8
(Q+
)/0.
289S
sssC
H –
0.0
357S
sOH
–
0.48
2IB
+ 0
.405
BR
+ 0
.834
HLM
WV
OC
s; H
MW
OC
sM
oss
et a
l. (2
002)
log
Kp
= 0
.44R
2–
0.49
– 1
.48
– 3.
44+
1.9
4Vx
– 5.
13H
LMW
VO
Cs;
HM
WO
Cs
Mos
s et
al.
(200
2)
log
Kp
= –
0.59
– 0
.63
– 3.
48+
1.7
9Vx
– 5.
05H
LMW
VO
Cs;
HM
WO
Cs
Mos
s et
al.
(200
2)
log
Kp
= –
5.33
– 0
.62
– 0
.38
– 3.
34+
1.8
5Vx
HA
lcoh
ols,
ste
roid
sG
hafo
uria
n an
d F
oola
di (
2001
)
Ste
ric
des
crip
tors
Kp
= (
b 1+
0.0
025/
(b2
+ b
3+
))–1
MW
b5H
LMW
VO
Cs;
HM
WO
Cs
Mos
s et
al.
(200
2)
Kp
= (
b 1+
b 2P
o:w)e
(b3M
W)
HLM
WV
OC
s; H
MW
OC
sM
oss
et a
l. (2
002)
log
Kp
= –
5.14
– 0
.47
Ca
+ 0
.23
Cd
+ 0
.038
Pol
HA
lcoh
ols,
ste
roid
sR
aevs
ky a
nd S
chap
er (
1998
)lo
gK
p=
–6.
14 –
0.4
2C
a +
0.2
3C
d +
0.2
1L –
0.1
1WH
Alc
ohol
s, s
tero
ids
Rae
vsky
and
Sch
aper
(19
98)
log
Kp
= –
7.29
+ 0
.15P
olH
Alc
ohol
sR
aevs
ky a
nd S
chap
er (
1998
)lo
gK
p=
b 1+
b 2lo
gP
o:w
+b 3
MW
0.5
HLM
WV
OC
s; H
MW
OC
sM
oss
et a
l. (2
002)
log
Kp
= –
0.42
8–
4.80
+ 2
8.06
HH
ydro
cort
icon
e es
ters
Gha
four
ian
and
Foo
ladi
(20
01)
log
Kp
= 0
.652
log
Po:
w–
0.00
603M
W –
0.6
23A
BS
Qon
– 0
.313
Sss
sCH
– 2
.3H
Der
mal
dru
gs; L
MW
VO
Cs;
H
MW
OC
sP
atel
et
al. (
2002
)
log
Kp
= 0
.77
log
Po:
w–
0.01
03M
W –
2.3
3H
LMW
VO
Cs;
HM
WO
Cs
Mos
s et
al.
(200
2)
log
Kp
= –
0.78
6OT
+ 0
.252
2–
1.61
7 –
5.7
67H
Alc
ohol
s, s
tero
ids
Gha
four
ian
and
Foo
ladi
(20
01)
log
Kp
= 0
.82
log
Po:
w–
0.00
93V
m–
0.03
9MP
t–
2.36
HS
tero
ids
Mos
s et
al.
(200
2)lo
gK
p=
0.8
4 lo
g P
o:w
– 0.
07(lo
g P
o:w)2
– 0.
27H
b –
1.84
log
MW
+ 4
.39
HLM
WV
OC
s; H
MW
OC
sM
oss
et a
l. (2
002)
IN SILICO APPROACHES FOR PBPK MODELING 515
log
Kp
= 2
8.4q
–+
0.0
18V
m+
2.8
24H
Bar
bitu
rate
s; I
soqu
inol
ine;
S
alic
yclic
aci
dG
hafo
uria
n an
d F
oola
di (
2001
)
log
Kp
= 3
.99
log
TA +
4.5
3 –
0.7
62O
T –
11.
364
HA
lcoh
ols,
Ste
roid
sG
hafo
uria
n an
d F
oola
di (
2001
)
Hyd
rop
ho
bic
des
crip
tors
Kp
= 1
.17
10–7
+ 2
.73
10–8
HP
harm
aceu
tical
sM
oss
et a
l. (2
002)
Kp
=b 1
(/(
b 3+
))H
HM
WO
Cs
Mos
s et
al.
(200
2)
log
Kp
= –
0.20
7 lo
g +
1.4
9 lo
g P
o:w
– 5.
42H
Ste
roid
sS
eyde
l and
Sch
aper
(19
82)
log
Kp
= –
0.37
log
+ 2
.39
log
Po:
w–
8.71
HP
heno
lsTe
sta
et a
l. (2
000)
log
Kp
= 0
.544
log
Po:
w–
2.88
HA
lipha
tic a
lcoh
ols
Sey
del a
nd S
chap
er (
1982
)lo
gK
p=
0.8
0 lo
g P
o:w
– 8.
