Alternative Toxicological Methods - H. Salem, S. Katz (CRC, 2003) WW

ALTERNATIVE
TOXICOLOGICAL
METHODS
ALTERNATIVE
TOXICOLOGICAL
METHODS
Edited by
Harry Salem
Sidney A. Katz
CRC PR E S S
Boca Raton London New York Washington, D.C.
This edition published in the Taylor & Francis e-Library, 2005.
To purchase your own copy of this or any of Taylor & Francis or Routledges
collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.
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Historical Developments in the Humane

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Table 1.1 Chronology for the Enactment of Animal Welfare Legislation

Year State Year State Year State
1828 New York 1864 Idaho 1880 Ohio
1835 Massachusetts 1864 Oregon 1881 North Carolina
1838 Connneticut 1867 New Jersey 1881 South Carolina
1938 Wisconsin 1868 California 1883 Alabama
1842 New Hampshire 1868 West Virginia 1883 Maine
1845 Missouri 1869 Illinois 1884 Hawaii
1848 Virginia 1871 District of Columbia 1887 New Mexico
1851 Iowa 1871 Michigan 1887 South Dakota
1851 Minnesota 1871 Montana 1889 Florida
1852 Kentucky 1872 Colorado 1890 Maryland
1854 Vermont 1873 Delaware 1891 North Dakota
1856 Texas 1873 Indiana 1893 Oklahoma
1857 Rhode Island 1873 Nebraska 1895 Wyoming
1858 Tennessee 1875 Georgia 1898 Utah
1859 Kansas 1879 Arkansas 1913 Alaska
1859 Washington 1879 Louisiana 1913 Arizona
1860 Pennsylvania 1880 Mississippi 1921 Virgin Islands
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A History of Interagency Approaches

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Table 3.1 Member Agencies

Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)
Consumer Product Safety Commission
Department of Defense
Department of Energy
Department of Health and Human Services
Agency for Toxic Substances and Disease Registry
Food and Drug Administration
National Institute for Occupational Safety and Health
National Institutes of Health, Ofce of the Director
National Cancer Institute
National Institute of Environmental Health Sciences
National Library of Medicine
Department of the Interior
Department of Labor
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Department of Transportation
Research and Special Programs Administration
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Table 3.2 Test Method Validation and Acceptance Criteriaa
Validation Criteria
Clear statement of proposed use
Biological basis/relationship to effect of interest provided
Formal detailed protocol
Reliability assessed
Relevance assessed
Limitations described
All data available for review
Data quality: Ideally good laboratory practices (GLPs)
Independent scientic peer review
Acceptance Criteria
Fits into the regulatory testing structure
Adequately predicts the toxic endpoint of interest
Generates data useful for risk assessment
Adequate data available for specied uses
Robust and transferable
Time and cost effective
Adequate animal welfare consideration (3 Rs)
a These are shortened versions of the adopted criteria. For the full text see: National Institute
of Environmental Health Sciences (NIEHS), Validation and Regulatory Acceptance of
Toxicological Test Methods: A Report of the ad hoc Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM), NIH publication 97-3981, Research
Triangle Park, NC, 1997.
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Table 3.3 The Purposes of the ICCVAMa

Increase the efciency and effectiveness of federal agency test method review
Eliminate unnecessary duplicative efforts and share experiences between federal regulatory
agencies
Optimize use of scientic expertise outside the federal government
Ensure that new and revised test methods are validated to meet the needs of federal
agencies
Reduce, rene, or replace the use of animals in testing where feasible
a ICCVAM Authorization Act (U.S. Code, 2000).
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 scientic validity
Coordinate the technical review and evaluation of new and revised test methods of
interagency interest
Submit ICCVAM test recommendations to each appropriate federal agency
Facilitate and provide guidance on validation criteria and processes
Facilitate:
Interagency and international harmonization of test protocols that encourage the reduction,
renement, and replacement of animal test methods
Acceptance of scientically valid test methods and awareness of accepted methods
Make ICCVAM nal test recommendations and agency responses available to the public
Prepare reports on the progress of this act and make these available to the public
a ICCVAM Authorization Act (U.S. Code, 2000).
THE NATIONAL TOXICOLOGY PROGRAM INTERAGENCY

CENTER FOR THE EVALUATION OF ALTERNATIVE
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Figure 3.1 ICCVAM test method evaluation process.
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INTEGRATED IN VITRO APPROACHES FOR ASSESSING SYSTEMIC TOXICITY 45
1. Experimental 2. Modeling 3. Validation

(literature data)
in vitro data on kinetics kinetic modeling kinetics in vivo
in vitro data on dynamics prediction of target tissue

(e.g., CNC, EC20, EC50) concentrations
(e.g., NOEL, LOEL)
prediction of dynamics
prediction of systemic in vivo systemic

toxic doses toxic doses
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LD50) LD50)
Figure 5.1 Building blocks of the ECITTS scheme.
Compounds and Test Battery
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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 cytotoxicity
Neurite degeneration (ND) Number of neurites per cell Morphology
Protein synthesis rate (PSR) [3H] leucine incorporation in Physiology
proteins during 2 hr
Basal intracellular free Ca2+ Fura-2/Ca2+, uorescence Physiology
concentration (basal Ca2+)
Voltage operated Ca2+ channels High potassium-induced Ca2+ Neurochemistry
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Phospholipase C-coupled Carbachol-activated, immediate Neurochemistry
acetylcholine receptor signal transient Ca2+ peak,
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Neuroblastoma SH-SY5Y Cells, on in Vitro Endpoints as Presented in
Table 5.1
Concentration
Compound In vitro effect QM or QM/hr)
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Caffeine CNC; inhibition of VOCC 10
Diazepam CNC; inhibition of VOCC 49
Lindane CNC; inhibition of VOCC 3.4
Lindane 20% increased basal [Ca2+]i 35
Lindane 50% cytotoxicity 150
Parathion/paraoxon CNC; neurite degeneration/time 0.5
Phenytoin CNC; inhibition of protein synthesis 87
CNC, critical cellular neurotoxic concentration; VOCC, voltage operated calcium channels.
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Abdominal rigidity Discharge, abnormal Pinna reex
Abortion Dyspnea (difcult breathing) Prostrate
Agalactia Epistaxis (nasal bleeding) Pruritis
Anemia Excitable Pupillary
Analgesia Eyelid closure constriction/dilation
Anuria Eyes xed/sunken Rales, pulmonary
Apathy Fractured bone Rectal prolapse
Ataxia/incoordination Gasping Recumbency, prolonged
Bleeding Groomingfailure to do Red eye(s)/nose
Blepharospasm Hunched/stiff posture Reexes
Blood in feces or urine Hyperreexia Retention of feces
Blood around nose, eyes Immobile/inactive Righting reex
Boarded abdomen Jaundice (icterus) Salivation, excessive or
Body temperature, abnormal Joints swollen abnormal
Body weight loss or Kyphosis Seizures
emaciation Lateral position Self-mutilation
Breathing difculties Limping/lameness Skin bruising/color/crepitus
(Dyspnea) Locomotory behavior Spasm
Cachexia Lordosis Staggering
Chewing, persistent Loss of condition, body Sunken anks
Chromodachryorrhea muscle Suppuration
Circling Mammary gland Swellings
Comatose abnormalities Tenesmus
Compulsive behavior Moribund Tetany
Constipation Motor excitation Tremor
Convulsions Not eating/drinking Urine retention
Corneal ulceration Oedema Vaginal prolapse
Coughing/sneezing Pale mucous membranes Vocalization
Cyanosis Paralysis Vomiting
Dehydration Paresis
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Table 7.1 Models and Areas of Research and Specic Techniques That Cause Distress
Research Models or Areas
Non-Pain-Induced Distress
Aggression
Anxiety (e.g., Vogel conict-drinking model)
Cancer (tumor burden, cachexia, carcinogenicity testing)
Depression (e.g., learned helplessness, forced swimming, maternal
deprivation)
Diabetes
Drug addiction and withdrawal
Environmental stress (e.g., hot, cold)
Fear
Immunological research (e.g., vaccine potency testing)
Infectious disease
Motion sickness
Nutrition research (e.g., nutrient deprivation)
Panic
Pharmacology (some) (e.g., tumor necrosis factor, capsaicin research)
Psychopathology (other than anxiety, depression, fear, etc., mentioned above)
Radiation research
Stress (psychological)
Toxicology (induced effects)
Transgenic research
Pain-Induced Distress
Arthritis
Burn research
Cancer research (tumor pain)
Chronic pain studies1
Dental studies
Inammation studies
Experimental surgery (e.g., organ transplantation/rejection)
Muricide (as a model of aggression, neophobia, etc.)
Orthopedic studies
Trauma research
Specic Techniques
Non-Pain-Induced and Pain-Induced Distress

Anesthesia aftereffects
Antibody production (polyclonal and monoclonal)
Aversive stimuli (e.g., electric shock)
Bleeding techniques (including retro-orbital bleeding)
Complete Freunds Adjuvant
Control group (animals denied experimental treatments)
Deprivation (e.g., water, food, sleep, or social partners/experiences)
Dosing techniques (e.g., gavage)
Granuloma techniques
Gut loop studies
(continued)
Table 7.1 (continued) Models and Areas of Research and Specic Techniques That
Cause Distress
Knockout technology
Restraint
Surgery sequelae
1 Acute pain should not be a problem if the guidelines of the International Association for the
Study of Pain (IASP, 1979) are followed.
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Table 7.2 The USDAs Current Pain and Distress Categories and a Proposed
Modication, as Well as Related Features of the Two Systems
A. Current Scheme
USDA Pain and/or Anesthesia/ Full ACUC Alternative
Category Distress Analgesia Review Literature Search
C (63%)1 Little or None No Yes No
D (29%) Yes or No2 Yes Yes Yes
E (8%) Yes No Yes Yes
B. Proposed Scheme
Pain and/or Anesthesia/ Full IACUC Alternative
Category Distress Analgesia Review Literature Search
I Minor or None No No No
II Minor or None Yes Perhaps Perhaps
III Moderate Yes or No Yes Yes
IV Severe Yes or No Yes Yes
1 Numbers in parentheses are USDA gures for 2000.
2 Animals listed in USDA category D were given pain- or distress-relieving drugs, but
these drugs may not have been sufcient to relieve all pain and distress throughout the
experiment.
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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
Percentage Percentage
of Animals of Animals
State in Column E State in Column E
Nationwide 8 Missouri 22
California 4 Nebraska 15
Delaware 15 New Jersey 6
Georgia 2 New York 11
Illinois 3 North Carolina 6
Indiana 18 Ohio 3
Iowa 28 Pennsylvania 8
Kansas 16 Texas 3
Maryland 12 Virginia 0
Massachusetts 1 Washington 20
Michigan 15 Wisconsin 9
Minnesota 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 animals
used across all pain categories.
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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. Not specied 3v/week Not specied 3v/week
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tumors
Priming As low as 0.1 Not specied 0.2 ml max 0.5 ml max
ml pristane pristane pristane
Number of taps Max 3 taps, last Not specied 2 taps, last after Not specied
terminal euthanasia
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postinoculation week, then
daily
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uid after subcutaneous
ascites harvest
Anesthesia Anesthesia can Anesthesia Not specied Not specied
during tap be used used for new
personnel
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References
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PART II
Development of Predictive Methods

Based on Mechanisms of Eye Irritation
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CHAPTER 8
Meeting Industry and Regulatory Needs for

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REFERENCES
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CHAPTER 9
The Ocular Surface: Barrier Function

and Mechanisms of Injury and Repair
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CONTENTS
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ELECTROLYTES AND METABOLITES IN CORNEAL

STROMA AND EPITHELIUM
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Table 9.1 Stroma of rabbit cornea, mean SD,

hydration = 3.2 0.4
QMol/g H2O (n = 20) QMol/g net weight (n = 15)
Na 125 42 Lactate 9.66 1.2
Cl 123 25 Glucose 3.63 0.8
K 24 4 ATP 0.20 0.05
S 105 21 ADP 0.07 0.03
P 12 4 ASC 4.72 1.44
Note: ASC = ascorbate. Levels of some metabolites and
electrolytes in the corneal stroma. Adenosine triph-
osphate (ATP) and adenosine diphosphate (ADP) are
strictly intracellular metabolites. Since the corneal
stroma contains keratocytes only in about 10% of its
volume, their levels are much lower than in the epi-
thelium (see Table 9.2). Lactate is present in the
extracellular as well as in the intracellular space and
consequently 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 QMol/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)
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corneal epithelium, which may be sig-
nicant to estimate vitality, to indicate
microtrauma, and to show levels of
radical scavengers (Hennighausen et
al., 1972; Reim et al., 1966, 1967,
1970, 1976, 1982).
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Table 9.3 Grading of Eye Burns

I II III IV
Immediate signs
Erosion Large erosion Surface defect Epithelia destroyed
Hyperemia Ischemia 1/3 Ischemia >1/2 Deep ischemia >3/4
Chemosis Rose chemosis Dense corneal opacity
Corneal opacity Conjunctival necroses
Sclera porcelain white
Discoloration and atrophy of iris
Fibrin exudate
Later signs
Regeneration Recirculation Persistent erosion Proliferation
Regeneration Ulceration Large ulcerations
Vascularization Melting of cataract
Scars Glaucoma
Scarication
Note: Grading of eye burns according to clinical signs. The upper part lists immediate
damage visible, the lower one later secondary events. The classication of signs
was 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 completely
lost, but the stroma remained undamaged and clear. The conjunctiva showed hype-
remia, but no swelling or ischemia. The damage healed within a few days.
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Figure 9.2 Lime burn stage II (Table 9.3). The whole corneal and some conjunctival epithelium
was destroyed. The corneal stroma exhibited little supercial turbidities. The lower
conjunctiva is demonstrated by upgaze. It was swollen (chemosis). Supercially
in the conjunctiva, ischemia is recognized by the interrupted blood columns. With
the lit lamp microscope, bloodstream could not be detected. Underneath the
otherwise pale conjunctiva, intact sclera appeared with a faint rose background.
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Figure 9.3 Clinical appearance of a severe chemical injury grade IV. The inner margin of the
upper lid showed a white line of necrosis. The conjunctiva appeared at and white,
also from necrosis, which presumably included visible parts of the sclera. In the
upper left region, some hemorrhages were deposited in necrotic conjunctiva.
Ischemia was evident. The cornea was completely turbid. The outlines of iris and
pupil could be hardly identied.
Figure 9.4 Melting of the anterior eye segment, nine days after most severe burn from liquid
metal. There were extended necroses of all conjunctival, subconjunctival, and
scleral tissues, appearing homogeneously white and slippery. Only in the right
upper region, some hemorrhages in necrotic tissues showed red color. The cornea
was opaque in the upper marginal parts. The lower and central cornea was melted
away and the iris and lens exposed. Since at that time (1977) corneal donor
material was not available, the eye was lost and had to be removed. In the melting
tissues 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).
PATHOPHYSIOLOGY OF CHEMICAL EYE INJURIES
Ischemia and Necrosis
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Inammatory Response
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Cornea and conjunctiva
Severe lesion, severe response
PGE2, Interleukins, LT 4, Subst-P, VIP, CGRP

Mild lesion
weak response
IL-1, IL-6 PMNs,
IL-8, TNF macrophages
_ +
T-lymphocytes O2 OH -radicals
PMNs
B-lymphocytes lysosomal enzymes
Plasma cells
Cellular and humoral antibodies

Restitution
Inflammation Scars Ulceration
Figure 9.5 Flow diagram of inammatory cascade following chemical and thermal injuries of
the eye. The inammatory response is a quantitative process produced by the
affected tissues and the leucocytes involved (Ghattacherjee et al., 1979; Reim et
al., 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).
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Cytokines and Growth Factors in Cornea and Tears
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Table 9.4 Cytokines in Tears

EGF p regeneration of epithelium
TGFbeta 2 p inhibits proliferation
TNFalpha, in inammation
Many others, but presumably released from damaged surface epithelia
Note: Cytokines and growth factors in tears inuencing the corneal
epithelium (Mishima et al., 1991; Kruse and Tseng, 1994; Soto-
zono and Kinshita, 1998).
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Levels of IL-1 and IL-6 (n=127)

pg/mg protein human corneal buttons
1000
100
10
Keratitis Decompensation
Inflammat.Cone Dystrophy, Scars
Ulceration Keratoconus
Figure 9.6 Interleukin-1F (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). Inamed corneas
revealed very high levels of IL-1 and IL-6. The levels in the uninamed, quiete
corneas were lower by an order of magnitude.
Chemical Alteration of Extracellular Matrix
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N-Acetylglucose aminidase in human tears

wmol/min/ml
10
Atopic conjunctivitis
1
Range of 8 samples
from human burns
Normal 0.24 s 0.09 (9)
0.1
Figure 9.7 Activity of N-acetylglucose aminidase (NAcGA, E.C.3.2.1.50) in human tears

collected from nine human cases with eye burns stage I and II and an atopic
patient. Please note that the ordinate is in logarithmic scale! The enzyme activity
(QMol/min/ml) increased considerably in surface diseases.
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Table 9.5 Stroma of Rabbit Cornea, QMol/g H2O, mean SD

Alkali Burn, Denuded,
Rinsed for 16 Days, 4v daily
EDXA Normal (n = 20) with 0.9% NaCl (n = 8)
Na 125 42 90 11
Cl 123 25 65 15
S 105 21 24 4
P 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).
Calcication and Contamination
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Table 9.6 Stroma of Rabbit Cornea, QMol/g H2O, Mean SD