883
HH
ydro
cort
icon
e es
ters
Gha
four
ian
and
Foo
ladi
(20
01)
log
Kp
= –
1.46
lo
gP
o:w
+ 0
.29
log
Po:
w–
3.75
HA
lcoh
ols,
ste
roid
sTe
sta
et a
l. (2
000)
lo
gK
p=
–4.
36 –
0.3
8C
a +
0.2
4C
dH
Ste
roid
sR
aevs
ky a
nd S
chap
er (
1998
)
Mec
han
isti
cally
bas
ed e
qu
atio
ns
Kp
=
(Pvo
:w*0.
028D
l/0.0
340)
+ (
Pp:
w*0.
88D
p/0.0
018)
HA
cids
; Alc
ohol
s; H
ydro
carb
ons
Pou
lin a
nd K
rishn
an (
2001
)
a=
sol
ubili
ty p
aram
eter
, =
dip
olar
ity/p
olar
izab
ility
, =
ove
rall
hydr
ogen
-bon
d ac
idity
, =
ove
rall
hydr
ogen
-bon
d ba
sici
ty,
Ca
= h
ydro
gen
bond
acce
ptor
fre
e en
ergy
in t
he m
olec
ule,
C
d =
hyd
roge
n bo
nd d
onor
in t
he m
olec
ule,
2=
mol
ecul
ar s
hape
inde
x,
= c
onne
ctiv
ity in
dice
s, A
BS
Qon
= s
um o
f ab
solu
te c
harg
es o
n ox
ygen
and
nitr
ogen
ato
ms,
b1,
b 2,
b 3,
b 4,
b 5=
reg
ress
ion
coef
ficie
nts
with
out
any
assi
gned
rol
e, B
R=
num
ber
of r
otat
able
bond
s, D
l=
coe
ffici
ent
for
diffu
sion
int
o th
e lip
id f
ract
ion
of s
trat
um c
orne
um,
Dp
= c
oeffi
cien
t fo
r di
ffusi
on i
nto
the
prot
ein
frac
tion
of s
trat
um c
orne
um,
Hb
= n
umbe
r of
hyd
roge
n bo
nds
form
ed b
y th
e su
bsta
nce,
IB
= B
alab
an in
dex,
L=
mol
ecul
ar le
ngth
, M
Pt=
mel
ting
poin
t, M
W =
mol
ecul
ar w
eigh
t, O
T=
num
ber
of h
ydro
gen
bond
ing
hete
roat
oms,
Po:
w=
n-o
ctan
ol:w
ater
par
titio
n co
effic
ient
(or
veg
etab
le o
il:w
ater
), P
ol =
des
crib
es b
ulk
or v
olum
e re
late
def
fect
s, P
p:w
= p
rote
in:w
ater
par
titio
n co
effic
ient
for
str
atum
cor
neum
, P
vo:w
= v
eget
able
oil:
wat
er p
artit
ion
coef
ficie
nt,
q–=
the
mos
t ne
gativ
e ch
arge
on
the
hydr
ogen
bon
d ac
cept
ing
hete
roat
oms,
Q+/
= p
ositi
ve c
harg
e pe
r un
it vo
lum
e,
= s
um o
f at
omic
cha
rges
on
hydr
ogen
bon
ding
het
eroa
tom
s, =
sum
of
atom
ic c
harg
es o
n hy
drog
en b
ondi
ng h
ydro
gens
, R
2=
exc
ess
mol
ar r
efra
ctio
n, S
sOH
= s
um o
f E
-sta
te i
ndic
es f
or a
ll hy
drox
y gr
oups
,S
sssC
H =
sum
of
E-s
tate
indi
ces
for
all m
ethy
l gro
ups,
TA
= t
otal
sol
vant
acc
essi
ble
surf
ace,
Vm
= m
olar
vol
ume,
Vx
= M
cGow
an c
hara
cter
istic
vol
ume,
and
W =
mol
ecul
ar w
idth
.b
F =
fish
, H
= h
uman
, an
d R
= r
ats.
cC
FC
s =
chl
orofl
uoro
carb
ons,
HM
WO
Cs
= h
igh
mol
ecul
ar w
eigh
t or
gani
c ch
emic
als,
LM
WV
OC
s =
low
mol
ecul
ar w
eigh
t vo
latil
e or
gani
c ch
emic
als,
and
VO
Cs
= v
olat
ile o
rgan
ic c
hem
ical
s.
516 ALTERNATIVE TOXICOLOGICAL METHODS
In Silico Approaches for Oral Absorption Constants
INTEGRATING IN SILICO APPROACHES INTO RISK ASSESSMENT
IN SILICO APPROACHES FOR PBPK MODELING 517
Tab
le 4
0.9
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e O
ral
Ab
sorp
tio
n C
on
stan
t (K
a) o
f C
hem
ical
s
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
Ref
eren
ce
QS
AR
s: L
FE
-typ
e eq
uat
ion
s
Ele
ctro
stat
ic d
escr
ipto
rs
log
Ka(
abso
rptio
n)=
–0.
58 lo
g P
o:w
+ 0
.35p
Ka
– 1.
77F
Bar
bitu
rate
sS
eyde
l and
Sch
aper
(19
82)
Hyd
rop
ho
bic
des
crip
tors
Ka(
abso
rptio
n)=
k m[(
1/R
f) –
1]n’/(
Q+
[(1
/Rf)
– 1]
n’)
RS
ulfo
nam
ides
Test
a et
al.
(200
0)
log
Ka(
abso
rptio
n)=
–0.
04 (
log
Po:
w)2
+ 0
.22
log
Po:
w+
0.0
4R
Sul
fona
mid
esS
eyde
l and
Sch
aper
(19
82)
log
Ka(
abso
rptio
n)=
0.0
67 +
log
Po:
w–
log(
1.4
+ P
o:w)
RP
harm
aceu
tical
sYa
mag
uchi
et
al. (
1996
) lo
gK
a(ab
sorp
tion)
= –
0.08
2(lo
g P
o:w)2
+ 0
.268
log
Po:
w+
3.9
6R
Org
anic
aci
dsS
eyde
l and
Sch
aper
(19
82)
log
Ka(
abso
rptio
n)=
–0.
09 (
log
Po:
w)2
+ 0
.44
log
Po:
w–
0.39
6R
Sul
fona
mid
esS
eyde
l and
Sch
aper
(19
82)
log
Ka(
abso
rptio
n)=
0.0
9 lo
g P
o:w
+ 0
.83
RX
anth
enes
Sey
del a
nd S
chap
er (
1982
)lo
gK
a(ab
sorp
tion)
= 0
.18
log
Po:
w+
0.2
3R
Car
bam
ates
Sey
del a
nd S
chap
er (
1982
)lo
gK
a(ab
sorp
tion)
= 0
.24
log
Po:
w–
1.37
RA
ntih
ista
min
esS
eyde
l and
Sch
aper
(19
82)
log
Ka(
abso
rptio
n)=
0.3
0 lo
g P
o:w
– 0.
07(lo
g P
o:w)2
– 2.
38R
Sul
fony
lure
asS
eyde
l and
Sch
aper
(19
82)
log
Ka(
abso
rptio
n)=
0.3
log
Po:
w–
0.57
log
(0.3
4 P
o:w
+ 1
) –
0.15
I–
0.74
RC
arba
mat
esS
eyde
l and
Sch
aper
(19
82)
log
Ka(
abso
rptio
n)=
0.4
6 lo
g P
o:w
– 0.
36 lo
g (0
.60P
o:w
+ 1
) –
0.23
RS
ulfo
nylu
reas
Sey
del a
nd S
chap
er (
1982
)lo
gK
a(ab
sorp
tion)
= 0
.5 lo
g P
o:w
–0.6
1 lo
g (0
.07P
o:w
+ 1
) –
0.39
RS
ulfo
nam
ides
Sey
del a
nd S
chap
er (
1982
)lo
gK
a(ab
sorp
tion)
= 0
.502
log
Po:
w–
log
(0.0
53P
o:w
0.08
62+
1)
– 0.
384
RS
ulfo
nam
ides
Sey
del a
nd S
chap
er (
1982
)lo
gK
a(ab
sorp
tion)
= 0
.56
log
Po:
w–
(0.0
4Po:
w0.
84+
1)
– 0.
63R
Phe
nols
; Ani
lines
; Est
ers
Sey
del a
nd S
chap
er (
1982
)lo
gK
a(ab
sorp
tion)
= 1
.36
log
Po:
w+
0.3
6R
Org
anic
ani
ons
Sey
del a
nd S
chap
er (
1982
)(c
ontin
ued)
518 ALTERNATIVE TOXICOLOGICAL METHODS
Tab
le 4
0.9
(co
nti
nu
ed)
In S
ilico
Ap
pro
ach
es f
or
Est
imat
ing
th
e O
ral
Ab
sorp
tio
n C
on
stan
t (K
a) o
f C
hem
ical
s
Ap
pro
ach
aS
pec
iesb
Ch
emic
al C
lass
Ref
eren
ce
QS
AR
s: F
ree–
Wils
on
typ
e eq
uat
ion
s
log
Ka(
abso
rptio
n)=
BS
(BE
N-S
O2-
NH
CO
NH
)(–2.
272)
+ n
H (
0) +
n2-
CH
3(0
.088
) +
n4-
CH
3(0
.074
) +
n 4-C
2H5(0
.163
) +
n4-
OC
H3
(–0.
229)
+ n
2-N
O2
(–0.
324)
+
n3-N
O2
(–0.
207)
+ n
4-N
O2
(–0.
323)
+ n
4-C
l (0.
198)
+ n
4-B
r(0.
122)
+ n
n-C
4H9(
0) +
nC
H3
(–0.
638)
+ n
C2H
5(–
0.36
1) +
nn-
C3H
7(–
0.14
5) +
ni-C
3H7
(–0.
244)
+ n
i-C4H
9(–0
.035
) +
nt-
C4H
9(0
.149
) +
ncy
-C6H
11(0
.135
) +
nal
lyl
(–0.