Alkali Burn, Denuded,
Rinsed for 16 Days, 4v Daily
EDXA Normal (n = 20) with Phosphate Buffer (n = 8)
Na 125 42 105 22
Cl 123 25 88 33
S 105 21 28 4
P 12 4 623 307
Ca 3 3 435 198
Note: Changes of the levels of some electrolytes in the corneal stroma after alkali burn
and rinsing with isotonic phosphate buffer, four times daily for 16 days. Na, Cl, and
especially S were decreased, but P and Ca were largely increased. Clinically,
calcication of the cornea was observed (Schrage, 1997; Fischern et al., 1998;
Haller, 2001).
Figure 9.8 Left eye of a 16-year-old boy six months after a most severe chemical injury. In
this accident, a highly alkaline etching uid used to work on electronic parts spilled
into both eyes of the patient. In this case, a severe inammatory response had
developed and remained for years. The conjunctiva-like proliferation tissue sur-
rounding the cornea was swollen and very hyperemic. The cornea was devoid of
epithelium. It showed extended ulceration especially in its marginal parts and was
generally thinned. The upper right cornea showed white calcication. To save the
eye from melting, a keratoplasty was performed. The excised cornea was examined
with electron dispersive x-ray analysis method (EDXA) (see Figures 9.9 and 9.10).
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Figure 9.9 Scanning electron microscopy (SEM) on a cross section of the cornea seen in
Figure 9.8. Magnication v200. The upper part shows calcication, the lower one
parallel corneal lamellae (Schrage et al., 1988, 1993, 1996).
Figure 9.10 Electron dispersive x-ray analysis (EDXA) of the calcied cornea as demonstrated
in Figures 9.8 and 9.9. The spectra of the x-rays backscattered at scanning
electron microscopy (SEM) showed as expected high peaks for calcium (Ca) and
phosphorus (P). But the most prominent peak from this sample was emitted from
silicon (Si). Thus, EDXA revealed an unexpected high contamination of the cornea
by silicone, which might have explained the severe and longstanding inammatory
response in this case (Schrage et al., 1988, 1993, 1996).
ASPECTS OF REPAIR
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Figure 9.11 Eye of a 42-year-old male 2 years after severe lime burn. Heavy scar formation
could not be prohibited. The cornea was covered with thick highly vascularized
proliferation tissue. The conjunctiva developed strong scars between the globe
and the lids, reducing eye motility. The conjunctival scars also deformed the lid
margins. The hyperemic, red scar tissue showed that the inammatory response
had not subsided after 2 years. The eye was practically blind and had bad
prognoses for surgical rehabilitation.
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CHAPTER 10
Evaluation and Renement of the Bovine

Cornea Opacity and Permeability Assay
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Figure 10.1 Cross-section of the new cornea holder. In contrast to the old holder, the new
holder clamps onto sclera rather than cornea. Also, the shape of the chamber ts
the normal curvature of the cornea in contrast to the at chamber of the current
BCOP holder.
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Table 10.1 Absorbance at 570 nm (A570) of Bovine Corneas Exposed to Various

Treatments and Incubated in MEM for 3 h
Exposure
Treatment None 10 min 1 min 30 sec
Intact, 35C, MEM 0.05 0.03
w/o Epi, 35C, MEM 0.11 0.03
w/o Epi/Endo, 4C, H2O 0.67 0.13
Isopropanol 0.59 0.08 0.23 0.04 0.24 0.07
Acetone 1.38 0.22 1.07 0.21 0.87 0.25
30% TCA 1.43 0.08 2.28 0.20 1.96 0.06
1% NaOH 1.69 0.22 1.28 0.29 0.36 0.19
30% 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 epithelium
removed, not exposed to chemical, and incubated in MEM for 3 h. w/o Epi/Endo is
cornea with both epithelium and endothelium removed, not exposed to chemical,
and incubated in H2O at 4C. Values are mean SD, n = 510. (Data cited from
Ubels et al. [2000]. With permission of Elsevier Science.)
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18
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30 sec
14 1 min
mg H20 / mg cornea
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Figure 10.2 Corneal hydration (mg H2O/mg cornea) following exposure to test substances for
30 sec, 1 min, or 10 min and incubation in MEM for 3 h. Intact is untreated cornea
incubated in MEM. w/o Epi is cornea with epithelium removed, not exposed to
chemical, and incubated in MEM for 3 h. w/o Epi-Endo is cornea with both
epithelium and endothelium removed, not exposed to chemical, incubated in H2O
at 4rC. Mean SD, n = 5. Values for intact, without Epi, and without Epi-Endo
were all signicantly different from each other. 30-sec values and unmarked 1-
min values are not different than intact value. # Signicantly different than 30-sec
and 1-min values within group. * Signicantly different than 30-sec and 10-min
values within group. (ANOVA and Dunnetts test, P e0.05). (Data cited from Ubels
et al. [2000]. With permission of Elsevier Science.)
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Figure 10.3 Corneal endothelial cell layers stained with Alizarin Red S and trypan blue. Twenty
percent of the endothelial layer is damaged after mounting in the old corneal
holder (left), and none of the endothelial layer is damaged after mounting in the
new holder (right). The streaks of damaged cells exhibited after mounting in the
old holder are characteristic of wrinkling caused by the holder. Magnication 35v.
(Reproduced from Ubels et al. [2002]. With permission of Elsevier Science.)
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CHAPTER 11
Corneal Organ Culture for

Ocular Toxicity Test of Commercial
Hair Care Products
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METHODS
Corneal Organ Culture
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Culture Treatment
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Figure 11.1 Biotin surface labeling to visualize epithelial barrier. Cultured bovine cornea was
incubated with sulfo-NHS-LC-biotin for 30 min and then embedded in OCT, snap-
frozen, and sectioned (6 Qm). Cryostat sections (8 Qm) were (A) stained directly
with 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 the
corneal surface indicates functional epithelial TJ barrier in cultured corneas. Ep,
epithelium; BM, basement membrane; St, stroma that consists of broblasts. A
and B are mirror orientations of the same corneal sections.
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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 epithelium
was assessed by surface biotinylation as described in Figure 11.1. Inserts: corneal
sections stained directly with hematoxylin to reveal corneal morphology. Note:
extended biotinylation of the corneal surface caused by GA and GB exposure in
a concentration dependent manner. However, no disruption of TJ was observed
in GC treated cornea.
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response to consumer product challenge. Panels showed cultured corneas were
treated with different concentrations of three hair care products for 5 min, untreated
cells were used as control (C). The corneas were then cultured for 10 min without
the presence of the chemicals. Cell extracts from corneal epithelial cells treatment
were probed with 32P-labeled AP-1 (upper panel) or NF-OB (lower panel) consen-
sus oligonucleotide. EMSA experiments were repeated two times, and gels pre-
sented in the gure are from a representative set.
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CHAPTER 12
Human Corneal Equivalents

for In Vitro Testing
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embryonic dorsal root ganglion (not shown). Neurites, labeled with nerve-specic
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corneal periphery and branching into the cornea (C). White dashed line, corneal
periphery; arrows, parallel neurites; arrowheads, branching neurites.
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REFERENCES
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CHAPTER 13
The EpiOcular Prediction Model:

A Reproducible In Vitro Means
of Assessing Ocular Irritancy
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Viability of the Initial 20-Min Exposure
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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. ET-50 Draize
# tested (min) (MMAS)
1 Benzalkonium chloride (10%) 2.0% 1.07 108.0
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
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 32
21 Body/hand wash 20% 14.8 35
22 Eye gel/colorant 20% 240 0
23 Eye gel/colorant 20% 240 2
24 Face/body wash 20% 240 2
25 Face/body wash 20% 6.5 25
26 Face/body wash 20% 10.2 40
27 Hand/body lotion 20% 240 2
30 Shampoobaby 20% 30.8 10
31 Shampoobaby 20% 25.7 18
32 Conditioner 20% 240 2
33 Shampooregular 20% 6.0 30
34 Shampooregular 20% 8.7 35
35 Na2-ricinoleadmido MEA 20% 108.9 0
sulfosuccinate
36 Sodium trideceth sulfate 20% 2.5 33
37 Cetrimonium chloride 20% 116.9 6.67
38 Stearalkonium chloride 20% 240 14
39 Cocamide DEA 20% 240 0
40 Disodium cocoamphodipropionate 20% 11.2 15.3
41 Surfactant blend 20% 19.2 6.0
42 Surfactant blend 20% 40.4 2.67
43 Final formulation shampoo 20% 26.1 4.0
(continued)
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 Data
from ECETOC Database or Commercial Sources
Conc. ET-50 Draize
# tested (min) (MMAS)
53 Hydro-alcohol (hair spray) solution 20% 84.1 6.0
54 10% fatty alcohol ethoxylate 20% 189 3.5
55 Eye makeup remover (surfactant sol.) 20% 240 0
56 Lactic acid (3% solution) 20% 240 0
57 Oleic acid 20% 240 2
58 Skin care emulsion 20% 240 0
59 Body spray 20% 240 0
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RESULTS
Histological Characterization of the EpiOcular Tissue Model
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Figure 13.1 Hematoxylin and Eosin (H&E) stained histological cross sections of (A) EpiOcular
tissue model and (B) rabbit cornea epithelium and underlying stroma. Tissues
were xed in 10% formalin, embedded in parafn, and stained with H&E. Final
magnication 360v.
OCL-200 Prediction Equation

150
Draize Score (MMAS)

125 r = 0.90
59 materials
100
75
50
25
0
1 10 100 300
ET-50 (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)
Figure 13.2 Graphical depiction of in vivo and in vitro data used to derive the prediction
equation. All materials tested that had specic gravity >0.95 were diluted to 20%
in ultrapure water. If actual ET-50 exceeded 240 min, ET-50 was set equal to 240
min. If ET was less than 1 min, ET-50 was set to 1 min.
Determination of Prediction Model
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Table 13.3 EpiOcular Prediction Model Testing: Comparison between In Vivo and In
Vitro Predicted Draize
Predicted Actual Draize
Code Product Draize (MMAS)
1 10599 A Body wash 14.6 16.7
2 10599 B Body wash 31.3 44.7
3 10599 C Dishwashing liquid 51.3 38.3
4 10599 D Hand soap liquid 28.0 24.7
5 10599 E Dishwashing liquid 25.2 39.3
6 10599 F Facial soap 15.6 9.3
7 10599 G Dishwashing liquid 50.8 39.0
8 10599 H Dishwashing liquid 60.5 50.3
9 10599 I Laundry detergent 37.3 37.3
10 10599 J Laundry detergent 1.8 0.7
11 10599 K Laundry detergent 33.4 37.7
12 10599 L Dishwashing liquid 96.9 37.7
13 10599 M Shampoo 6.5 4.0
14 10599 N Shampoo 32.9 41.7
15 10599 O Shampoo 8.4 3.3
16 10599 P Hand soap liquid 18.1 13.3
17 10599 Q Skin lotion 1.8 0.7
18 10599 R Shampoo 46.9 33.7
19 10599 S Skin lotion 1.8 0.0
20 10599 T Shampoo 39.3 37.7
21 10599 U Body wash 41.2 33.0
22 10599 V Laundry detergent 1.8 0.7
23 10599 W Laundry detergent 39.9 44.0
24 10599 X Skin lotion 1.8 0.7
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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 on
the ET-50 using the prediction equation shown in Figure 13.2.
Table 13.4 EpiOcular QC Testing Results for Positive (0.3% Triton X-100) and
Negative (ultrapure water) Controls
Triton ET-50 Neg. Control Avg. Lot CV
Calendar Year Tissue Lots (min) (OD) (%)
2000 60 23.1s6.0 1.441 5.5
1999 84 22.6s5.0 1.433 5.6
1998 85 25.2s5.6 1.354 5.5
1997 81 22.9s4.7 1.343 5.4
1996 47 24.9s6.3 1.274 5.2
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Table 13.5 Results of Interlaboratory Testing

ET-50 (mins): Intralaboratory
Laboratory: BAK SDS Triton Reproducibility Avg. CV (%)
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
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 1.1
5.00% 83.8 1.0
1.00% 45.3 5.9
0.30% 8.67 28.9
0.10% 0 212.7
0.03% 0 2053.0
Sodium dodecyl sulfate (SDS)

30.00% 60.5 2.1
15.00% 59.2 5.1
3.00% 16.0 9.0
1.00% 0.67 29.5
0.30% 0 740.1
0.10% 0 1938.3
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Figure 13.4 Use of EpiOcular to differentiate between very mild materials that cannot be
distinguished by the in vivo Draize test (MMAS < 1.0).
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REFERENCES
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CHAPTER 14
Three-Dimensional Construct of the Human

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RESULTS
Histologic Characteristics of the Three-Dimensional Corneal

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Figure 14.1 Light micrograph of a plastic section from a construct after 24 hr equilibration at
37C. The tissue organization reveals a stratied appearance with columnar basal
cells, dened wing cells, and attened supercial cells. Toluidine blue and basic
fuchsin, original magnication 160v.
Figure 14.2 Transmission electron micrograph of the construct after 24 hr equilibration at 37C.
The supercial cells give rise to numerous villus processes. The nucleus is oriented
parallel to the surface. Some small, spot-like junction arrangements are seen
between cells at the surface. Desmosomes are present between adjacent cells.
Original magnication 12,600v.
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 extensive
cytoskeletal network. Mitochondria surround the nucleus and desmosomes join
adjacent cells. Original magnication 10,000v.
Figure 14.4 High-power transmission electron micrograph of the basal membrane of a basal
cell revealing numerous mature hemidesmosomes. Also visible are the compo-
nents of the hemidesmosomes: the intracellular membrane placode, laments
radiating through the membrane, and the typical band in what would be the lamina
lucida. The amorphous band at the bottom is the polycarbonate support membrane
of the construct. Original magnication 75,000v.
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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-dimensional
construct (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 (Lane
1) 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 (Lane
4). The molecular weights of several cytokeratin isoforms recognized by the
respective antibodies are indicated below under AE1 and AE3, respectively.
Figure 14.6 Western blot of the 64 kDa cornea-specic keratin AE5 in the three-dimensional
construct. Lanes show the construct on Day 0 (immediately upon arrival), Day 1
(24 hr equilibration at 37C), and Day 2 (48 hr at 37C), as well as the fresh
human donor corneal epithelial cell positive control (+) and the rabbit lacrimal
gland negative control ().
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Figure 14.7 Western blot of laminin in the three-dimensional construct. One band is seen at
220 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.
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Figure 14.9 Top: Western blot analysis for phosphorylated (active) p42/p44 mitogen-activated
protein 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 intensity
level (activity) of p42/p44 with increasing concentrations of benzalkonium chloride
(BAK). The absence of activity with 0.1% benzalkonium chloride is likely due to
cell death resulting from toxic insult.
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CHAPTER 15
The Human Corneal Epithelial HCE-T TEP

Assay for Eye Irritation: Scientic Relevance
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Table 15.1 Similarities and Differences in the Endpoint Measured in the HCE-T TEP Assay and in the Draize Eye Irritation Test
Test Exposure Exposure Concentration Mechanism of Tissue
Species Test Tissue Route Time Tested Endpoint Measured Damage
Human Corneal Supercial to 5 min 5 concentrations Concentration of test material Cytotoxicity and junctional
(in vitro) epithelium cell cultures with highest equal causing 15% of uorescein disruption in the corneal
to that evaluated in to penetrate through the epithelium
Draize test corneal epithelium (FR85)
Rabbit Ocular Supercial to Until washed Concentration Cornea: area of damage and Area of corneal damage is
THE HUMAN CORNEAL HCE-T TEP ASSAY
(in vivo) surface eye out by dened by opacity related to epithelial

(cornea, blinking and toxicologist (neat Conjunctiva: cytotoxicity and junctional
conjunctiva tearing or diluted) chemosis, redness and disruption; opacity is related
and iris) (<5 min) discharge to penetration of test
Iris: degree of effect material into stroma.
Conjunctiva damage similar
to corneaepithelial toxicity
and penetration.
Irismaterial has
penetrated to cause more
severe damage.
167
tear film tight

junction
desmosomal
junction
stroma
Figure 15.2 Cross-sectional diagram of the human corneal epithelium. A drawing illustrating
the multiple cell layers, the apical tight junctions, and the intercellular desmosomal
junctions of the corneal epithelium.
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Figure 15.3 Cross sections of stratied HCE-T cultures visualized by transmission electron
microscopy following 5-min supercial exposures to (A) cell culture medium (KGM)
(5000v); and (B) 0.01% benzalkonium chloride (BAC) (6000v). Ap = apical surface
of the culture; M = collagen membrane. [Photos contributed by S.D. Dimitrijevich.]
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Figure 15.4 Cross sections of stratied HCE-T cultures, visualized by transmission electron
microscopy, which were xed in a special buffer to retain the mucin layer. The
dark-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 airliquid interface in serum-free medium containing 1.15 mM
calcium) in which the TER is high (tight junctions are intact); and (B) mucin is
found between the cells in this non-differentiated culture (maintained submerged
in serum-free medium containing 0.15 mM calcium) in which the TER is low (tight
junctions not intact).
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Day 1 Day 3
100
80
% of Control
60
40
TEP
20 MTT
TER
0
0.00 0.02 0.04 0.06 0.00 0.02 0.04 0.06
SDS(%) SDS(%)
Figure 15.5 Three assays were used to evaluate the dose-dependent effects of sodium dodecyl
sulfate (SDS) on HCE-T cultures on the day of treatment (day 1), and 48 hr later
(day 3). TEP, transepithelial permeability to uorescein; 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 triplicate
cultures, and the error bars are the standard deviations.
The Mechanism of Action of Fluorescein TEP in the Test System