419)
+ n
C6H
5(–
0.08
8)
RS
ulfo
nylu
reas
Sey
del a
nd S
chap
er (
1982
)
aal
lyl
= a
llyl
grou
p, B
r =
bro
mid
e, B
S =
bas
ic s
truc
ture
, C
2H5
= e
thyl
gro
up,
C6H
5=
aro
mat
ic r
ing
grou
p, C
H3
= m
ethy
l gr
oup,
Cl
= c
hlor
ide
grou
p, c
y-C
6H11
= c
yclo
hexy
l gr
oup,
H =
hyd
roge
n gr
oup,
I=
fam
ily i
ndic
ator
var
iabl
e, i
-C3H
7=
iso
prop
yl g
roup
, i-C
4H9
= i
sobu
tyl
grou
p, k
m=
rat
e co
nsta
nt f
ortr
ansf
er o
ut o
f th
e m
embr
ane,
n=
gro
up o
ccur
renc
e in
mol
ecul
e, n
’= c
onst
ant
spec
ific
to t
he e
quat
ion
with
out
any
give
n ro
le,
n-C
3H7
=n-
prop
yl g
roup
,n-
C4H
9=
n-bu
tyl g
roup
, N
O2
= n
itroo
xide
gro
up,
OC
H3
= m
ethy
l eth
er g
roup
, pK
a=
log
diss
ocia
tion
cons
tant
of
an a
cid
in w
ater
, P
o:w
=n-
octa
nol:w
ater
part
ition
coe
ffici
ent (
or v
eget
able
oil:
wat
er),
Q=
con
stan
t spe
cific
to th
e eq
uatio
n w
ithou
t any
giv
en r
ole,
Rf=
rev
erse
-pha
se T
LC li
poph
ilici
ty p
aram
eter
,an
dt-
C4H
9=
tert
-but
yl g
roup
.b
F =
fish
and
R =
rat
s.
IN SILICO APPROACHES FOR PBPK MODELING 519
Free–Wilson QSARs for Chloroethanes
Table 40.10Frequency of Occurrence of Molecular Fragments for Each Chloroethane of the Series
Chemical BSa H3 Cl Cl2 Cl3
Chloroethane 1 1 1 0 01,1-dichloroethane 1 1 0 1 01,2-dichloroethane 1 0 2 0 01,1,1-trichloroethane 1 1 0 0 11,1,2-trichloroethane 1 0 1 1 01,1,1,2-tetrachloroethane 1 0 1 0 11,1,2,2-tetrachloroethane 1 0 0 2 0Pentachloroethane 1 0 0 1 1Hexachloroethane 1 0 0 0 2
a BS = basic structure (C-C).
520 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 40.1 Chemical description methodology used in this study. The chemicals are repre-sented as a basic structure (C-C) with substituents on the two carbons. Examplesof the description of 1,1,1 trichloroethane and 1,1,2,2 tetrachloroethane are pre-sented.
Cl
Cl
Cl
H
H
H
Cl
Cl
H
Cl
Cl
H
1,1,1-trichloroethane 1,1,2,2-tetrachloroethane
Basic structure:(-C-C-)
Molecular fragments:1 x (-C-C-)
1 x H3
1 x Cl3
Molecular fragments:1 x (-C-C-)
2 x Cl2H
IN SILICO APPROACHES FOR PBPK MODELING 521
Table 40.11 Contributionsa of Chloroethane Structural Features to Rat Partition Coefficientsb and Metabolic Constantsc
Fragments Pb Pl Ps Pf Vmaxc Km
BS 56.8 2.02 0.746 28.9 52.7 3.75Cl2 42.7 –0.319 –0.0181 –1.16 9.40 0.863Cl3 7.00 1.60 0.233 14.1 –15.3 –0.0932Cl –9.60 –0.506 0.00710 –7.22 –7.22 –0.234H3 –50.1 –0.653 –0.0770 –8.56 12.9 –1.65r 2 0.98 0.91 0.99 0.96 0.82 0.88
a Contributions were obtained by multiple linear regression from experimental data onchloroethane, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,1,2-tetra-chloroethane, 1,1,2,2-tetrachloroethane, pentachloroethane, and hexachloroethane. BS =basic structure (C-C).
b Pb, Pl,, Ps, and Pf refer to blood:air, liver:blood, slowly perfused tissue:blood and fat:bloodpartition coefficients, respectively.
c Vmaxc ( mol/hr/kg) and Km ( M) refer to maximal velocity of metabolism and affinity con-stant, respectively.
Table 40.12 Contributionsa of Chloroethane Structural Features to Human Partition Coefficientsb
Fragments Pb Pl Ps Pf
BS 37.4 2.72 1.099 38.9Cl2 29.6 –0.365 –0.163 0.105Cl3 7.53 2.16 0.166 12.2Cl –8.92 –0.446 0.0510 –10.6H3 –39.3 –0.699 0.0450 –1.05r 2 0.83 0.98 0.91 0.94
a Contributions were obtained by multiple linear regression from experimental data on chlo-roethane, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,1,2-tetrachlo-roethane, 1,1,2,2-tetrachloroethane, and hexachloroethane. BS = basic structure (C-C).
b Pb, Pl, Ps, and Pf refer to blood:air, liver:blood, slowly perfused tissue:blood and fat:bloodpartition coefficients, respectively.
522 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 40.2 Comparison of rat experimental and predicted parameter values. Experimentalvalues from Gargas et al. (1988, 1989).
Figure 40.3 Comparison of human experimental and predicted parameter values. Experimen-tal values were derived from Gargas et al. (1988, 1989).
y = 1.0089xR2 = 0.9159
0.1
1
10
100
1000
0.1 1 10 100 1000
Experimental parameter value
Est
imat
ed p
aram
eter
val
ue
y = 0.8984xR2 = 0.8557
0.1
1
10
100
1000
0.1 1 10 100 1000
Experimental parameter value
Est
imat
ed p
aram
eter
val
ue
IN SILICO APPROACHES FOR PBPK MODELING 523
Integrating Free–Wilson QSARs into PBPK Models
Table 40.13 Comparison of Experimentala (Exp) and QSAR-Estimated (Est) Values of Rat Partition Coefficientsb and Metabolic Constantsc for1,1,1-Trichloroethane
Parameter Exp Est
Pb 5.67 13.7Pl 1.52 2.97Ps 0.56 0.90Pf 46.4 34.5
Vmaxc 43.1 50.3Km 3.14 2.01
a Experimental data from Gargas et al. (1988, 1989).b Pb, Pl,, Ps, and Pf refer to blood:air, liver:blood, slowly perfused
tissue:blood and fat:blood partition coefficients, respectively.c Vmaxc ( mol/hr/kg) and Km ( M) refer to maximal velocity of
metabolism and affinity constant, respectively.
Table 40.14 Comparison of Experimentala (Exp) and QSAR-Estimated (Est) Values of Human Partition Coefficientsb for 1,1,1-Trichloroethane
Parameter Exp Est
Pb 2.53 5.56Pl 3.40 4.18Ps 1.25 1.31Pf 104 50.1
a Data derived from Gargas et al. (1989).b Pb, Pl, Ps, and Pf refer to blood:air, liver:blood, slowly perfused
tissue:blood and fat:blood partition coefficients, respectively.
524 ALTERNATIVE TOXICOLOGICAL METHODS
QSAR-Based Risk Assessment of Methyl Chloroform
IN SILICO APPROACHES FOR PBPK MODELING 525
CONCLUSIONS AND FUTURE DIRECTIONS
Figure 40.4 Quantitative structure-activity relationship (QSAR) physiologically based pharma-cokinetic (PBPK) modeling framework. User input consists of the exposure sce-nario and chemical structure information such as the number of fragmentsconstituting the molecule. This information is fed to the program that contains themodel constants, the Free–Wilson type SPR, the contribution values of eachmolecular fragment (Cs) and of the basic structure (BS) to the model parameters(P), and the simulation algorithms. The model can then simulate the pharmaco-kinetics of the chemical in biota and then provide its profile as output. The exampleof 1,1,1-trichloroethane is shown.
526 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 40.5 Comparison of steady-state blood and tissue concentrations of chloroethanes inrats exposed to 1 ppm, as simulated by conventional and QSAR PBPK models.
Table 40.15 Steady-State Tissue Concentrations ( g/L) of 1,1,1-Trichloroethane in Rat and Humans Estimated Using the Conventional (PBPK) and QSAR-Based (QSAR) Physiologic Model Following a Continuous Exposure to 1 ppm
TissueRat Human
QSAR PBPK QSAR PBPK
Blood 22.9 16.6 12.8 8.5Liver 6.77 4.22 6.23 5.61Slowly perfused 20.7 9.31 16.7 10.7Fat 790 770 502 472Richly perfused 68.1 25.2 53.4 28.9
Table 40.16 Steady-State Arterial Blood Concentration (Cass) Obtained Using the Conventional (PBPK) and QSAR-Based (QSAR) Physiological Model in Rats Exposed to the NOAEL of 1,1,1-Trichloroethane (875 ppm) and the Corresponding Environmental Concentration (Ci) in Humans Derived Using the Human Conventional (PBPK) and QSAR-Based (QSAR) Physiological Models
Endpoint QSAR PBPK
Rat Cass (mg/L) 59.3 24.9Human Ci (ppm)a 6342 4252
a Calculated using the QSAR-derived Cass (59.3 mg/L).
y = 1.2184xR2 = 0.9746
0.0001
0.001
0.01
0.1
1
10
0.0001 0.001 0.01 0.1 1 10
Tissue concentrations obtained using QSPR-PBPK type model (mg/L)
Tis
sue
con
cen
trat
ion
s o
bta
ined
usi
ng
co
nven
tio
nal
-typ
e P
BP
Km
od
el (
mg
/L)
IN SILICO APPROACHES FOR PBPK MODELING 527
528 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
IN SILICO APPROACHES FOR PBPK MODELING 529
530 ALTERNATIVE TOXICOLOGICAL METHODS
IN SILICO APPROACHES FOR PBPK MODELING 531
532 ALTERNATIVE TOXICOLOGICAL METHODS
533
CHAPTER 41
In Silico Application of Quantum ChemicalMethods for Relating Toxicity
to Chemical Reactivity
CONTENTS
INTRODUCTION
534 ALTERNATIVE TOXICOLOGICAL METHODS
IN SILICO APPLICATION OF QUANTUM CHEMICAL METHODS 535
Figure 41.1 Structures of commercial pesticides.