Compared to the Human Eye
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30.0
Draize Data
25.0
20.0
15.0
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5.0
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20.000 0.000 20.000 40.000 60.000 80.000 100.000 120.000
24 Hr Fluorescein
Figure 15.6 Fluorescein staining data from rabbit eyes following surfactant-containing formu-
lation exposure correlates well with Draize tissue score data (modied maximum
average score, MMAS and modied maximum average corneal score, MMCS)
from the same animals. The error bars are the standard deviations; n = 56 rabbits
evaluated per Draize test; and n = 24 rabbits evaluated for uorescein staining
at 24 hr. [Data from Gettings et al., 1996.]
120
MMAS R2 = 0.632
MMCS R2 = 0.7057
100 24 Hr R2 = 0.7361
Fluorescein
80
Draize Data
60
40
20
0
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logFR85
Figure 15.7 The relationship between the in vivo 24-hour uorescein staining data and the in
vitro log FR85 values for the subset of the CTFA formulations that are represented
in the HCE-T TEP assay Prediction Model. The error bars are the standard
deviations; n = 2 TEP assays; n = 56 rabbits per Draize test; and n = 24 rabbits
per uorescein data.
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Table 15.2 Prevalidation Study Test Materials and Draize Data
Percent
Conc. Formula Surfactant Draize FHSA
Product Type Testeda Surfactant Ingredients (w/w) Class Scoresb Classc
Bubble bath 2.5% sodium laureth sulfate 25.0 anionic MAS 4.8
cocamidopropyl betaine 5.0 amphoteric CS 0.0
COS 0.0
Hair conditioner 100% stearalkonium chloride 1.14 cationic MAS 14.2
ceteth-24 1.0 nonionic CS 0.0
dimethyl stearamine 0.67 cationic COS 0.0
glyceryl stearate 0.44 nonionic
Cleansing gel 100% cocoamphodiacetate 15.0 amphoteric MAS 22.0 /+
THE HUMAN CORNEAL HCE-T TEP ASSAY
sodium nonoxynol-6- 6.0 anionic CS 10.0

phosphate 1.5 cationic COS 0.5
quaternium-26 1.5 nonionic
PEG-120-methyl glucose
dioleate
Shampoo 1 100% sodium lauryl sulfate 25.0 anionic MAS 37.8 +
disodium laureth 15.0 anionic CS 20.0
sulfosuccinate nonionic COS 1.0
butylene gylcol 5.0 nonionic
lauramide DEA 0.5
Shampoo 2 100% ammonium lauryl sulfate 12.0 anionic MAS 57.4 +
lauramide DEA 2.0 nonionic CS 38.0
ethoxydiglycol 0.4 COS 2.0
hydroxypropyl methylcellulose 0.15
a Concentration used in Draize test, and initial concentration for dilution in TEP assay.
b MAS is the maximum average score; CS is the corneal score; COS is the corneal opacity score.
c Federal Hazardous Substances Act (FHSA) classications: , negative; +, positive; /+ repeat test (FHSA, 1979).
177
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70
65
60
A
55
50
Draize MAS
45
40
35
30
25
20
15
10
5
0
0.8 0.4 0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2
LogFR85
70
65
60 B
55
50
Draize MAS
45
40
35
30
25
20
15
10
5
0
0.8 0.4 0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2
LogFR85
Figure 15.8 Overlay of the prevalidation study test results for ve test materials in the MAS
Prediction Model (PM). The solid line represents the nonlinear regression curve
of the MAS PM, and the dashed lines are the 95% condence intervals. (A)
Overlay of the 60 log FR85 values in the MAS PM. There are 12 values for each
of the ve test materials on the plot, but, due to overlap in the data, all 12 points
may not be distinct. (B) Fit of the average log FR85 value for each of the ve
test materials in the MAS PM. The result for each test material is the average of
12 assays.
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Table 15.3 The Nonirritant/Irritant Classication of the Five Test Materials as

Determined by the Draize Test and by the HCE-T TEP Assay
Draize Score TEP Assay TEP Assay TEP Assay
Test Material Classicationa MAS PMb CS PMb COS PMb
Bubble bath 1 1 1 1
Hair conditioner 1 1 1 1
Cleansing gel 2 2 2 1
Shampoo 1 2 2 2 2
Shampoo 2 2 2 2 2
a Draize classication was the same across the MAS, CS, and COS scores, except for the
cleansing gel which was an irritant by the MAS and CS, but a nonirritant by the COS.
b Draize scores for the classications: nonirritant MAS e 15; nonirritant CS < 5; nonirritant
COS < 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
Draize Predicted Predicted
Test Material Draize MASa Classb MASa Classb
Bubble bath 4.8 1 4.20 1
Hair conditionerc 14.2 1 1.82 1
Cleansing gel 22.0 2 16.15 2
Shampoo 1 37.8 3 34.62 3
Shampoo 2 57.4 4 41.18 3
a MAS, maximum average score.
b The four classication cutoffs for MAS are based on the scheme proposed by Kay and
Calandra (1962): MAS 015, minimal (class 1); MAS 15.125, mild (class 2); MAS
25.155, moderate (class 3); MAS > 55, severe (class 4).
c Hair conditioner was less water soluble than other test materials, which may account for
its underprediction when it was diluted.
SUMMARY
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QXPEHURIGLIIHUHQWH[SHULPHQWVFRQGXFWHGXVLQJWKHWHVWV\VWHPDQGZLWKUHOHYDQW
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KXPDQFRUQHDOUHVSRQVHWRH\HLUULWDQWV
Table 15.5 Variability in the Draize MAS Compared to the Predicted MAS for the
HCE-T TEP Assay Prevalidation Study Data
Intralab CV (%) for Intralab CV (%) for Interlab CV (%) for
Test Material Draize MASa Predicted MASb Predicted MASc
Bubble bath 22.82 9.94 6.03
10.06
4.94
Hair conditionerd 19.67 33.19 15.59
15.80
54.09
Cleansing gel 53.24 17.50 15.10
3.01
25.90
Shampoo 1 10.31 12.54 12.49
21.21
20.42
Shampoo 2 8.32 14.60 2.83
14.62
29.49
a Evaluated using ve 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). Coefcient of variation, CV; maximum average score,
MAS.
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CHAPTER 16
Alternative Methods
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Figure 17.2 Illustration of contact hypersensitivity (CH) mechanisms: induction and elicitation
phase. 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 cytokine
mediators which assist the LCs in the migration to the draining lymph node and
maturation into effective antigen-presenting cells. In the lymph node, LC to T
lymphocyte interactions occur, which is followed by lymphocyte proliferation. The
proliferation results in primed effector lymphocytes which maintain a memory for
the specic allergen. The elicitation phase of CH appears to involve two mech-
anisms of action. Initially, allergens cause direct cellular action releasing a series
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of the cellular inux into the site of allergen challenge. Additionally, the primed
lymphocytes are called into the area in a very specic response. These responses
and the cytokines, costimulatory factors, and adhesion molecules act to produce
the end results of erythema and edema. (Modied from Selgrade et al., 2001.)
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TEST METHODS
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Table 18.1 Acetochlor In Vivo (Mean dose distribution as Qg equivalents of acetochlor per cm2. Mean of four male rats.)
214
Carbon Skin Cage Absorbed

Exposure (hr) Wash Cover Filter Qg/cm2 % Urine Feces Wash Carcass Qg/cm2 %
3 Qg/cm2
0.5 0.22 0.2 0.1 0.1 4.00 0.01 0.001 0.001 0.1 0.1 3.79
1 2.2 0.2 0.1 0.2 6.00 0.01 0.001 0.001 0.1 0.1 4.28
2 2.0 0.2 0.2 0.2 7.17 0.01 0.001 0.001 0.2 0.2 7.32
4 1.8 0.2 0.3 0.2 7.93 0.02 0.001 0.01 0.3 0.3 9.93
10 1.2 0.3 0.4 0.3 9.63 0.2 0.01 0.01 0.4 0.6 19.55
24 0.9 0.4 0.7 0.4 12.53 0.5 0.2 0.02 0.2 0.9 31.37
42 Qg/cm2
0.5 34.1 3.0 1.4 3.18 0.004 0.001 0.01 1.7 1.7 3.95
1 32.3 3.2 1.3 2.99 0.02 0.001 0.01 2.5 2.5 5.98
2 34.9 4.1 1.2 2.78 0.02 0.005 0.3 1.8 1.9 4.38
4 31.1 4.9 1.2 2.81 0.2 0.003 0.01 3.6 3.9 9.24
10 22.1 5.6 1.2 2.66 2.1 0.3 0.4 7.0 9.8 23.06
24 15.1 8.1 0.9 1.83 5.5 2.1 0.6 4.4 12.6 29.64
270 Qg/cm2
0.5 212.0 24.2 9.2 3.40 0.01 0.01 0.04 12.9 12.9 4.79
1 204.9 21.7 8.7 3.21 0.02 0.01 0.05 15.1 15.2 5.63
2 216.9 23.7 13.3 4.91 0.02 0.01 0.05 8.1 8.2 3.03
4 218.5 21.0 8.7 3.21 0.7 0.01 0.06 12.3 13.0 4.83
10 190.4 28.2 10.8 3.99 3.5 0.1 0.56 21.1 25.2 17.52
2934 Qg/cm2
0.5 2571.9 285.9 74.2 2.53 0.1 0.3 0.4 40.3 40.9 1.39
1 2175.7 405.4 95.2 3.24 0.2 0.3 0.2 150.9 153.04 5.22
2 2322.7 355.2 98.9 3.37 0.5 0.1 0.6 98.5 99.6 3.39
4 2260.1 502.7 93.3 2.18 2.3 0.1 1.1 74.8 78.3 2.67
10 2261.4 435.5 88.3 3.01 15.2 0.2 1.6 114.3 131.3 4.47
24 1788.9 627.2 86.0 2.92 97.1 47.9 8.4 216.7 369.9 12.61
aAbsorbed is sum of urine, feces, cage wash, and carcass.
Source: Lythgoe, 1990a.
ALTERNATIVE TOXICOLOGICAL METHODS
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 Qg/cm2.)
Exposure Absorbed Skin Total

(hr) Qg/cm2 % Wash Qg/cm2 % Donor Loop Recovered
3.02 Qg/cm2
0.05 0.41 13.68 1.73 0.11 3.60 0.08 0.55 2.89
1 0.82 27.12 1.32 0.11 3.77 0.04 0.44 2.73
2 1.14 37.78 0.95 0.12 3.84 0.03 0.50 2.73
4 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 2.99
24 2.12 70.13 0.13 0.08 2.75 0.02 0.41 2.77
47.3 Qg/cm2
0.5 2.02 4.27 25.6 1.09 2.30 1.41 1.03 31.1
1 3.90 8.25 28.2 1.17 2.47 0.97 1.02 35.3
2 10.3 21.78 18.7 1.46 3.09 0.57 1.25 32.2
4 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 30.6
24 28.7 60.68 2.9 0.79 1.73 0.14 1.21 33.7
318 Qg/cm2
0.5 6.58 2.07 283 12.9 4.06 10.5 13.4 325
1 21.4 6.73 383 16.6 5.22 14.3 16.7 452
2 33.4 10.50 261 28.1 8.84 19.4 8.25 350
4 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 11.6 379
24 246 77.36 67 14.4 4.53 5.52 13.3 347
3095 Qg/cm2
1 17 0.55 2411 143 4.62 311 118 2999
2 104 3.36 3146 187 6.04 313 138 3858
4 137 4.43 2103 111 3.59 316 161 2828
10 264 8.53 2344 196 6.33 294 156 3253
24 757 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.
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Table 18.3 Acetochlor In Vitro (Mean dose distribution in vitro dermal absorption in
human skin. Mean of six skin samples. Results expressed as Qg/cm2.)
Exposure Absorbed Skin Total

(hr) Qg/cm2 % Wash Qg/cm2 % Donor Loop Recovered
3.02 Qg/cm2
2 0.015 0.486 2.36 0.273 9.04 0.035 0.359 3.04
10 0.074 2.44 2.61 0.271 9.98 0.136 0.194 3.28
24 0.261 8.63 1.54 0.092 3.03 0.098 0.244 2.24
47.3 Qg/cm2
2 0.140 0.296 34.2 1.89 3.99 3.12 2.61 41.9
10 1.70 3.59 30.4 4.43 9.37 3.86 2.31 42.7
24 3.63 7.68 26.2 1.63 3.45 2.80 3.09 37.4
318 Qg/cm2
2 0.816 0.256 239 18.9 5.94 70.4 22.4 352
10 5.69 1.79 221 12.1 3.82 91.8 15.8 347
24 43.6 13.7 245 18.2 5.72 62.3 15.8 384
3095 Qg/cm2
2 4.57 0.148 2796 51.5 1.67 772 260 3884
10 22.4 0.722 2057 80.5 2.60 896 259 3314
24 32.5 1.05 1669 38.4 1.24 671 217 2628
Source: Clowes and Scott, 1990b.
DISCUSSION
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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
Qg/cm2)
(Q 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 31.4
3.02 in vitro 13.7 27.1 37.8 45.3 71.2 70.1
42.5 in vivo 4.0 6.0 4.4 9.2 23.1 29.6
47.3 in vitro 4.3 8.2 21.8 33.8 43.8 60.7
270 in vivo 4.8 5.6 3.0 4.8 9.3 17.5
318 in vitro 2.1 6.7 10.5 25.4 57.2 77.4
2934 in vivo 1.4 5.2 3.4 2.7 4.5 12.6
3095 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 2.2
47.3/42.4 1.1 1.4 5.0 3.7 1.9 2.1
318/270 0.4 1.2 3.5 5.3 6.2 4.4
3095/2934 0.1 1.0 1.6 1.9 1.9
Dose 1 Dose 3
Dose 2 Dose 4
7
5
Ratio vitro/vivo
0
0 5 10 15 20 25
Exposure (h)
Figure 18.1 The ratio of the in vitro absorption to the in vivo absorption with exposure duration
in the rat. Dose ratios 1, 2, 3, and 4 are in order of low dose ratio to high dose
ratio. Data are from Table 18.4.
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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
Qg/cm2)
(Q 2.0 10.0 24.0
Exposure Duration (hr)

3.03 rat 37.8 71.2 70.1
human 0.49 2.44 8.63
47.3 rat 21.8 43.8 60.7
human 0.30 3.59 7.68
318 rat 10.5 57.2 77.4
human 0.26 1.79 13.7
3095 rat 3.4 8.5 24.5
human 0.15 0.72 1.05
Ratio Rat/Human
3.03 77.1 29.2 8.1
47.3 72.7 12.2 7.9
318 40.4 32.0 5.6
3095 22.7 11.8 23.3
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vitro R/H 47.3 wg/cm2

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vitro R/H 3095 wg/cm2
80
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30
20
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Duration of Exposure (h)
Figure 18.2 The ratio of the rat in vitro absorption to the human in vitro absorption with
exposure duration. Dose ratios 1, 2, 3, and 4 are in order of low dose ratio to high
dose ratio. Data are from Table 18.5.
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CHAPTER 19
A Molecular Diagnostic Approach

to Irritant or Allergic Patch
Testing Using the DermPatch
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Table 19.1 Skin Cytokins (adapted from Gerberick et al., 1998)

Constitutive or inducible expression in
Cytokines Langerhans cells Keratinocytes Fibroblasts
IL-1E + +
IL-1F + +
IL-3 +
IL-6 + + +
IL-7 +
IL-8 + +
IL-10 +
IL-12 +
IL-15 +
G-CSF + +
M-CSF +
GM-CSF + +
TGF-E +
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Table 19.2 mRNA Cytokine Proles from Human Skin Biopsy

or Human Cell Samples
ACD ICD
TNF-E increased increased
IFN-K increased increased
IL-2 increased increased
GM-CSF increased increased
IL-1E (human cells) dependent on allergen increased
IL-1F (human cells) increased increased
IL-4 increased not determined
IL-6 increased not determined
IL-10 increased no change
IL-12 p35 (human cells) no change no change
IL-12 p40 (human cells) increased no change
Source: Adapted from Wakem and Gaspari, 2000.
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Table 19.3 Mechanisms of Irritant versus Allergic Contact Dermatitis

Feature Allergic Irritant
Chemical agents low molecular weight, lipid acids, alkalies, surfactants,
soluble solvents, oxidants, enzymes
Concentration of the agent less critical more critical
Genetic predisposition ++++ ++
Sensitization and lag period necessary not necessary
Trigger interaction of antigen with damage to keratinocytes
primed T cells
Cytokine release ++++ +++
T-cell activation early ++++ later ++++
Mast-cell activation ++ ++
Langerhans cells increased decreased
Eicosanoid production ++ ++
Source: Adapted from Marks and DeLeo.
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 yes
Reaction delay after contact days minutes to hours
Spongiosis ++++ ++++
Dermal edema ++++ ++++
Necrotic keratinocytes ++++ ++++
Ballooning degeneration + +++
Lymphocytic inltrate ++++ ++++
Neurotrophilic inltrate + +++
Source: Adapted from Marks and DeLeo.
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CHAPTER 20
In Vitro Skin Equivalent Models

for Toxicity Testing
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INTRODUCTION
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IN VITRO SKIN EQUIVALENT MODELS FOR TOXICITY TESTING 231
Tissue Culture Well

Culture Insert
ALI Tissue
Membrane
Medium
Figure 20.1 Schematic representation of the airliquid interface (ALI) tissue culture technique.
Table 20.1 Epithelial Tissues Successfully Cultured at the ALI

Skin equivalentsa
Keratinocytes only
Keratinocytes plus broblastsb
Keratinocytes plus melanocytes
Keratinocytes plus Langerhans cells
Ocular corneal epitheliuma
Tracheal/bronchiala epithelium
Tracheal/bronchial submucosal glands
Vaginal epitheliuma,b
Gingival epitheliuma,b
a Cultured at MatTek Corp.
b In development.
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(A)
(B)
Figure 20.2 (A) Histological cross section of H&E stained EpiDerm-200. Magnication = 440v.
(B) Transmission electron micrograph of ruthenium tetroxide stained intercellular
lamellar lipid sheets (150,000v).
Figure 20.3 Top macroscopic view of MelanoDerm tissues containing normal human melano-
cytes derived from Black donor skin (400v magnication).
Asian (A)
Black (B)
Caucasian (C)
Figure 20.4 Development of pigmentation in MelanoDerm tissue produced with melanocytes

derived from various skin phototypes. The gure shows the top macroscopic view
of tissue inserts. Day 0 indicates the day of shipment of a fully differentiated tissue.
Pigmentation develops during additional culture of the fully differentiated tissue
by the end user.
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Figure 20.5 Pigmentation of MelanoDerm tissue (Black melanocytes) induced by E-MSH and
F-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 20
days following shipment of commercial MelanoDerm tissue.
Day: 3 7 10 14
Kojic Acidtopical
Control
Figure 20.6 Lightening effect of topical kojic acid on MelanoDerm tissue. Tissues containing
Black melanocytes were treated topically with 1% kojic acid for the indicated
number of days (25 Ql applied topically every other day). The top macroscopic
view of the tissues reveals the visually observable lightening effect.
Table 20.2 Lightening of MelanoDerm Tissue by Topical Treatment with

1% Kojic Acid
Qg/tissue)
Melanin content (Q
Treatment Day 10 Day 14
H 2O 19.9 35.3
1% kojic acid 11.4 18.3
Figure 20.7 EpiDerm-FT tissue contains normal human broblasts cultured within a collagen
type I dermal matrix. A fully differentiated epidermis derived from normal human
keratinocytes is cultured on the top of the dermis.
Figure 20.8 Immunohistochemical staining of HLA-DR on human Langerhans cells within (A)
ImmunoDerm tissue and (B) excised human skin.
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Figure 20.9 Phototoxicity of chlorpromazine. EpiDerm tissues were topically treated with the indi-
cated concentrations of chlorpromazine and incubated overnight. On the following
day tissues were either irradiated with 6 J/cm2 of UVA or kept in the dark. Tissues
were then rinsed, fed with fresh medium, and incubated overnight. Finally, tissue
viability was determined by the MTT assay. A decrease in tissue viability of 30% or
greater between the irradiated and nonirradiated tissues indicates a phototoxic effect.
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Table 20.3 EpiDerm-200 Triton X-100 ET50 Database Summary

EPI-200 EPI-200
Year Triton ET-50 Triton C.V. Lots Avg. C.V.
2000 a 6.76 16.4 89 6.2
1999 6.75 18.2 146 5.7
1998 7.24 17.9 175 9.2
1997 6.78 15.9 228 9.9
1996 6.74 14.6 184 9.6
1995 6.65 77.8 112 4.9
a Through September 2000.
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Figure 20.10 Changes in cancer-related gene expression following UVB-irradiation of Epi-

Derm tissue. AtlasTM human cancer cDNA expression array analysis of gene
expression changes induced in EpiDerm tissues 6 hr after irradiation with 175
mJ/cm2 UVB.
Table 20.4 UVB-Irradiation of EpiDerm Tissue

WAF-1 (+)
MAP Kinase p38 (+)
Growth-arrest-specic 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 ()
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20 25 30 35
Cycles
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 isolated
from EpiDerm tissue 20 hours following UVB-irradiation. () no irradiation. (+)
UVB-irradiated. The expected PlGF PCR product is 273 bp.
High-Throughput ALI Tissue Formats
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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 reservoir
to avoid cross-contamination of samples.
Figure 20.13 Total RNA-96 isolation kit designed for high throughput total RNA isolation from
EpiDerm-296.
Figure 20.14 Agarose gel electrophoresis of total RNA isolated from EpiDerm-296 with the
Total RNA-96 isolation kit. Average yield is approximately 10 Qg DNA-free total
RNA/EpiDerm-296 tissue.
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REFERENCES
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CHAPTER 21
Cellular Resistance of Tetrahymena

to Sulfur Mustard
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CELLULAR RESISTANCE OF TETRAHYMENA TO SULFUR MUSTARD 253
RESULTS
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100
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60
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CHO and HeLa

PBL
20
HEK
Tetrahymena
Yeast
0
10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1
SM Concentration [M]
Figure 21.1 Cell viability vs. [SM]. Viability of six different cell lines was analyzed over a range
of SM concentrations. Symbols are represented within the graph.
60
8.0 mM
4.0 mM
50 2.0 mM
0.5 - 1.0 mM
0.3 mM & Positive Control
40
Cell Number
30
20
10
0
0 10 20 30 40 50 60 70 80
Time (h)
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 72
hr. Symbols are represented within the graph.
DISCUSSION
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REFERENCES
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CHAPTER 22
Studies of Cellular Biochemical Changes

Induced in Human Cells by Sulfur Mustard
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Table 22.1 Problems Seen with DNA Isolation from HEK

Purity Level of DNA
DNA (260/280 Yield
Procedure Date Sample Ratio) (mg/ml)
Purication of HEK DNA using 8/4/98 Control 1.35 60
Boehringer Mannheim Kit for 8/18/98 Control 1.65 45
mammalian cells 8/24/98 Control 1.35 25
Purication of lymphocyte DNA 9/2/98 Control 1.82 189
using Boehringer Mannheim Kit 9/8/98 Control 1.81 185
for mammalian blood 9/29/98 Control 1.93 194
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10/13/98 Control 1.47 67
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mammalian cells (modied 11/5/98 Control 1.86 98
procedure)
Table 22.2 Purication of HEK DNA using Boehringer

Mannheim Kit for Mammalian Cells Exposed to
Sulfur Mustard (HD)
Purity Level of DNA DNA
Date Sample (260/280 Ratio) Yield
11/19/98 Control 1.81 122
50 mM HD 2.10 163
100 mM HD 1.89 150
300 mM HD 1.82 199
11/26/98 Control 1.85 194
50 mM HD 1.99 213
100 mM HD 1.86 198
300 mM HD 1.81 172
12/8/98 Control 1.98 183
50 mM HD 1.96 179
100 mM HD 1.91 176
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120
% of maximum response 100
80
60
40
20
0
1 2 4 6 9 24
Time post-HD (h)
100 M HD
10 M HD
HEK PARP enzyme activity

140
120
% of maximum response
100
80
60
40
20
0
1 2 4 6 24
Time post-HD (h)
x-axis vs. 100 M HD
x-axis vs. 10 M HD
Figure 22.1 Time-dependent response of PARP activity following HD exposure of (top) HeLa
cells and (bottom) HEK cells. Two concentrations of HD were used, 10 and 100 QM,
and PARP activities are presented as percent maximal response.
DISCUSSION
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1.8
1.7
0 200 400 600 800 1000 1200 1400
Time (s)
Figure 22.2 Increase in intracellular calcium in HEK (passage 2) exposed to 300 QM HD as

measured by 340:380 nm ratio of Fura-2 AM.
Table 22.3 Binding in HD-Exposed HEKa

Hrs post HD QM)
Dose HD (Q
C 1q 0 100 200 300
8
16 NT ++
24 + +++ +++
CD32 0 50 100 200

8 + + +
24 + ++ +++
a + = weak; ++ = moderate; +++ = intense; NT = not tested. Grading of staining
was judged visually by uorescence microscope and HEK controls not exposed
to HD were negative for uorescence.
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2000
1500
1000
500
0
-50 0 50 100 150 200 250 300 350
HD Concentration
HD-exposed HEK
Figure 22.3 Concentration-dependent increase in the secretion of IL-8 from HEK exposed to HD.
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REFERENCES
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CHAPTER 23
Human Keratinocyte Inammatory

Transcript Gene Activity
Following Sulfur Mustard*
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Table 23.1 HEK Housekeeping Gene Transcripts at 16 Hr Following Sulfur Mustard

Gene Ratioa
Control
Gene Transcript Intensityb 25 QM SM 200 QM SM
14-3-3 zeta protein 317 0.7 0.44
23 kDa Highly Basic Protein 1689 2.0 2.3
E-Tubulin 307 0.57 1.4
F-Actin 236 1.2 1.2
Glyceraldehyde 3-phosphate dehydrogenase 612 0.44 1.1
HLA class I histocompatibility antigen C-4 487 0.94 1.0
alpha chain
Hypoxanthine-guanine 59 0.9 3.7
phosphoribosyltransferase
Ribosomal Protein S9 978 1.2 2.1
Ubiquitin 2064 0.8 0.73
a Induction ratio from phosphorimage densitometeric measurements of listed gene transcript
normalized to the sum total of the detected transcript intensity/pixel density.
b Control intensity (grayscale pixel density) adjusted from background. Background was set
at the median intensity of the blank space between the six array panels. The subtracted
background intensity ranged from 19 to 43.
Table 23.2 HEK Inammation-Associated Transcripts at 16 Hr Following Sulfur Mustard

Gene Ratioa
Control
Gene Transcript Intensityb 25 QM SM 200 QM SM
CD40 86 4.9 0.3
Interleukin 1 E 67 0.1 1.4
Interleukin 1 F 101 0.6 3.5
Interleukin 2 receptor E subunit 148 0.3 0.4
Interleukin 6 494 0.7 0.6
Interleukin 7 receptor E subunit 131 0.4 0.7
Interleukin 8 16 c 20
Interleukin 13 349 3.0 0.7
Interleukin 15 215 0.03
Macrophage inammatory protein 2E 11 13
S19 ribosomal protein 886 0.9 2.1
Tumor necrosis factor E 50 1.4
a Induction ratio from phosphorimage densitometeric measurements of listed gene transcript
normalized to the sum total of the detected transcript density.
b Control intensity (grayscale pixel density) adjusted from background. Background was set
at the median intensity of the blank space between the six array panels. The subtracted
background intensity ranged from 19 to 43.
c Transcript not detected above background.
Figure 23.1 Atlas cDNA array nylon blot image. HEK were exposed to 200 QM sulfur mustard
for 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 housekeeping
genes at the bottom of the array (see 3 housekeeping genes in rectangular box).
Boxes 1 and 2 show expression of macrophage inammatory protein 2E and
interleukin 8, respectively. These inammatory transcripts were at very low expres-
sion levels in control HEK.
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24 hr at 37SC. Viabilities were determined by using these dyes as described in
the Experimental section.
DISCUSSION
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CHAPTER 25
Comparison of Spectrophotometric and

Fluorometric Assays of Proteolysis in
Cultured Human Cells Exposed
to Sulfur Mustard
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INTRODUCTION
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Chromozym Substrates
Chromozym TH Tosyl-glycyl-prolyl-arginine-4-nitranilide acetate
Chromozym t-PA N-Methylsulfonyl-D-Phe-Gly-Arg-4-nitranilide acetate
Chromozym TRY Carbobenzoxy- valyl-glycyl-arginine-4-nitrilanilide acetate
Chromozym U Benzoyl--alanyl-glycyl-arginine-4-nitranilide acetate
CellProbe Enzyme Substrates:

Nonfluorescent (aa)x -Fl Fluorescent Dye +
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Substrate leaving group
Enzyme Substrate
AAPV- Elastase (AAPV) Dl-Alanyl-Alanyl-Prolyl-Rho 110
D-aminopeptidase A (DAA) Asp-Asp-Rho 110
K-Aminopeptidase B (KAB) Lysine-Lysine-Rho 110
G-Aminopeptidase (GA) Gly-Gly-Rho 100
L- Aminopeptidase (LA) Leu-Leu-Rho
A- Aminopeptidase M (AAM) Alanine-Alanine-Rho 110
R- Aminopeptidase B (RAB) Arginine-Arginine-Rho 110
Figure 25.1 Substrate descriptions.
Chromozym Assay
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Figure 25.2 Protease activity was measured in two donors as a function of cell number.
Increasing the cell concentration above 2 v 106/well did not increase protease
activity and this concentration was used in all subsequent studies.
Figure 25.3 PBL from ve different donors were exposed to 250 QM HD and then incubated
for 24 hr. Protease activity using the Chromozym TH substrate was measured
as described in Material and Methods. Signicant protease activity occurred
reproducibly. All assays were run in triplicate and all donors were evaluated
multiple times on different days.
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 only
the Chromozym U and Chromozym t-PA substrates were signicantly hydrolyzed.
Figure 25.5 HEK were exposed to HD at the indicated concentrations, and protease activity
was measured uorometrically 24 hr later as described in the Material and Meth-
ods section. KAB and GA substrates were signicantly hydrolyzed above the
control values while elastase (AAPV) and DAA were decreased.
SUMMARY
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REFERENCES
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CHAPTER 26
Effects of Low Dose Sulfur Mustard

on Growth and DNA Damage
in Human Cells in Culture
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10
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0 10 20 30 40 50 60
M HD
HD
HD-HP
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 concentrations
of 5 QM or greater inhibited expression of SSB in the comet assay. Points represent
mean SEM from two experiments.
40
30
Comet Moment
20
10
0
0 10 20 30 40 50 60
M HD
HD
HD-HP
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 similar
to those seen with PBLs. Points represent mean SEM from two experiments.
40
30
Comet Moment
20
10
0
0 10 20 30 40 50 60
M CEES
CEES
CEES-HP
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 not
inhibit expression of H2O2-induced SSB. Points represent mean SEM from two
experiments.
40
30
Comet Moment
20
10
0
0 200 400 600 800 1000 1200
M CEES
CEES
CEES-HP
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 to
those seen with PBLs. Points represent mean SEM from two experiments.
70
60
50
Comet Moment
40
30
20
10
0 10 20 30 40 50 60
M HD
HD
HD and H2O2
Figure 26.5 HEKs were exposed to buffer or to buffer plus HD. Eighteen hours following
exposure, cells were harvested and treated with 0.002% H2O2 or buffer. Points
represent mean SEM from two experiments.
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Figure 26.6 HEKs were exposed to buffer or to buffer plus HD. Twenty-four hours following
exposure, cells were harvested and treated with 0.002% H2O2 or buffer. Points
represent mean SEM from two experiments.
10x10 6
Viable Cells/mL
1x10 6
100x10 3
20 30 40 50 60 70 80 90 100
Time (h)
0.00 M HD
0.01 M HD
0.10 M HD
0.50 M HD
1.00 M HD
5.00 M HD
Figure 26.7 Growth curves for HEKs following exposure to HD in culture. Lowest line is for
5 QM concentration.
REFERENCES
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CHAPTER 27
Imaging Sulfur Mustard Lesions in Basal

Cells and Human Epidermal Tissues
by Confocal and Multiphoton
Laser Scanning Microscopy
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INTRODUCTION
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Figure 27.1 Basal cell adhesion complex. A microvesicle (lower left panel) showing details of
the dermalepidermal separations characteristic of a sulfur mustard blister.
Hemidesmosomes (arrowhead), at the roof of the blister, are well displaced from
the basement membrane (bm) and lamina densa (ld). An expanded model of the
intact adhesion complex (circumscribed area) includes the intracellular keratin
laments K5 and K14 and their facilitated attachments to the transmembrane E6F4
integrin receptors. The exodomains of E6F4 are shown linked by laminin 5 to the
basement membrane zone.
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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 laments
within the cell cytoplasm. Image intensity and K5 concentration were greatest
around the nucleus of each cell, and a lacy network of delicate laments projected
out toward the cell extremities. Analysis of confocal images from K5 controls (B)
and HD-exposed populations (C) showed a statistically signicant (p < 0.01) 29.2%
decrease in intensity at 1 hr postexposure to sulfur mustard.
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Figure 27.3 Multiphoton keratin 14 images from control HEK cultures (A) showed elaborate
cytoskeletal distribution comparable to that of keratin 5. Image intensity and K14
concentration were greatest around the nucleus. Lacy networks of laments pro-
jected out to the cell extremities where they interfaced closely with those of
adjacent cells (arrow). At 1 hr postexposure to sulfur mustard, K14 images (B)
indicated a disruption of organization, resulting in withdrawal of laments from the
plasma membrane margins, appearance of punctate nodules (arrows), and a
substantial loss of cytoskeletal denition.
Figure 27.4 Analysis of K14 confocal images from replicate cultures of HEK sham-treated
controls (A and C) and HD-exposed populations (B and D) showed a statistically
signicant (p < 0.01) 30.14% decrease in image intensity (K14 expression) at 1
hr postexposure and a nearly complete loss of expression (79% decrease in image
intensity) at 2 hr.
Figure 27.5 A multiphoton montage showing the organization of E6 integrins on an explant of

human epidermis. Serial slices (A and B) illustrate the receptor outline on suc-
cessive cross sections through the epidermal rete pegs. The three-dimensional
reconstruction (C) and the stereo image (D) are from the same Z-series of serial
slices. Together, they show the topographic complexity of ventral epidermis plus
the circular shape and extensive distribution of E6F4-integrin receptors.
Figure 27.6 A multiphoton image (slice) of

human epidermis showing in
cross section the distribution
and circular shape of F4 inte-
grins (arrows) on the basal
cell surface.
Figure 27.7 Multiphoton images of E6F4 receptors in human epidermis exposed to sulfur
mustard indicated unraveling and loss of circular shape at 1 hr postexposure (A)
and an almost total loss of E6F4 expression at the basal cell surface by 2 hr
postexposure (B, C). At 2 hr postexposure, only a basolateral pattern of residual
uorescence remained to outline the constituent basal cells. Loss of E6 and F4
integrin also occurred spontaneously in epidermal tissues following dermalepi-
dermal separation; therefore, the effects may not be strictly related to sulfur
mustard exposure.
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Figure 27.8 Analysis of confocal images from HEK in replicate control and HD-exposed cul-
tures indicated a statistically signicant (p < 0.01) decrease of 27.3% and 26.3%
in image intensity of E6 and F4 integrins, respectively, at 1 hr postexposure. The
decrease was characterized by a loss of uorescence from the surface of attached
basal cells, resulting in a honeycomb pattern of residual, basolateral uorescence.
Postexposure image patterns from cultures were very similar to those recorded
from intact epidermal tissues (see Figures 27.7B,C).
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CHAPTER 28
Suppression of Sulfur Mustard-Increased