N
CH3
OCH
O
P SCH3O
S
OCH3
CH C
CH2 C
O
OCH2CH3
OCH2CH3
O
MalathionCarbaryl
536 ALTERNATIVE TOXICOLOGICAL METHODS
CHEMICAL REACTIVITY
PROGRESS OF THE REACTION
Figure 41.2 Hydrolysis of acetylcholine.
+
Choline
HOCH2CH2N CH3
CH3
CH3+
Acetic acid
CH3C
O
OHAChE
H2O
Acetylcholine
CH3C OCH2CH2N
O
CH3
CH3
CH3+
IN SILICO APPLICATION OF QUANTUM CHEMICAL METHODS 537
STRUCTURE/TOXICITY RELATIONS BASED ON CHEMICAL REACTIVITY
538 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 41.3 Progress of the reaction (x-axis) mapped against energy (y-axis).
ENERGY
P R2R1
O
F
PR1R2
O
OH
F
- P R2R1
OH
O
++ F-
OH-
PROGRESS OF THE REACTION
IN SILICO APPLICATION OF QUANTUM CHEMICAL METHODS 539
REFERENCES
540 ALTERNATIVE TOXICOLOGICAL METHODS
IN SILICO APPLICATION OF QUANTUM CHEMICAL METHODS 541
543
CHAPTER 42
In Silico Cardiac Toxicity: Increasingthe Discovery of Therapeutics through
High-Performance Computing
CONTENTS
INTRODUCTION
544 ALTERNATIVE TOXICOLOGICAL METHODS
IN SILICO CARDIAC TOXICITY 545
IN SILICO MODELS
546 ALTERNATIVE TOXICOLOGICAL METHODS
E-Cell
Virtual Cell Environment
IN SILICO CARDIAC TOXICITY 547
CellML Language and Other Resources
DATA MINING
548 ALTERNATIVE TOXICOLOGICAL METHODS
A PRACTICAL EXAMPLE
IN SILICO CARDIAC TOXICITY 549
In Silico Simulation of Atrial Action Potential Propagation in the Presence of Soman
550 ALTERNATIVE TOXICOLOGICAL METHODS
IN SILICO CARDIAC TOXICITY 551
o
552 ALTERNATIVE TOXICOLOGICAL METHODS
Solution of the Monodomain Equations on the MSRC Assets
RESULTS
DISCUSSION
IN SILICO CARDIAC TOXICITY 553
(a)
(b)
Figure 42.1 Frame sequence showing the evolution of the action potential in an atrial tissueusing the Nygren model for the electrophysiology of the tissue. The times of theframes going clockwise are (a) 50 ms, (b) 100 ms, (c) 150 ms, and (d) 200 ms.The stimulus consisted of a current pulse of 40 cm2 delivered to the leftmostcolumn of atrial cells. The stimulus lasted for 2 ms and VM in the legend is thevoltage in mV. (continued)
554 ALTERNATIVE TOXICOLOGICAL METHODS
(c)
(d)
Figure 42.1 (continued) Frame sequence showing the evolution of the action potential in anatrial tissue using the Nygren model for the electrophysiology of the tissue. Thetimes of the frames going clockwise are (a) 50 ms, (b) 100 ms, (c) 150 ms, and(d) 200 ms. The stimulus consisted of a current pulse of 40 cm2 delivered tothe leftmost column of atrial cells. The stimulus lasted for 2 ms and VM in thelegend is the voltage in mV.
IN SILICO CARDIAC TOXICITY 555
Figure 42.2 Action potential, entering from the left and encountering a region of high potassiumconcentration region at t = 40 and 250 ms, respectively. The hyperkalemic regionis along the tissue central axis, in the center taking up 1 cm2 of the 3 cm 3 cmtissue.
556 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 42.3 This figure contrasts the effect of potassium current blockers on the action poten-tial. The left-hand panel shows the voltage, that is, the action potential in thetissue without modulation of the currents. The effect of reducing the iKr and iKs,such as is effected by newer antiarrhythmic drugs such as azimilide, is shown inthe right-hand panel. Modulation of the wave distortion and erratic behavior shouldbe noted.
IN SILICO CARDIAC TOXICITY 557
ACKNOWLEDGMENTS
REFERENCES
558 ALTERNATIVE TOXICOLOGICAL METHODS
559
CHAPTER 43
Submillimeter-Wave FrequencyStudies of the Vibrational Modes
of Deoxyribonucleic Acid:A Metric for Mutagenicity?