IL-8 in Human Keratinocyte Cell Cultures
by Serine Protease Inhibitors: Implications
for Toxicity and Medical Countermeasures*
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MUSTARD-INDUCED IL-8 315
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-Inammatory Drugs
Fluphenazine dihydrochloride 50
Indomethacin 63 96
Olvanil 53 91
Hydrocortisone 65 71
Olvanil (saturated) 53
Retro olvanil 62 84
Olvanil (urea analog) 81
Octyl homovanillamide 65 100
Dexamethasone 72
Scavenger Drugs
2-Mercaptopyridine-1-oxide 65
6-Methyl-2-Mercaptopyridine-1-oxide 56
4-Methyl-2-Mercaptopyridine-1-oxide 57 94
Dimercaprol 43 92
Protease Inhibitor
1-(4-aminophenyl)-3-(4-chlorophenyl)urea HCl 54
N-(OP)-L-Ala-L-Ala-benzy ester hydrate 63
1(G-T)-4-(4-methyl phenylsemithiocarbazide) 50
PADPRP Inhibitor
3-(4d-Bromophenyl)ureidobenzamide 74
Benzoylene urea 54
Other
Hydrogen peroxide gel, 3% 58
Data generated by the U.S. Army Medical Research Institute of Chemical Defense
Anti-vesicant Drug Screen.
Sulfur Mustard Exposure
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Figure 28.2 Percent IL-8 of HD-exposed HEK following treatment with 1579.
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Proteins
Inflammatory
Cells
Membranes HD DNA Strand Breaks PARP
Ca++ Inflam NAD+

depletion
Protease Metabolic Inhibition of

release Disruption glycolysis
CELLULAR
TISSUE
Breakdown of Toxicity
epithelial Memb
attachment
Ca++
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separation
Inflam Hyd
VESICATION
Figure 29.1 The cellular and tissue alterations induced by HD that are proposed to result in
blister formation. HD can have many direct effects such as alkylation of proteins
and membrane components (Memb), as well as activation of inammatory cells.
One of the main macromolecular targets is DNA, with subsequent activation of
poly(ADP-ribose) polymerase (PARP). Activation of PARP can initiate a series of
metabolic changes culminating in protease activation. Within the tissue, the pen-
ultimate event is the epidermaldermal separation that occurs in the lamina lucida
of the basement membrane zone. Accompanied by a major inammatory response
and changes in the tissue hydrodynamics (Hyd), uid lls the cavity formed at this
cleavage 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 cysteine
DNA strand breaks Cell cycle inhibitors Mimosine
PARP activation PARP inhibitors Niacinamide
Disruption of calcium Calcium modulators BAPTAa
Proteolytic activation Protease inhibitors AEBSFa
Inammation Antiinammatories Indomethacin; Olvanil
a BAPTA is a calcium chelator; AEBSF is a sulfonyl uoride compound.
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Table 29.2 Candidate Countermeasures with Greater Than 50%

Efcacy in Mouse Ear Model
Percentage Reduction
in Pathology
Anti-Inammatory Drugs
Fluphenazine dihydrochloride 50
Indomethacin 96
Olvanil 91
Olvanil (saturated) 53
Retro olvanil 84
Olvanil (urea analog) 81
Octyl homovanillamide 100
Scavenger Drugs
2-Mercaptopyridine-1-oxide 66
6-Methyl-2-mercaptopyridine-1-oxide 56
4-Methyl-2-mercaptopyridine-1-oxide 94
Dimercaprol 78
Na 3-sulfonatopropyl glutathionyl disulde 64
Hydrogen peroxide gel, 3% 58
Protease Inhibitors
1-(4-aminophenyl)-3-(4-chlorophenyl) urea 54
N-(OP)-L-Ala-L-Ala-benzy ester hydrate 62
Ethyl p-guanidino benzoate hydrochloride 62
PARP Inhibitors
3-(4d-Bromophenyl)ureidobenzamide 74
Benzoylene urea 54
4-Amino-1-naphthol hydrochloride tech 80
Total number of positive compounds = 19
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REFERENCES
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CHAPTER 30
Molecular Neurotoxicology
of 6-Hydroxydopamine
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CHAPTER 31
A Microassay Method Using

a Neuroblastoma Cell Line to Examine
Neurotoxicity of Organophosphate Mixtures
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0.400 31,000
0.200 16,000
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Time (min)
Figure 31.1 Determination of optimal cell number per well and reaction time for AChE activity.
Concentration Dichlorvos (M)

Control 1.E-02 5.E-02 1.E-01 5.E-01 1.E+00 5.E+00 1.E+01
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Absorbance (405 nm)
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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 (M)
Paraoxon Dichlorvos
Figure 31.2 Effect of dichlorvos or paraoxon on AChE activity of SH-SY5Y neuroblastomas.

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1.0 D/10-4 Px
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10-1 D/10-3 Px
10-1 D/10-4 Px
10-1 D/10-5 Px
10-2 D/10-3 Px
10-2 D/10-4 Px
10-2 D/10-5 Px
M Dichlorvos (D) or Paraoxon (Px)
Figure 31.3 Effect of dichlorvos (D) or paraoxon (Px) alone or in combination on AChE activity
of SH-SY5Y neuroblastomas. *p < 0.05.
IN VITRO ASSESSMENT OF ORGANOPHOSPHATE NEUROTOXICITY 345
Table 31.1 Effect of Paraoxon (Px) or Dichlorvos (D) on AChE Activity of SH-SY5Y
Neuroblastomas, Signicance Level 0.05
OP Absorbance
concentration at 405 nm Measured Predicted Signicant
QM)
(Q SEM inhibition (%) inhibition (%) vs. control?
control 1.269 0.031
102 D 1.373 0.091 8
101 D 1.198 0.033 6
1.0 D 0.831 0.023 34 x
105 Px 1.352 0.081 7
104 Px 0.811 0.086 36 x
103 Px 0.362 0.024 72 x
102 D/105 Px 1.332 0.042 5 15
102 D/104 Px 0.630 0.077 50 28 x
102 D/103 Px 0.418 0.017 67 63 x
101 D/105 Px 1.370 0.167 8 1
101 D/104 Px 0.647 0.104 49 42 x
101 D/103 Px 0.453 0.027 64 77 x
1.0 D/105 Px 0.432 0.151 66 28 x
1.0 D/104 Px 0.590 0.156 54 71 x
1.0 D/103 Px 0.339 0.052 73 106 x
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REFERENCES
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CHAPTER 32
Development of Integrin Expression

as a Molecular Biomarker for Early,
Sensitive Detection of Neurotoxicity
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INTEGRIN EXPRESSION AS A BIOMARKER FOR NEUROTOXICITY 349
Figure 32.1 Integrin heterodimers transduce signals both into and out of the cell via interactions
with extracellular ligands and cytoplasmic accessory or regulatory proteins, the
cytoskeleton, 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).
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Ligand Accessory Protein
Receptor
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Cell Re-Organization of Activation of Signal Gene

Migration Cytoskeletal Proteins Transduction Pathways Expression
Tissue Cell
Organization Neurite Cell Survival
Elongation Cell - Cell Cycle
Communication
Figure 32.2 Integrin-mediated cell functions.
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Co-Cultures
Neuropathic TMT, MeHg
Test
Known Axonpathic Acrylamide
Agents
Synaptopathic MDMA
Validate
Known In Vivo TMT, MeHg
Agents
Unknown
In Vitro
Test Mechanisms
New
Compounds Suspected
In Vivo
Neurotoxicants
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).
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REFERENCES
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CHAPTER 33
Two-Photon Fluorescence Microscopy:

A Review of Recent Advances
in Deep-Tissue Imaging
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Figure 33.1 Jablonski diagrams for (a) one-photon and (b) two-photon excitation. One-photon
excitation occurs through the absorption of a single photon. The two-photon
process occurs through the simultaneous absorption of two lower energy photons.
After either excitation process, the uorophore relaxes to the lowest energy level
of the rst excited electronic state. The subsequent uorescence emission process
is independent of the mode of excitation.
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Figure 33.5 Two independent spectral components isolated in ex vivo human skin based on
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prole of this component.
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CHAPTER 34
The Application of Genomics and

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posttranslational modification
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Protein identity mRNA expressed
Research lead Toxicological marker Toxicant signature
Figure 34.1 Schematic of genomic and proteomic technologies: The rst step in toxicoge-
nomics (right) is the construction of a microarray that involves the amplication
by PCR and the immobilization of known DNA sequences (either cDNA or
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model can be labeled and hybridized to the microarray and visualized using
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software allows determination of the extent of hybridization of the labeled probes
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isoelectric point (rst dimension) and molecular weight (second dimension),
allowing bioinformatic analysis of differences in protein expression of treated
versus untreated samples. Therefore, there is no preliminary work such as array
construction for proteomic studies (left), but proteins with altered expression
have to be identied subsequently. Individual proteins of interest are excised
from two-dimensional-gels, digested with trypsin, and applied to a mass spec-
trometer. Identication of these proteins is obtained by searching protein data-
bases with mass spectrometry data.
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Table 35.1 IARC Class I or 2A Human Carcinogen, or NTP Reasonably Anticipated

Human Carcinogen
Tg.AC
Topical TgrasH2 p53+/ XPA//p53+/
Genotoxic
Benzene + + + nd*
Benzo(a)pyrene + nd + +
Cyclophosphamide + + nd
7,12-Dimethylbenzanthracene + nd + +**
Melphalan +/ + nd
Phenacetin +
Procarbazine nd + nd nd
Nongenotoxic
Cyclosporin A + +/ + +
Diethylstilbestrol + + + +
17-F-estradiol (or ethinyl estradiol#) +# +/ +
Oxymetholone + nd nd
2,3,7,8-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.AC
Topical TgrasH2 P53+/ XPA//P53+/
p-Cresidine + + +
2,4-Diaminotoluene + nd nd
Diethylnitrosamine nd + nd nd
Dimethylnitrosamine nd + + nd
N-Ethylnitrosourea nd + + nd
Glycidol + nd
N-Methylnitrosourea nd + + nd
Phenolphthalein nd + nd
Thiotepa nd + nd nd
Urethane nd + + nd
4-Vinyl-1-cyclohexene-diepoxide* + + nd
* Applied dermally to each model tested.
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Table 35.3 Nongenotoxic Rodent Carcinogens and Human

Carcinogenicity Unlikely or Uncertain
Tg.AC
Chlorpromazine nd nd nd
Clobrate + + nd
Dieldrin nd nd nd
Diethylhexylphthalate + +/
Haloperidol nd
D-Limonene nd nd nd
Metaproterenol nd nd
Pentachlorophenol + nd nd
Phenobarbital
Reserpine
Sulfamethoxazole
WY-14643 + +*
* Positive in 6 month XPA/, not tested in XPA//p53+/ bitransgenic.
Table 35.4 Rodent Noncarcinogens

Tg.AC
Genotoxic
p-Anisidine nd
2-Chloroethanol nd nd nd
1-Chloro-2-propanol nd nd
2,6-Diaminotoluene nd nd
8-Hydroxy-quinoline nd
Nongenotoxic
Ampicillin nd nd nd
Benzethonium chloride nd nd nd
D-Mannitol nd nd
Oleic acid diethanolamine nd nd
Phenol nd nd nd
Resorcinol + nd
Rotenone nd
Sulsoxazole nd nd
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CHAPTER 36
Changes in Gene Expression after Exposure

to Organophosphorus (OP) Agents
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Figure 36.1 Rat toxicology U34 gene array. This gene expression display was created by
GeneSpring (Silicon Genetics) gene array analysis software from our data (rat
brain RNA 1 h postexposure to CPF) read from an Affymetrix Rat Toxicology U34
GeneChip. In the original display (depicted in gray tones here), red and purple
blocks represented up-regulated genes and the blue represented down-regulated
genes. Gray blocks represented genes whose expression is essentially the same
as in the control animal.
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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, then
return 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.
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CPF-exposed rats.
Figure 36.4 Scatterplot of data from rat toxicology U34 GeneChip.
Identication of Up-Regulated Genes Involved in Key Cellular Functions

and Biochemical Pathways
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DISCUSSION
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CHAPTER 37
Archival Data in Toxicology: Minimizing

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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 the
effective dosage, after detoxication and loss of the 30% of inhaled HCN that is
exhaled (Moore and Gates, 1946), leaving 0.7 retained. HCN concentrations up
to 30 mg/m3 are normally neutralized by human detoxication systems before
effects become observable (Prentiss, 1937).
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Figure 37.2 Contrasting plots of data from the same rat exposures to HCN (after Ballantyne,
1987). One plot (left) reects use of exposures with xed duration and varied HCN
concentrations. The steep initial slope shows that HCN must be very concentrated
for a small number of breaths to deliver a lethal dosage. The second plot presents
the data results as customarily displayed, given a xed toxic gas concentration
and varied exposure durations. LCt50 values tend to increase as more time
becomes available for detoxication.
Figure 37.3 Comparison of responses from different strains of rats under similar HCN exposure
conditions (after Levin et al., 1985). Loglog plots of the data reveal differences
of effective HCN concentrations and probit slopes of lines but do not show which
strain better represents the human race.
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Figure 37.4 Relationships of anatomical and pharmacological factors leading to lethality for
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move the short distance from alveoli to the sensor. The sensor response (or lack
of a signal) communicates a need for oxygen to respiratory neurones that initiate
hyperventilation. Increased minute volumes multiply HCN intake until respiratory
center poisoning leads to apnea.
Mechanisms and Rationales
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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., 1985
9,300b Men, 11/11 hyperventilate Bodansky and Helm, 1944
7,740b Men, most hyperventilate Cope and Abramovitz, 1959
4,000a Rat, inhalation LC50 Ballantyne, 1987
3,200b Men, 50% hyperventilate Wexler et al., 1947
2,230 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.
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pigs exposed to HCN for 2 min (after Stemler et al., 1994). Values for blood
concentrations of cyanide, at the top and right side of Figure 37.5, are presented
with the corresponding respiratory rate values. Although data acquired after the
onset of HCN exposure for 2 min are very limited, they are consistent with other
indicators that an aortic blood HCN concentration of 4 mg/m3 at 5 min is a threshold
value for a lethal outcome.
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slow infusion trials (1 mg/kg/min) with NaCN solution at 4.0 mg/ml (after Vick
et al., 2000). RA indicates respiratory arrest and cessation of NaCN infusion.
Methemoglobin formation by 20 mg/ml hydroxylamine hydrochloride solution was
initiated 30 sec after RA. Although 3.6 Qg/kg was the average value observed
at RA, it appears that survival is dependent upon avoidance of blood cyanide
concentrations above 4 Qg/kg.
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Figure 37.8 Bar chart designed to illustrate relative severity of enteric disease effects as
correlated with time after arrival of a mixed population in Mexico City. Each of the
19 represented cases involved isolation of enterotoxigenic Escherichia coli organ-
isms that accounted for 45% of travelers diarrhea cases observed during this
prospective study (Merson et al., 1976).
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 coli
Bacteriaa
Percentage of cases with degraded military performance
Day of % New Recoveredg
Onsetb Casesc No.d (25%)c No.e (50%)c No.f (100%)c (%)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 coli
infection 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 travelers 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.
SUMMARY
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Figure 37.9 This cartoon is included to suggest that modeling represents a combination of art
and science. The art of modeling methodology is applied when conventional testing
methods are not applicable for collection of experimental results. In each case,
previously collected data were reacquired and analyzed to provide a best possible
estimate of reality.
REFERENCES
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CHAPTER 38
Information Management at the Library of

Congress: An Overview with Special
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010 _a sh 85064594 Record Number
053 _a RC582.17 Class Number
150 _a Immunotoxicology Subject Heading
450 _a Immunologic toxicology UF (Use For Term)
450 _a Immunotoxicity UF (Use For Term)
550 _w g _a Immunopathology BT (Broader Term)
550 _w g _a Toxicology BT (Broader Term)
550 _a Immunopharmacology RT (Related Term)
Figure 38.1 LC authority record for Immunotoxicology, supplemented with explanatory notations.
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Genetic toxicology / editors, Albert P. Li, Robert H. Heich. --

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. Heich, 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/DLC
for Library of Congress
Figure 38.2 Traditional LC catalog card for Genetic Toxicology, Li, AP, and Heich, RH,
editors, 1991.
0$5&:HEVLWHKWWSOFZHEORFJRYPDUFXPE-DQXDU\
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. Heich.
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 Heich, 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 Heich, RH, editors, 1991.
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Genetic toxicology / editors, Albert P. Li, Robert H. Heich
LC Control Number: 90011279

Main Title: Genetic toxicology / editors, Albert P. Li, Robert H. Heich
Published/Created: Boca Raton : CRC Press, c1991.
Related Names: Li, A. P.
Heich, Robert H., 1946-
Description: x, 493 p.,: ill.; 24 cm.
ISBN: 0849388153
Notes: Includes index.
Includes bibliographical references.
Subjects: Genetic toxicology.
Carcinogens.
Chromosome Abnormalities--chemically induced.
Mutagens--adverse effects.
Mutation.
LC Classication: RA1224.3 .G457 1991
NLM 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 Heich,
editors, 1991.
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World Wide Web Biomedical, Chemical,

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In Silico Approaches for Physiologically

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IN SILICO APPROACHES FOR PBPK MODEL PARAMETERS
In Silico Approaches for Tissue:Air Partition Coefcients
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Table 40.1 In Silico Approaches for Estimating the Tissue:Air Partition Coefcients (P) of Chemicals
Approacha Speciesb Chemical Classc Reference
QSARs: LFE-type equations
Electrostatic descriptors
H Inert Gases; LMWVOCs Abraham and Weathersby
log Padipose:air = 0.294 0.172R2 + 0.729 T + + 1.7474 E + + 0.219 F + +
(1994)
0.895 log Phe:a
log Pbrain:air = 1.074 + 0.427R2 + 0.286 T + + 2.781 E + + 2.787 F + +
(1994)
0.609 log Phe:a
log Pheart:air = 1.208 + 0.128R2 + 0.987 T + + 0.643 E + + 1.783 F + +
(1994)
0.597 log Phe:a
log Pkidney:air = 1.084 + 0.417R2 + 0.226 T + + 3.624 E + + 2.926 F + +
(1994)
0.534 log Phe:a
IN SILICO APPROACHES FOR PBPK MODELING

log Pliver:air = 1.031 + 0.059R2 + 0.774 T + + 0.593 E + + 1.049 F + +
(1994)
0.654 log Phe:a
log Plung:air = 1.300 + 0.667R2 + 0.680 T + + 3.539 E + + 3.35 F + + 0.458
(1994)
log Phe:a
log Pmuscle:air = 1.14 + 0.544R2 + 0.216 T + + 3.4714 E + + 2.924 F + +
(1994)
0.578 log Phe:a
Steric descriptors
R Haloalkanes Gargas et al. (1988)
log Padipose:air = (0.7341xv) (0.029 [ VY ) (1.57(1/1x)) (0.559(1/1xv))
0.0983 [ FY + 2.213
(continued)
485
Table 40.1 (continued) In Silico Approaches for Estimating the Tissue:Air Partition Coefcients (P) of Chemicals
486

R Haloalkanes Csanady and Laib (1990)
log Padipose:air = 0.7341Xv 0.0291 [ VY 1.570/1Xv 0.5591Xv 0.0984 [ FY
+ 2.213
log Padipose:air = 0.563NCl + 1.028NBr + 0.467NC + 0.270QH 0.199NF R Haloalkanes Gargas et al. (1988)
0.097
log Padipose:air = 1.0371xv (0.007(1/ [ VY )) + 0.022QH 0.1773 [ FY 0.199NF
0.0036
log Pliver:air = (1.0721xv) (0.021(1/ [ VY )) + (0.647(1/1xv)) (0.3044 [ FY )
1.212
log Pliver:air = 0.366NCl 0.588NBr + 0.345QH 0.179NF 0.007 R Haloalkanes Gargas et al. (1988)
Y R Haloalkanes Gargas et al. (1988)
log Pliver:air = 0.6851xv (0.020(1/ [ )) + 0.232QH + (0.298(1/1xv)) +
V
0.104NCl 0.726
R Haloalkanes Csanady and Laib (1990)
log Pliver:air = 1.0721Xv 0.021/ [ VY + 0.647/1Xv 0.3044 [ FY 1.212
log Pmuscle:air = 0.379QH 0.278NCl + 0.536NBr 0.190NF + 0.169NCl R Haloalkanes Gargas et al. (1988)
0.439
log Pmuscle:air = 0.3991xv (0.007(1/ [ VY )) + 0.295QH + 0.2594 [ SF 0.194NF
0.217
log Pmuscle:air = (0.9951xv) (0.018(1/ [ VY )) (0.4244 [ FY ) (0.559(1/1xv) +
(0.602(1/1xv)) 1.334
Hydrophobic descriptors
log(Padipose:water Vwt) = 0.9Po:w + 0.31 F Chloroethanes; Benzene Bertelsen et al. (1988)
log(Pkidney:water Vwt) = 0.72Po:w 0.56 F Chloroethanes; Benzene Bertelsen et al. (1988)
log(Pliver:water Vwt) = 1.06Po:w 1.43 F Chloroethanes; Benzene Bertelsen et al. (1988)
log(Pmuscle:water Vwt) = 0.63Po:w 0.60 F Chloroethanes; Benzene Bertelsen et al. (1988)
ln Padipose:air = 0.032Tb 5.456 H Haloalkanes Csanady and Laib (1990)
ln Pliver:air = 0.022Tb 4.638 H Haloalkanes Csanady and Laib (1990)
log Padipose:air = 0.209 + 0.0628 log Pw:a + 0.8868 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Padipose:air = 0.21 log Po:a + 0.24 log Pw:a H Hydrophilic VOCs Tichy (1991b)
log Padipose:air = 0.782 log Po:a + 0.201 log Pw:a + 0.432 H Hydrophobic VOCs Tichy (1991a)
log Padipose:air = 0.901 log Po:a + 0.150 H LMWVOCs Fiserova-Bergerova et al.
(1984)
log Padipose:air = 0.174 + 0.910 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pbrain:air = 0.16 log Po:a + 0.82 log Pw:a + 0.47 H Hydrophilic VOCs Tichy (1991b)
log Pbrain:air = 0.274 + 0.537 log Pw:a + 0.444 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pbrain:air = 0.394 + 1.096 log Pw:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pbrain:air = 0.471 log Po:a + 0.630 log Pw:a 0.305 H Hydrophobic VOCs Tichy (1991a)
log Pbrain:air = 0.844 log Po:a 1.124 H LMWVOCs Fiserova-Bergerova et al.
(1984)
log Pbrain:air = 0.850 + 0.773 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pbrain:air = 3.692 + 1.253RG H Inert gases; LMWVOCs Abraham et al. (1985)
log Pkidney:air = 0.18 log Po:a + 0.82 log Pw:a + 0.53 H Hydrophilic VOCs Tichy (1991b)
log Pkidney:air = 0.277 + 1.111 log Pw:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pkidney:air = 0.466 log Po:a + 0.379 log Pw:a 0.332 H Hydrophobic VOCs Tichy (1991a)
log Pkidney:air = 0.700 log Po:a 0.877 H LMWVOCs Fiserova-Bergerova et al.
(1984)
log Pkidney:air = 0.920 + 0.764 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pliver:air = 0.388 + 0.502 log Pw:a + 0.497 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pliver:air = 0.432 + 1.064 log Pw:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pliver:air = 0.746 log Po:a + 0.178 log Pw:a 0.767 H Hydrophobic VOCs Tichy (1991a)
log Pliver:air = 0.871 log Po:a 1.044 H LMWVOCs Fiserova-Bergerova et al.
(1984)
log Pliver:air = 0.875 + 0.773 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Plung:air = 0.21 log Po:a + 0.91 log Pw:a + 0.41 H Hydrophilic VOCs Tichy (1991b)
log Plung:air = 0.057 + 0.870 log Pw:a + 0.146 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Plung:air = 0.057 + 0.978 log Pw:a H Inert gases; LMWVOCs Abraham et al. (1985)
(continued)
487
Table 40.1 (continued) In Silico Approaches for Estimating the Tissue:Air Partition Coefcients (P) of Chemicals
488

log Plung:air = 0.373 log Po:a + 0.416 log Pw:a 0.216 H Hydrophobic VOCs Tichy (1991a)
log Plung:air = 0.644 log Po:a 0.815 H LMWVOCs Fiserova-Bergerova et al.
(1984)
log Plung:air = 0.833 + 0.911 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pmuscle:air = 0.19 log Po:a + 0.82 log Pw:a + 0.54 H Hydrophilic VOCs Tichy (1991b)
log Pmuscle:air = 0.49 log Po:a + 0.39 log Pw:a 0.31 H Hydrophobic VOCs Tichy (1991b)
log Pmuscle:air = 0.263 + 0.575 log Pw:a + 0.423 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pmuscle:air = 0.351 + 1.108 log Pw:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pmuscle:air = 0.652 log Po:a 0.702 H LMWVOCs Fiserova-Bergerova et al.
(1984)
log Pmuscle:air = 0.852 + 0.768 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pmuscle:air = 3.247 + 0.965RG H Inert gases; LMWVOCs Abraham et al. (1985)
Padipose:air = 0.447Po:a + 0.075Pw:a + 6.59 H LMWVOCs; CFCs Meulenberg and Vijverberg
(2000)
Pbrain:air = (0.026So + 0.51Sw)/Sa H LMWVOCs Paterson and Mackay (1989)
Pbrain:air = 0.020Po:a + 0.380Pw:a + 0.94 H LMWVOCs; CFCs Meulenberg and Vijverberg
(2000)
Pkidney:air = (0.014So + 0.51Sw)/Sa H LMWVOCs Paterson and Mackay (1989)
Pkidney:air = 0.011Po:a + 0.400Pw:a + 0.69 H LMWVOCs; CFCs Meulenberg and Vijverberg
(2000)
Pkidney:air = 0.391 + 0.550 log Pw:a + 0.440 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
Pliver:air = (0.028So + 0.51Sw)/Sa H LMWVOCs Paterson and Mackay (1989)
Pliver:air = 0.028Po:a + 0.79 H LMWVOCs; CFCs Meulenberg and Vijverberg
(2000)
Pmuscle:air = 0.014Po:a + 0.384Pw:a + 0.94 H LMWVOCs; CFCs Meulenberg and Vijverberg
(2000)
ln Padipose:air = 0.032Tb 5.456 R LMWVOCs Csanady and Laib (1990)
ln Pliver:air = 0.022Tb 4.638 R LMWVOCs Csanady and Laib (1990)
log Padipose:air = 0.920 log Po:a + 0.136 R LMWVOCs Gargas et al. (1989)
log Padipose:air = 0.927 log Po:a 0.032 log Pw:a + 0.120 R LMWVOCs Gargas et al. (1989)
log Padipose:air = 1.027 log Po:a 0.046 log Pw:a 0.119 R Haloalkanes Gargas et al. (1988)
log Pliver:air = 0.574 log Po:a + 0.302 log Pw:a 0.278 R Haloalkanes Gargas et al. (1988)
log Pliver:air = 0.730 log Po:a + 0.128 log Pw:a 0.550 R LMWVOCs Gargas et al. (1989)
log Pmuscle:air = 0.477 log Po:a + 0.365 log Pw:a 0.374 R Haloalkanes Gargas et al. (1988)
log Pmuscle:air = 0.644 log Po:a + 0.180 log Pw:a 0.725 R LMWVOCs Gargas et al. (1989)
Padipose:air = 0.594Po:a + 0.085Pw:a + 9.40 R LMWVOCs; CFCs Meulenberg and Vijverberg
(2000)
Pbrain:air = 0.054Po:a + 0.832Pw:a R LMWVOCs; CFCs Meulenberg and Vijverberg
(2000)
Pkidney:air = 0.097Po:a + 0.826Pw:a R LMWVOCs; CFCs Meulenberg and Vijverberg
(2000)
Pliver:air = 0.026Po:a + 0.878Pw:a + 2.36 R LMWVOCs; CFCs Meulenberg and Vijverberg
(2000)
Pmuscle:air = 0.010Po:a + 0.772Pw:a + 0.29 R LMWVOCs; CFCs Meulenberg and Vijverberg
(2000)
Biologically based algorithms

Ptissue:air = (SsVwt + SvVnt + 0.7SsVpt + 0.3SvVpt)/Sa R, H LMWVOCs Poulin and Krishnan (1996a)
Ptissue:air = Po:wPw:a(Vnt + 0.3Vpt) + Pw:a(Vwt + 0.7Vpt) R, H LMWVOCs Poulin and Krishnan (1996c)
a T + = dipolarity/polarizability, E + = overall hydrogen-bond acidity, F + = overall hydrogen-bond basicity, 1Xv, [ VY , 1X, 4 [ FY , 3 [ FY , 4Xvpc = connectivity
indices, NBr = number of bromide atoms in the molecule, NC = number of carbon atoms in the molecule, NCl = number of chloride atoms in the molecule,
NF = number of uoride atoms in the molecule, Phe:a = hexadecane:air partition coefcient, Po:a = n-octanol:air partition coefcient (or vegetable oil:air),
Po:w = n-octanol:water partition coefcient (or vegetable oil:water), Pw:a = water:air partition coefcient, QH = variable dependant on the polarity of the
molecule due to the presence of hydrogen atoms, R2 = Excess molar refraction, Rg = average solubility in entire set of solvent systems, Sa = solubility
in air, So = solubility in n-octanol (or vegetable oil), Ss = solubility in saline, Sv = solubility in vegetable oil, Sw = solubility in water, Tb = boiling point,
Vnt = volume fraction of neutral lipids in tissues, Vpt = volume fraction of phospholipids in tissues, and Vwt = volume fraction of water in tissues.
b F = sh, H = human, and R = rats.
c CFCs = chlorouorocarbons, LMWVOCs = low molecular weight volatile organic chemicals, and VOCs = volatile organic chemicals.
489
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In Silico Approaches for Blood:Air PCs
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Table 40.2 In Silico Approaches for Estimating the Blood:Air Partition Coefcients (P) of Chemicals
log Pblood:air = 1.269 + 0.612R2 + 0.916 T + + 3.614 E + + 3.381 F + +
(1994)
0.362 log Phe:a
log Pplasma:air = 1.48 + 0.490R2 + 2.04 T + + 3.5074 E + + 3.911 F + +
(1994)
0.157 log Phe:a
Steric descriptors
log Pblood:air = 0.0072MW + 0.197 H Trihalomethanes Batterman et al. (2002)
log Pblood:air = 0.321NBr + 1.06 H Trihalomethanes Batterman et al. (2002)
Pblood:air = 0.07MW + 5.59 H Aliphatic hydrocarbons Perbellini et al. (1985)

log Pblood:air = 0.443QH 0.303NF + 0.225NCl + 0.510NBR + 0.155NC 0.104 R Haloalkanes Gargas et al. (1988)
log (Pblood:water Vwb) = 0.7Po:w 0.75 F Chloroethanes; benzene Bertelsen et al. (1998)
ln Pblood:air = 0.038Tb 13.3 H Aliphatic hydrocarbons Csanady and Laib (1990)
log Pblood:air = 0.0109Tb 2.584 H Trihalomethanes Batterman et al. (2002)
log Pblood:air = 0.14 log Po:a + 0.86 log Pw:a + 0.47 H Hydrophilic VOCs Tichy (1991b)
log Pblood:air = 0.685 log Po:a 0.6565 H Trihalomethanes Batterman et al. (2002)
log Pblood:air = 0.45 log Pw:a + 1.21 H VOCs Laass (1987)
log Pblood:air = 0.003 log Pw:a + 1.47 H VOCs Laass (1987)
log pblood:air = 0.074 + 0.802 log Pw:a + 0.218 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pblood:air = 0.07 log Sw + 1.21 H VOCs Laass (1987)
(continued)
491
Table 40.2 (continued) In Silico Approaches for Estimating the Blood:Air Partition Coefcients (P) of Chemicals
492

log Pblood:air = 0.09 log Po:a + 2.45 H VOCs Laass (1987)
log Pblood:air = 0.102 + 0.675 log Pw:a + 0.315 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pblood:air = 0.295 + 0.588 log Pw:a + 0.411 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pblood:air = 0.338 log Po:a + 3.121 H Halogenated hydrocarbons Tichy et al. (1984)
log Pblood:air = 0.6737 + 0.5319 log Po:a log Pw:a H VOCs Sato and Nakajima (1979)
log Pblood:air = 0.695 log Po:a 1.076 H LMWVOCs Fiserova-Bergerova et al.
(1984)
log Pblood:air = 0.820 + 0.754 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pblood:air = 0.09 log Sw + 8.25 log Vo 11.09 H VOCs Laass (1987)
log Pblood:air = 0.180 log Po:a + 0.889 log Pw:a + 0.054 H Hydrophobic VOCs Tichy (1991a)
log Pblood:air = 0.22 log Pw:a + 0.67 log Po:a 0.98 H VOCs Laass (1987)
log Pblood:air = 0.22 log Sw + 10.78 log Vw 40.99 H VOCs Laass (1987)
log Pblood:air = 0.262 + 0.996 log Pw:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pblood:air = 0.27 log 1000/P + 5.10 log Vo 6.67 H VOCs Laass (1987)
log Pblood:air = 0.31 log Sw + 3.90 log Vo 4.53 H VOCs Laass (1987)
log Pblood:air = 0.35 log 1000/P + 1.01 H VOCs Laass (1987)
log Pblood:air = 0.35 log Sw + 0.79 log 1000/P + 1.34 log Vo 2.23 H VOCs Laass (1987)
log Pblood:air = 0.37 log Sw + 10.09 log Vw 38.40 H VOCs Laass (1987)
log Pblood:air = 0.38 log Sw + 0.91 log 1000/P 0.45 H VOCs Laass (1987)
log Pblood:air = 0.45 log Sw + 0.81 log 1000/P 0.40 H VOCs Laass (1987)
log Pblood:air = 0.48 log Sw + 0.75 log 1000/P + 1.67 log Vo 2.77 H VOCs Laass (1987)
log Pblood:air = 0.581 log Po:a + 0.332 log Pw:a 0.599 H LMWVOCs Gargas et al. (1989)
log Pblood:air = 0.65 log Po:a 0.84 H VOCs Laass (1987)
log Pblood:air = 0.984 log Pw:a + 0.053 H Ketones; ethers; gases Tichy et al. (1984)
log Pblood:air = 1.07 log Pw:a + 0.27 log Po:a 0.79 H VOCs Laass (1987)
log Pblood:air = 1.21 log Vo 0.17 H VOCs Laass (1987)
log Pblood:air = 3.05 0.34Po:n H Ketones Cabala et al. (1992)
log Pblood:air = 3.922 + 1.369RG H Inert gases; LMWVOCs Abraham et al. (1985)
log Pblood:air = 5.89 log Vw 21.43 H VOCs Laass (1987)
log Pblood:air = 7.86 log Vo 10.40 H VOCs Laass (1987)
log Pblood:air = 8.90 log Vw 33.40 H VOCs Laass (1987)
log Pmilk:air = 0.900 log Po:a 1.095 H Trihalomethanes Batterman et al. (2002)
log Pplasma:air = 0.079 + 0.896 log Pw:a + 0.149 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pplasma:air = 0.082 + 0.894 log Pw:a + 0.152 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pplasma:air = 0.848 + 0.890 log Po:a H Inert gases; LMWVOCs Abraham et al. (1985)
log Pplasma:air = 3.696 + 1.208RG H Inert gases; LMWVOCs Abraham et al. (1985)
log Pplasma:air = 0.038 + 1.019 log Pw:a H Inert gases; LMWVOCs Abraham et al. (1985)
Pblood:air = 0.0072Po:a + 0.898Pw:a + 0.03 H LMWVOCs; CFCs Meulenberg and Vijverberg
(2000)
Pblood:air = 0.08e0.0308Tb H Aliphatic hydrocarbons Perbellini et al. (1985)
Pblood:air = 0.00442Po:a H Aliphatic hydrocarbons Perbellini et al. (1985)