CONTENTS
INTRODUCTION
560 ALTERNATIVE TOXICOLOGICAL METHODS
METHODS AND MATERIALS
Computational Studies
Experimental Measurements
FREQUENCY STUDIES OF THE VIBRATIONAL MODES OF DNA 561
RESULTS AND DISCUSSION
Computational Studies
Figure 43.1 Calculated spectra of Poly(dA)Poly(dT) and of Poly(dAdT)Poly(dTdA) at two line-widths: 2-wavenumber and 7-wavenumber.
0 100 200 300 400 500
Poly(dA)Poly(dT)
γ = 7 cm -1
γ = 2 cm -1
0 100 200 300 400 500
Poly(dAdT)Poly(dTdA)
0 100 200 300 400 5000 100 200 300 400 500
Abs
orpt
ion
Frequency (cm-1)
562 ALTERNATIVE TOXICOLOGICAL METHODS
Experimental Studies
Table 43.1 Comparison of Predicted Vibrational Band Positions (in Wavenumbers) of Poly(dA)Poly(dT) with Values Observed by Experiments in the Literature
CalculationsExperiment
(Powell, 1987) CalculationsExperiment
(Powell, 1987)
20 Unknown 172 17043 Unknown 199 20057 62 211 21471 80 — 23894 95 270 —
106 106 348 —131 136 400 —160 — 459 —
Figure 43.2 Experimental absorbance spectra of herring DNA in the mid-infrared through thevery far infrared region.
3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 250 0
IIIIII
Rel
ativ
e A
bsor
banc
e
Wavenumber (cm-1)
FREQUENCY STUDIES OF THE VIBRATIONAL MODES OF DNA 563
Figure 43.3 Fine structure in the absorbance spectra of herring and salmon DNA in the veryfar infrared region.
24 22 20 18 16 14 12 100.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60 Herring DNA Salmon DNA
Tran
smis
sion
Wavenumber (cm-1)
564 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
Figure 43.4 Transmission scattering parameter (S12) measurement of herring and salmon DNAin the microwave (W-band) region.
75.0 80.0 85.0 90.0 95.0 100.0 105.0 110.00.6
0.7
0.8
0.9
SalmonDNA HerringDNA
Sca
tterin
g P
aram
eter
, S12
Frequency, GHz
FREQUENCY STUDIES OF THE VIBRATIONAL MODES OF DNA 565
567
CHAPTER 44
Trophoblast Toxicity Assay (TTA):A Gestational Toxicity Test Using
Human Placental Trophoblasts
CONTENTS
HUMAN TROPHOBLASTS IN VIVO:THREE DIFFERENTIATION PATHWAYS
568 ALTERNATIVE TOXICOLOGICAL METHODS
Figure 44.1 Pathways of trophoblast differentiation. Just as the basal layer of the skin givesrise to keratinocytes, the cytotrophoblast — the stem cell of the placenta — givesrise to the differentiated forms of trophoblasts. (Left) Within the chorionic villi,cytotrophoblasts fuse to form the overlying syncytiotrophoblast. The villous syn-cytiotrophoblast makes the majority of the placental hormones, the most studiedbeing hCG. cAMP, EGF, and even hCG itself have been implicated as stimulatorsof this differentiation pathway. In addition to upregulating hCG secretion, cAMPhas also been shown to down-regulate trophouteronectin (TUN) synthesis. (Cen-ter) At the point where chorionic villi make contact with external extracellularmatrix (decidual stromal ECM in the case of intrauterine pregnancies), a popu-lation of trophoblasts proliferates from the cytotrophoblast layer to form the secondtype of trophoblast — the junctional trophoblast. These cells form the anchoringcell columns that can be seen at the junction of the placenta and endometriumthroughout gestation. Similar trophoblasts can be seen at the junction of thechorion layer of the external membranes and the decidua. The junctional tropho-blasts make a unique fibronectin — trophouteronectin — that appears to mediatethe attachment of the placenta to the uterus. TGF and LIF have been shown toinduce cultured trophoblasts to secrete increased levels of trophouteronectin,while down-regulating hCG secretion. (Right) Finally, a third type of trophoblastdifferentiates toward an invasive phenotype and leaves the placenta entirely —the invasive intermediate trophoblast. In addition to making human placentallactogen, these cells also make urokinase and plasminogen activator inhibitor-1(PAI-1). Phorbol esters have been shown to increase trophoblast invasiveness inin vitro model systems and to upregulate PAI-1 in cultured trophoblasts. Thegeneral theme that comes from these observations is that specific factors arecapable of shifting the differentiation pathway of the cytotrophoblast toward oneof the above directions while turning off differentiation toward the other pathways.See text for details.