Pblood:air = 0.88Pw:a + 0.012 H VOCs Feingold (1976)
Pblood:air = 0.89Pw:a + 0.011Po:a H LMWVOCs Tichy et al. (1984)
Pblood:air = 0.90 log Pw:a 461 H Esters; alcohols Kaneko et al. (1994)
Pblood:air = Pw:a + (Po:a/100) H Anaesthetics Eger and Larson (1964)
Pblood:air = Sw(1 + 0.0035Po:w)/Sa H LMWVOCs Paterson and Mackay
(1989)
log Pblood:air = Pw:a[VlbPo:w0.85 + Vprb(86.2/Po:w + 3.70)] + Vwb H, R LMWVOCs Connell et al. (1993)
log Pblood:air = 0.426 log Po:a + 0.515 log Pw:a 0.070 R Haloalkanes Gargas et al. (1988)
log Pblood:air = 0.553 log Po:a + 0.351Pw:a 0.286 R LMWVOCs Gargas et al. (1989)
Pblood:air = 0.0054Po:a + 0.931Pw:a + 1.16 R LMWVOCs; CFCs Meulenberg and Vijverberg
(2000)
(continued)
493
Table 40.2 (continued) In Silico Approaches for Estimating the Blood:Air Partition Coefcients (P) of Chemicals
494
QSARs: FreeWilson-type equations

Pblood:water = BS (C-C) (28.4) + nCL2(12.9) + nCL3(12.9) F Chloroethanes Fouchcourt et al. (2000)
Pblood:air = BS (C-C) (26.2) + nH3(34.9) + nCL(4.51) + nCL2(29.4) + H Chloroethanes Fouchcourt and Krishnan
nCL3(11.5) (2000)
Pblood:air = BS(C-C) (45.6) + nH3(51.5) + nCL(8.86) + nCL2(36.4) + R Chloroethanes Fouchcourt and Krishnan
nCL3(11.1) (2000)

Pblood:air = Po:wPw:a(Vnb + 0.3Vpb) + Pw:a(Vwb + 0.7Vpb) R, H LMWVOCs Poulin and Krishnan (1996c)
Pblood:air = [fe(SsVwe + SvVne + 0.7SsVpe + 0.3SvVpe) + fp(SsVwp + SvVnp + R, H LMWVOCs Poulin and Krishnan (1996b)
0.7SsVpp + 0.3SvVpp)]/Sa
a T + = dipolarity/polarizability, E + = overall hydrogen-bond acidity, F + = overall hydrogen-bond basicity, BS = Basic structure, fe = fraction of erythrocytes
in blood, fp = fraction of plasma in blood, MW = molecular weight, NBr = number of bromide atoms in the molecule, NC = number of carbon atoms in
the molecule, NCl = number of chloride atoms in the molecule, nCL = number of CL fragments, nCL2 = number of CL2 fragments, nCL3 = number of
CL3 fragments, NF = number of uoride atoms in the molecule, nH3 = number of H3 fragments, P = vapor pressure, Phe:a = hexadecane:air partition
coefcient, Po:a = n-octanol:air partition coefcient (or vegetable oil:air), Po:n = vegetable oil:nitrogen partition coefcient, Po:w = n-octanol:water partition
coefcient (or vegetable oil:water), Pw:a = water:air partition coefcient, QH = variable dependant on the polarity of the molecule due to the presence
of hydrogen atoms, R2 = excess molar refraction, Rg = parameters relative to the solvent, Sa = solubility in air, Ss = solubility in saline, Sv = solubility
in vegetable oil, Sw = solubility in water, Tb = boiling point, Vlb = volume fraction of lipids in blood, Vnb = volume fraction of neutral lipids in blood, Vne =
volume fraction of neutral lipids in erythrocytes, Vnp = volume fraction of neutral lipids in plasma, Vo = surface tension, Vpb = volume fraction of phospholipids
in blood, Vpe = volume fraction of phospholipids in erythrocytes, Vpp = volume fraction of phospholipids in plasma, Vprb = volume fraction of proteins in
blood, Vw = heat released due to evaporation of the substance at boiling temperature, Vwb = volume fraction of water in blood, Vwe = volume fraction of
water in erythrocytes, and Vwp = volume fraction of water in plasma.
c CFCs = chlorouorocarbons, LMWVOCs = low molecular weight volatile organic chemicals, and VOCs = volatile organic chemicals.
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Table 40.3 In Silico Approaches for Estimating the Tissue:Blood Partition Coefcients (P) of Chemicals
Steric descriptors
H Inert gases; LMWVOCs; CFCs Abraham and Weathersby
log Padipose:blood = 0.168 + 0.198R2 + 0.130 T + 1.211 E +
(1994)
3.267 F + + 2.275Vx
log Pbrain:blood = 0.166 + 0.239R2 0.626 T + 0.368 E + 0.615 F +
(1994)
+ 1.072Vx
log Pbrain:blood = 0.0148PSA + 0.152 log Po:w + 0.139 H Inert gases; HMWOCs; LMWVOCs Clark (1999)
log Pbrain:blood = 1.359 + 0.338 log Pcyh 0.00618Vm H H2-R antagonists Kalizan and Markuszewski
(1996)
log Pheart:blood = 0.346 + 0.204 T + 2.150 E + 0.853 F + + 0.931Vx
(1994)

log Pkidney:blood = 0.188 + 0.226R2 0.559 T + 0.433 F + + 0.832Vx
(1994)
log Pliver:blood = 0.270 + 0.233R2 0.375 T + 1.004 E + 1.118 F +
(1994)
+ 0.832Vx
log Plung:blood = 0.150 0.195 T + + 0.389Vx
(1994)
log Pmuscle:blood = 0.222 0.479 T + 0.517 F + + 0.999Vx
(1994)
Padipose:plasma = 1.9988 0.5004UNS + 0.1793NPL + 0.05931DIFF2 H PCBs (48)
+ + R H2-R antagonists Norinder and Haeberlein
log Pbrain:blood = 0.088 + 0.264R2 0.966 T 0.7057 E
(2002)
+
0.7567 F + 1.189Vx
(continued)
497
Table 40.3 (continued) In Silico Approaches for Estimating the Tissue:Blood Partition Coefcients (P) of Chemicals
498

log Pbrain:blood = 0.088 + 0.272 log Po:w 0.00116MW R H2-R antagonists Kalizan and Markuszewski
(1996)
log Pbrain:blood = 0.00116MW + 0.272 log Po:w 0.088 R Inert gases; volatile hydrocarbons Norinder and Haeberlein
(2002)
log Pbrain:blood = 0.01Vm + 0.35 log Po:w + 0.99I3 + 1.25 R Drug-like molecules Norinder and Haeberlein
(2002)
log Pbrain:blood = 0.021PSA 0.003MV + 1.643 R Inert gases; HMWOCs; LMWVOCs Clark (1999)
log Pbrain:blood = 0.0322DPSA + 1.33 R HMWOCs Norinder and Haeberlein
(2002)
R H2-R antagonists; Inert gases; SOMs Norinder and Haeberlein
log Pbrain:blood = 0.038 + 0.198R2 0.687 T + 0.7157 E +
(2002)
0.6987 F + + 0.995Vx
log Pbrain:blood = 0.218(NN + NO) + 0.235 log Po:w 0.027 R HMWOCs Norinder and Haeberlein
(2002)
log Pbrain:blood = 0.476 + 0.541 log Po:w 0.00794MW R H2-R antagonists Kalizan and Markuszewski
(1996)
log Pbrain:blood = 1.296 + 0.309 log Pcyh 0.00570MW R H2-R antagonists Kalizan and Markuszewski
(1996)
log Pbrain:blood = 0.39 log Po:w + 0.68 H Drugs, hormones Seydel and Schaper (1982)
log Pbrain:blood = 0.054Go + 0.43 H H2-R antagonists; LMWVOCs Lombardo et al. (1996)
$ $ H, R LMWVOCs DeJongh et al. (1997)
Padipose:blood = ?(Vlt 3RZ + Vwt)/(Vlb 3RZ + Vwb)A+ B
Pbrain:blood = ?(Vlt 3RZ + Vwt)/(Vlb 3RZ + Vwb)A+ B
Pkidney:blood = ?(Vlt 3RZ + Vwt)/(Vlb 3RZ + Vwb)A+ B
Pliver:blood = ?(Vlt 3RZ +Vwt)/(Vlb 3RZ + Vwb)A+ B
Pmuscle:blood = ?(Vlt 3RZ + Vwt)/(Vlb 3RZ + Vwb)A+ B
Ln Pkidney:blood = 0.0065o R HMWOCs Yamaguchi et al. (1996)
Ln Pliver:blood = 0.025i R HMWOCs Yamaguchi et al. (1996)
Ln Pmuscle:blood = 0.0069i R HMWOCs Yamaguchi et al. (1996)
log Pbrain:blood = 0.035(Gsolv + 0.259 R H2-R antagonists; LMWVOCs Norinder and Haeberlein
(2002)
log Pbrain:blood = 0.4275 0.3873nacc,solv + 0.1092 log Po:w 0.0017Apol R Drugs; LMWVOCs; anaesthetics Feher et al. (2000)
log Pbrain:blood = 1.979 + 0.373 log Pcyh 0.00275Vwav R H2-R antagonists Kalizan and Markuszewski
(1996)
log Pbrain:plasma = 0.48 (log Poct-cyc + 0.89 R H2-R antagonists Testa et al. (2000)
ln Padipose:blood = 0.05i + 0.021 R HMWOCs Yamaguchi et al. (1996)
Rb Basic drugs Yokogawa et al. (1990)
Padipose:blood = 0.915 3RZ
Padipose:plasma = 0.016 3RZ
Pbone marrow:blood = 1.975 3RZ
Pbone:plasma = 0.036 3RZ

Pbrain:blood = 3.157 3RZ
Pbrain:plasma = 0.062 3RZ
Pgut:blood = 3.002 3RZ
Pgut:plasma = 0.058 3RZ
Pheart:blood = 1.678 3RZ
Pheart:plasma = 0.032 3RZ
Pkidney:plasma = 0.075 3RZ
(continued)
499
Table 40.3 (continued) In Silico Approaches for Estimating the Tissue:Blood Partition Coefcients (P) of Chemicals
500

Pliver:plasma = 0.064 3RZ
Plung:blood = 1.158 3RZ
Plung:plasma = 0.031 3RZ
Pmuscle:blood = 4.928 3RZ
Pmuscle:plasma = 0.099 3RZ
Pskin:blood = 2.997 3RZ
Pskin:plasma = 0.058 3RZ
Pspleen:blood = 3.002 3RZ

Padipose:blood = BS(C-C)(94.5) + nCL2(29.2) + nCL3(29.2) F Chloroethanes Fouchcourt et al. (2000)
Pliver:blood = BS(C-C)(2.93) + nCL2(0.238) + nCL3(0.238) F Chloroethanes Fouchcourt et al. (2000)
Pmuscle:blood = BS(C-C)(3.02) + nCL2(0.175) + nCL3(0.175) F Chloroethanes Fouchcourt et al. (2000)
Padipose:blood = BS(C-C)(49.2) + nH3(0.440) + nCL(14.54) + H Chloroethanes Fouchcourt and Krishnan
nCL2(6.65) + nCL3(26.5) (2000)
Pliver:blood = BS(C-C)(2.64) + nH3(0.61) + nCL(0.66) + nCL2(0.18) H Chloroethanes Fouchcourt and Krishnan
+ nCL3(1.68) (2000)
Pmuscle:blood = BS(C-C)(1.11) + nH3(0.08) + nCL(0.02) + nCL2(0.21) H Chloroethanes Fouchcourt and Krishnan
+ nCL3(0.15) (2000)
Padipose:blood = BS(C-C)(30.1) + nH3(9.88) + nCL(6.02) + nCL2(3.90) R Chloroethanes Fouchcourt and Krishnan
+ nCL3(17.3) (2000)
Pliver:blood = BS(C-C)(1.79) + nH3(0.9) + nCL(0.38) + nCL2(0.21) + R Chloroethanes Fouchcourt and Krishnan
nCL3(1.27) (2000)
Pmuscle:blood = BS(C-C)(0.69) + nH3(0.12) + nCL(0.04) + nCL2(0.12) R Chloroethanes Fouchcourt and Krishnan
+ nCL3(0.17) (2000)

Ptissue:blood = (SoVnt + Sw0.7Vpt + So0.3Vpt + SwVwt)/(SoVnb + Sw0.7Vpb H LMWVOCs Poulin and Krishnan (1995a)
+ So0.3Vpb + SwVwb)
Ptissue:blood = (Po:wVnt + Vwt + Po:w0.3Vpt + 0.7Vpt)/[fe(Po:wVne + Vwe + R Ketones; Alcohols; Esters Poulin and Krishnan (1995b)
Po:w0.3Vpe + 0.7Vpe) + fp(Po:wVnp + Vwp + Po:w0.3Vpp + 0.7Vpp)]
Ptissue:blood = [Po:w(Vnt + 0.3Vpt) + (Vwt + 0.7Vpt)]/[Po:w(Vnb + 0.3Vpb) + R, H LMWVOCs Poulin and Krishnan (1996b)
(Vwb + 0.7Vpe)]
a T + = dipolarity/polarizability, E + = overall hydrogen-bond acidity, F + = overall hydrogen-bond basicity, (Gsolv = free energy of solvation in hexadecane,
7i = molecular structure Fujita value, 7o = molecular structure Fujita value, A1, A2 = Collander-type coefcient, Apol = polar surface area, B = correction
factor, BS = basic structure, DIFF = variable dependant on the number of chloride atoms in the aromatic cycle, DPSA = dynamic polar surface area,
fe = fraction of erythrocytes in blood, fp = fraction of plasma in blood, I3 = variable dependant on the presence of an amino nitrogen or carboxyl group,
MV = molecular volume, MW = molecular weight, nacc,solv = number of solvated hydrogen-bond acceptors, nCL = number of CL fragments, nCL2 =
number of CL2 fragments, nCL3 = number of CL3 fragments, nH3 = number of H3 fragments, NN = number of nitrogens, NO = number of oxygens, NPL
= variable dependant on the number of chloride atoms in the molecule in ortho position, oG = Gibbs free energy related to the solvation of the substance
in water, Pcyh = cyclohexane:water partition coefcient, Po:w = n-octanol:water partition coefcient (or vegetable oil:water), Poct-cyc = octanol-cyclohexane,
PSA = polar surface area, R2 = Excess molar refraction, So = solubility in n-octanol (or vegetable oil), Sw = solubility in water, UNS = variable dependant
on the number of atoms in the molecule that are not chlorides, Vlb = volume fraction of lipids in blood, Vlt = volume fraction of lipids in tissue, Vm = molar
volume, Vnb = volume fraction of neutral lipids in blood, Vne = volume fraction of neutral lipids in erythrocytes, Vnp = volume fraction of neutral lipids in
plasma, Vnt = volume fraction of neutral lipids in tissues, Vpb = volume fraction of phospholipids in blood, Vpe = volume fraction of phospholipids in
erythrocytes, Vpp = volume fraction of phospholipids in plasma, Vpt = volume fraction of phospholipids in tissues, Vwav = volume of water needed in order
to solubilize the substance, Vwb = volume fraction of water in blood, Vwb = volume fraction of water in blood, Vwe = volume fraction of water in erythrocytes,
Vwp = volume fraction of water in plasma, Vwt = volume fraction of water in tissue, Vwt = volume fraction of water in tissues, and Vx = McGowan
characteristic volume.
c CFCs = chlorouorocarbons, HMWOCs = high molecular weight organic chemicals, LMWVOCs = low molecular weight volatile organic chemicals, PCBs
= polychlorobiphenyls, and VOCs = volatile organic chemicals.
501
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Table 40.4 In Silico Approaches for Estimating Protein Binding of Chemicalsa
Approachb Speciesc Chemical Class Reference
log (1/fu(plasma) 1) = 0.994 log Po:w 1.10 H Aromatic acids Testa et al. (2000)
log (1/fu(plasma) 1) = 0.994 log Po:w 1.10 H Organic acids Laznicek et al. (1987)
log (1/Ka(plasma)) = 3.91 log Po:w 2 + 13 log P
o:w 13.7 H Cephalosporins Testa et al. (2000)
log (1 fu(brain)) = 0.36 log Po:w 1.07 H Barbiturates Seydel and Schaper (1982)
log (1 fu(plasma)) = 0.276 log Po:w + 1.2 H Penicillins Seydel and Schaper (1982)
log (1 fu(plasma)) = 0.30 log Po:w 1.03 H Barbiturates Seydel and Schaper (1982)
log (1 fu(plasma)) = 0.33 log Po:w + 1.94 H Tetracyclines Seydel and Schaper (1982)
log 1/Ka(albumin binding) = 0.85 log Po:w + 2.73 H Sulfapyrimidines; sulfapyridines Seydel and Schaper (1982)
log 1/Ka(albumin binding) = 0.97 log Po:w + 3.24 H Sulfapyridines Seydel and Schaper (1982)
log 1/Ka(albumin binding) = 0.97 log Po:w 0.70I + 3.24 H Sulfapyrimidines; sulfapyridines Seydel and Schaper (1982)
log 1/Ka(albumin binding) = 0.99 log Po:w + 2.49 H Sulfapyrimidines Seydel and Schaper (1982)
log Kalbumin binding = 0.89 log Po:w + 1.47 H Sulfapyrimidines Seydel and Schaper (1982)
log Kalbumin binding = 1.15 log Po:w + 1.23 H Sulfonamides Seydel and Schaper (1982)
log Kalbumin binding = 1.23 log Po:w 0.056 H Steroid bisguanylhydrazones Seydel and Schaper (1982)
log Kalbumin binding = 1.32 log Po:w + 0.37 H Penicillins Seydel and Schaper (1982)
log Kalbumin binding = 1.39 log Po:w 1.19 H Cardenolides Seydel and Schaper (1982)
log Kalbumin binding = 1.65 log Po:w 2.57 H Steroid hormones Seydel and Schaper (1982)
log Kplasma protein binding = 0.73(Rmui + 1.46 H Sulfapyridines Seydel and Schaper (1982)
log Ka(blood protein binding) = 0.5047T 0.665 H Penicillins Bird and Marshall (1967)
log (1/fu(plasma) 1) = 1.011 log Po:w 1.745 R Organic acids Laznicek et al. (1987)
log (1 fu)/fu(adipose) = log 0.750 + 0.936 log Po:w R Barbituric acids Nesterov et al. (1998)
log (1 fu)/fu(brain) = log 0.073 + 0.860 log Po:w R Barbituric acids Nesterov et al. (1998)
(continued)
503
Table 40.4 (continued) In Silico Approaches for Estimating Protein Binding of Chemicalsa
504
Approachb Speciesc Chemical Class Reference