Cytotrophoblast
AnchoringTrophoblasts
VillousSyncytiotrophoblast
hCG TUN
Invading Trophoblasts
PAI-1
cAMPhCG
PhorbolEsters
LIFTGFß
HJK
TROPHOBLAST TOXICITY ASSAY (TTA) 569
Villous Syncytiotrophoblast
570 ALTERNATIVE TOXICOLOGICAL METHODS
Anchoring Trophoblasts
Invading Trophoblasts
IN VITRO MODEL SYSTEMS TO STUDY TROPHOBLAST DIFFERENTIATION
TROPHOBLAST TOXICITY ASSAY (TTA) 571
TROPHOBLASTS AS ENDOCRINE CELLS
Figure 44.2 Purification of cytotrophoblasts from term placenta. A term placenta is minced anddigested with trypsin and DNAse. The supernatant is passed through calf serumto inactivate the digestive enzymes; then these pellets are pooled and placed ona Percoll gradient to separate out the cytotrophoblasts. (From Kliman et al. (1986)Endocrinology, 118(4), 1567–1582. With permission.)
572 ALTERNATIVE TOXICOLOGICAL METHODS
PROTEIN HORMONES
Chorionic Gonadotropin
Figure 44.3 In vitro morphologic differentiation of cytotrophoblasts. After purification, thecytotrophoblasts are dispersed as individual cells (left). When plated in culturemedia containing serum, these cells flatten out and begin to move toward eachother. After 24 hr in culture, aggregates begin to appear, with some evidence ofcell fusion (center). After 72 hr in culture, most of the trophoblasts have fused andformed large, multinucleated syncytiotrophoblasts. (From Kliman et al. (1986)Endocrinology, 118(4), 1567–1582. With permission.)
TROPHOBLAST TOXICITY ASSAY (TTA) 573
Figure 44.4 hCG secretion by trophoblasts in culture. Percoll-gradient purified cytotrophoblastswere cultured in DMEM media for four days. Media was changed daily and assayedfor hCG by radioimmunoassay. hCG was not detectable at the time of initial plating.(From Kliman et al. (1986) Endocrinology, 118(4), 1567–1582. With permission.)
Table 44.1 Regulation of Trophoblast hCG Secretion
FactorTrophoblasts (Trimester)
Effect on hCG Secretion References
CAMP Term Stimulates (Feinman et al., 1986)HCG Term Stimulates (Shi et al., 1993)GnRH Term Stimulates (Belisle et al., 1989;
Szilagyi et al., 1992)GnRH First, Term Not clear (Kelly et al., 1991)-Adrenergic agonists First Stimulates (Oike et al., 1990)
Dexamethasone Term Stimulates (Ringler et al., 1989a)Inhibin Term Inhibits (Petraglia et al., 1987,
1989, 1991)Activin Term Potentiates GnRH
stimulation of hCG secretion
(Petraglia et al., 1991)
Activin First Stimulates (Steele et al., 1993)EGF First, Term Stimulates (Maruo et al., 1987)Thyroid hormone First, Term Stimulates (Maruo et al., 1991)Thyroid stimulating hormone
Term Inhibits (Beckmann et al., 1992)
Interleukin-1 First Stimulates (Yagel et al., 1989b)Interleukin-6 First Stimulates (Nishino et al., 1990)Basement membrane First Stimulates (Truman and Ford,
1986)Decidual protein Term Inhibits (Ren and Braunstein,
1991)Prolactin Term Inhibits (Yuen et al., 1986)
574 ALTERNATIVE TOXICOLOGICAL METHODS
Human Placental Lactogen (hPL)
Figure 44.5 Effects of 8-bromo-cAMP on hCG and progesterone secretion by cultured cytotro-phoblasts. Percoll-gradient purified cytotrophoblasts were cultured for 48 hr in theabsence (�) or presence (�) of 8-bromo-cAMP. hCG (A) and progesterone (B)were quantitated in the medium at 24-hr intervals. Values presented are the mean± SE from six separate experiments. At each time point, 8-bromo-cAMP-treatedcultures secreted significantly more (p < 0.014, by the Wilcoxon signed rank test)progesterone and hCG than did control cultures. (From Feinman et al. (1986) J.Clin. Endocrinol. Metab., 63(5), 1211. With permission.)
TROPHOBLAST TOXICITY ASSAY (TTA) 575
TROPHOBLAST TOXICITY ASSAY
CONCLUSIONS
576 ALTERNATIVE TOXICOLOGICAL METHODS
REFERENCES
TROPHOBLAST TOXICITY ASSAY (TTA) 577
578 ALTERNATIVE TOXICOLOGICAL METHODS
TROPHOBLAST TOXICITY ASSAY (TTA) 579
580 ALTERNATIVE TOXICOLOGICAL METHODS
581
Index
A
582 ALTERNATIVE TOXICOLOGICAL METHODS
B
C
INDEX 583
D
584 ALTERNATIVE TOXICOLOGICAL METHODS
E
INDEX 585
F
G
H
586 ALTERNATIVE TOXICOLOGICAL METHODS
I
J
K
L
INDEX 587
M
N
588 ALTERNATIVE TOXICOLOGICAL METHODS
O
P
Q
INDEX 589
R
S
590 ALTERNATIVE TOXICOLOGICAL METHODS
T
U
V
INDEX 591
W Z