log (1 fu)/fu(gut) = log 0.099 + 0.824 log Po:w R Barbituric acids Nesterov et al. (1998)
log (1 fu)/fu(heart) = log 0.135 + 0.780 log Po:w R Barbituric acids Nesterov et al. (1998)
log (1 fu)/fu(kidney) = log 0.676 + 0.619 log Po:w R Barbituric acids Nesterov et al. (1998)
log (1 fu)/fu(liver) = log 1.775 + 0.504 log Po:w R Barbituric acids Nesterov et al. (1998)
log (1 fu)/fu(lung) = log 0.164 + 0.841 log Po:w R Barbituric acids Nesterov et al. (1998)
log (1 fu)/fu(muscle) = log 0.080 + 0.835 log Po:w R Barbituric acids Nesterov et al. (1998)
log (1 fu)/fu(pancreas) = log 0.022 + 1.095 log Po:w R Barbituric acids Nesterov et al. (1998)
log (1 fu)/fu(plasma) = log 0.016 + 0.975 log Po:w R Barbituric acids Nesterov et al. (1998)
log (1 fu)/fu(red blood cell) = log 0.178 + 0.677 log Po:w R Barbituric acids Nesterov et al. (1998)
log (1 fu)/fu(skin) = log 0.271 + 0.736 log Po:w R Barbituric acids Nesterov et al. (1998)
log (1 fu)/fu(spleen) = log 0.126 + 0.841 log Po:w R Barbituric acids Nesterov et al. (1998)
log (1 fu)/fu(stomach) = log 0.058 + 0.939 log Po:w R Barbituric acids Nesterov et al. (1998)
log (1 fu)/fu(testis) = log 0.120 + 0.747 log Po:w R Barbituric acids Nesterov et al. (1998)
log Kplasma protein binding = 0.33(Rmui 0.53I + 4.08 R Sulfapyridines Seydel and Schaper (1982)
log (1/fu(plasma) 1) = 1.016 log Po:w 1.275 Rb Organic acids Laznicek et al. (1987)
a K = protein afnity constant (Freundlich isotherm), Ka = protein afnity constant (Scatchard isotherm) and fu = unbound fraction.
b Po:w = octanol:water partition coefcient, T = molecular hydrophobicity constant, I = family indicator variable, (Rmui = variable dependant on the
resistance constant due to diffusion of the nonionized form in the lipid membrane.
c H = humans, Rb = rabbit, and R = rat.
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Table 40.5 In Silico Approaches for Estimating Clearances (CL) of Chemicals
log CL(hepatic) = 0.64 log Po:w 0.98IP + 9.33 H Benzodiazepines Lewis (2000)
log CL(hepatic) = 0.055Energy 0.95IP 0.53HBD + 10.63 H Benzodiazepines Lewis (2000)
log CL(hepatic) = 0.067Energy 1.01IP 0.34HBD 0.43(E + 14.66 H Benzodiazepines Lewis (2000)
log CL(hepatic) = 0.094Energy 1.18IP 0.74(E + 18.65 H Benzodiazepines Lewis (2000)
log CL(hepatic) = 0.65 log Po:w 0.40IP 0.37HBD + 0.0025Hf + 3.63 H Benzodiazepines Lewis (2000)
Metabolic ratio = 2.72Q6 + 1.96EH + 0.014SN + 6.43 R Dichlorobiphenyls Lewis and Dickins (2002)
Steric descriptors
1/log CL(intrinsic; hepatic) = 3.58 0.058S_sCl 0.57S_aaO 0.47Shadow Z length H Commercially available Ekins and Obach (2000)
0.75CIC drugs
1/log CL(intrinsic; hepatic) = 3.11 0.10Dipole mag + 13.25Jurs RPCG + 0.57Jurs H Commercially available Ekins and Obach (2000)
RPCS + 0.00013Apol drugs
CL(intrinsic; hepatic) = 25Steric + 44Electrostatic + 20LUMO + 11HINT R Haloalkanes Waller et al. (1996)
CL (intrinsic; hepatic) = 0.0555 Po:w1.05 H Basic drugs Yokogawa et al. (2002)
log CL(renal) = 0.24(log Po:w)2 0.04 log Po:w + 0.58 H Probenecid analogs Seydel and Schaper (1982)
H Probenecid analogs Seydel and Schaper (1982)
log CL(renal) = log(0.35 + 0.013 3RZ )
log CL(renal) = 0.5 log Po:w + 3 H NSAID Smith et al. (1996)
log CL(renal) = 0.5 log Po:w + 13 H -blockers Smith et al. (1996)
log CL(hepatic) = 0.54(Rmui 0.51 R Sulfonamides Seydel and Schaper (1982)
(continued)
507
Table 40.5 (continued) In Silico Approaches for Estimating Clearances (CL) of Chemicals
508

log CL(renal) = 0.41(Rmui 0.80 R Sulfonamides Seydel and Schaper (1982)
log CL(renal) = 0.51 log Po:w 0.33 R Sulfapyridines Yamaguchi et al. (1996)
log CL(renal) = log [0.048 + 6.98 v 104(107T)1.394] R Xylidines Seydel and Schaper (1982)
log CL(total) = 0.74(Rmui + 0.22pKa 1.73 R Sulfonamides Seydel and Schaper (1982)
log E(hepatic) = 0.045 log Po:w 0.32 R HMWOCs Yamaguchi et al. (1996)
Rb Basic drugs Ishizaki et al. (1997)
CL(intrinsic; hepatic) = 3.828 3RZ
CL(intrinsic; hepatic) = 0.0875 3RZ
CL(intrinsic; hepatic) = 0.248 3RZDSS
QSARS: FreeWilson-type equations

log CL(renal) = 0.417R2(CH3) 0.744R1(OC3H7) + 1.33 R Xylidines Seydel and Schaper (1982)
log CL(total) = 0.49R2(CH3) + 0.57 R2(C2H5) + 0.25R1(OC3H7) + 1.76 R Xylidines Seydel and Schaper (1982)
a (E = variable related to molecular orbitals, (Rmui = variable dependant on the resistance constant due to diffusion of the nonionized form in the lipid
membrane, T = molecular hydrophobicity constant, Apol = polar surface area, CIC = complementary information content, Dipole-mag = dipole moment,
EH = HOMO energy, Electrostatic = Coulombic interaction energy, Energy = minimum internal energy, HBD = potential hydrogen bond donor atoms in
the molecule, Hf = enthalpy of formation, HINT = hydrophobic eld energy, IP = ionization potential, Jurs-RPCG = relative positive charge, Jurs-RPCS
= relative positive charge surface area, LUMO = lowest unoccupied molecular orbital energy, Po:w = n-octanol:water partition coefcient (or vegetable
oil:water), Po:w, app = apparent octanol:water partition coefcient, Q6 = net atomic charge on carbon atom at biphenyl ring position, R2(CH3) = methyl
fragment at R2 position, R2(C2H5) = ethyl fragment at R2 position, R1(OC3H7) = propyl ether fragment at R1 position, S_aaO = E-state indices for oxygen
atoms with two aromatic bonds, S_sCl = E-state indice for chlorine atoms with a single bond, Shadow Z length = length of the molecule in Z dimension,
SN = total nucleophillic superdelocalizability, and Steric = Van der Waals interaction energy.
c CFCs = chlorouorocarbons, HMWOCs = high molecular weight organic chemicals, LMWVOCs = low molecular weight volatile organic chemicals, and
VOCs = volatile organic chemicals.
Table 40.6 In Silico Approaches for Estimating Reaction Rates of Chemicalsa
Approachb Speciesc Chemical Classd Reference
log V(oxidation) = 0.894 0.111diameter 0.007(E H Nitriles Lewis and Dickins (2002)
log kcat (oxidation) = 19.97 0.024(H{ 0.95IP H Toluenes Lewis and Dickins (2002)
log V(oxidation) = 26.90 2.58IP H Halothanes Lewis and Dickins (2002)
log kcat (oxidation) = 0.024Vol 0.23Q 1.14 H Barbiturates Lewis and Dickins (2002)
log kcat (oxidation) = 1.33 0.15Q H Anilines Lewis and Dickins (2002)
log kcat (demethylation) = 0.68 W + 1.06 R X-C6H4N(CH3)2 Hansch and Leo (1995)
Steric descriptors
log kcat/Km(Oxidation) = 0.0347SA 2.29(E + 1.92 H Toluenes Lewis and Dickins (2002)
log Vmax (n-demethylation) = 0.18 Length 1.94 H Ethylamines Lewis (2001)

log Vmax (n-demethylation) = 3.50 Length 0.13 Length2 23.9 H Ethylamines Lewis (2001)
log V(oxidation) = 2.4861 0.1364NPL * NSIDE + 0.5694UNS 0.2433NOM * NMC R PCBs Parham and Portier (1998)
+ 0.001227MW * NUNSTOT + 0.8242IND 1.1493MOD
log Vmax (oxidation) = 1.6764 [ FY + 0.4243 [ FY 0.134 [ SF + 1.622
V(n-demethylation) = 0.005SA 0.52 R Amines Lewis and Dickins (2002)
V(n-demethylation) = 0.038SA 0.00001SA2 25.64 R Amines Lewis and Dickins (2002)
log kcat/Km(demethylation) = 0.53 log Po:w + 3.47 R X-C6H4N(CH3)2 Hansch and Leo (1995)
log V = 0.55 log Po:w R Barbiturates Hansch and Leo (1995)
(continued)
509
Table 40.6 (continued) In Silico Approaches for Estimating Reaction Rates of Chemicalsa
510
Approachb Speciesc Chemical Classd Reference
QSARS: FreeWilson-type equations

Vmaxc = BS(C-C)(51.6) + nH3(14.6) + nCL(4.84) + nCL2(10.2) + nCL3(16.9) R, H Chloroethanes Fouchcourt and Krishnan
(2000)
a kcat = catalytic rate, Km = enzyme afnity constant, V = metabolic rate, Vmax = maximal velocity of metabolism, and Vmaxc = body weight normalized
maximal velocity of metabolism. Y Y Y
b (E = LUMO energy HOMO energy, (H{ = hydrogen abstraction energy, Q = dipolar moment of the molecule, 4 [ F , 3 [ F , [ SF = connectivity indices,
BS = basic structure, diameter = diameter of the molecule, IND = variable dependant on experimental data used, IP = ionization potential, Length =
length of the molecule, MOD = variable dependant on experimental data used, MW = molecular weight, nCL = number of CL fragments, nCL2 = number
of CL2 fragments, nCL3 = number of CL3 fragments, nH3 = number of H3 fragments, NMC = number of meta chlorines, NOM = number of adjacent
unsubstituted ortho-meta carbon pairs, NPL = variable dependant on the number of chloride atoms in the molecule in ortho position, NSIDE = variable
dependant on the number of chloride atoms in the molecule in meta position, NUNSTOT = variable dependant on the number of chloride atoms in
the molecule, Po:w = n-octanol:water partition coefcient (or vegetable oil:water), SA = surface area, UNS = variable dependant on the number of atoms
in the molecule that are not chloride, Vol = volume of the molecule, and W = Hammet constant.
c F = sh, H = human, and R = rats.
d PCBs = polychlorobiphenyls.
Table 40.7 In Silico Approaches for Estimating the MichaelisMenten Afnity Constant (Km) of Chemicals
log 1/Km(demethylation) = 0.46 log Po:w + 0.63W + 2.62 H X-C6H4N(CH3)2 Hansch and Leo (1995)
log 1/Km(sulfation) = 0.92 log Po:w 1.48MR4 0.64MR3 + 1.04MR2 + 0.67W + 4.01 H Phenols Hansch and Leo (1995)
log Km(acetylation) = 0.42 log Pui + 0.14pKa 2.89 H Sulfonamides Seydel and Schaper (1982)
Km(oxidation) = [(Qiad(T)i)/|IPi b|] + c R Alkenes Csanady et al. (1995)
log Km(acetylation) = 0.17pKa 0.69 R Sulfonamides Seydel and Schaper (1982)
log Km(acetylation) = 0.42(Rmu:i + 0.15pKa 1.39 R Sulfonamides Seydel and Schaper (1982)
log Km(acetylation) = 0.07pKa + 0.31 log Po:w 0.33 Rb Sulfonamides Seydel and Schaper (1982)
2 0.50
log 1/Km(oxidation) = 1.39 log Po:w 0.22 log Po:w H Barbiturates Lewis and Dickins (2002)
log 1/Km(sulfation) = 2.93F2 + 1.16T2 + 0.91T3 + 0.82MR2 0.59IFOH + 1.29IET + 2.59 H Phenols Hansch and Leo (1995)
log Km(oxidation) = 43.27 4.03(E 0.60 log Po:w H Toluenes Lewis and Dickins (2002)
log 1/Km(glucoronidation) = 0.83 log Po:w + 1.37 R Phenols Hansch and Leo (1995)
log 1/Km(hydrolysis) = 0.056Z1H2O + 0.051Z2H2O + 0.026Z3H2O + 0.04Z4H2O + 4.616 R Phenylhippurates Kim (1993)
log 1/Km(hydrolysis) = 0.066Z1H2O + 4.259 R Phenylhippurates Kim (1993)
log 1/Km(hydrolysis) = 0.44T+ 4.08 R Phenylhippurates Kim (1993)
log 1/Km(hydrolysis) = 0.40T+ 4.40 R Phenylhippurates Kim (1993)
log 1/Km(NADP-oxidation) = 0.69 log Po:w + 2.90 R Drugs Seydel and Schaper (1982)
log Km(n-demethylation) = 0.55 log Po:w + 2.67 R Morphines Hansch and Leo (1995)
log Km(oxidation) = 0.61 log Po:w + 2.23 R Carbamates Hansch and Leo (1995)
log Km(oxidation) = 1.02 log Po:w + 2.98 R Pyrazoles Hansch and Leo (1995)
(continued)
511
Table 40.7 (continued) In Silico Approaches for Estimating the MichaelisMenten Afnity Constant (Km) of Chemicals
512

log Km(oxidation) = 1.05 log Po:w + 1.22 R 4-nitrophenyl alkyl Hansch and Leo (1995)
ethers
log Km(oxidation) = 0.79 log Po:w + 1.46 R Alkylbenzenes Hansch and Leo (1995)
log Km(oxidation) = 1.04 log Po:w + 1.10 Rb Toluenes Hansch and Leo (1995)

Km = BS(C-C)(3.8) + nH3(2.59) + nCL(0.37) + nCL2(0.79) + nCL3(0.19) R, H Chloroethanes Fouchcourt and Krishnan
(2000)
a (E = LUMO energy HOMO energy, W = Hammet constant, Q = dipolar moment of the molecule, T,T2,T3 = molecular hydrophobicity constants, (Rmui
= variable dependant on the resistance constant due to diffusion of the nonionized form in the lipid membrane, a = orbital availability, b = LUMO energy,
BS = basic structure, c = variable dependant on the molecular size, d(T) = normalized electron density, F2 = variable dependant on the electrical eld
induced by ortho positioned atoms, lFOH = variable dependant on the number of F OH groups in the molecule, lET = variable dependant on the family
of the substance, IP = ionization potential, MR2,3,4 = molar refractivity indices, nCL = number of CL fragments, nCL2 = number of CL2 fragments, nCL3
= number of CL3 fragments, nH3 = number of H3 fragments, pKa = log dissociation constant of an acid in water, Po:w = n-octanol:water partition coefcient
(or vegetable oil:water), Pui = n-octanol:water partition coefcient for the nonionized form, and Z1, 2, 3, 4H2O = variables corresponding to the potential
energy for the interaction between the molecule and water.
c PCBs = polychlorobiphenyls.
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Table 40.8 In Silico Approaches for Estimating the Skin Permeability Coefcient (Kp) of Chemicals
514
log Kp = 0.6267Ca 23.87(Q+)/E 0.289SsssCH 0.0357SsOH H LMWVOCs; HMWOCs Moss et al. (2002)
0.482IB + 0.405BR + 0.834
H LMWVOCs; HMWOCs Moss et al. (2002)
log Kp = 0.44R2 0.49 T + 1.487 E + 3.447 F + + 1.94Vx 5.13
H LMWVOCs; HMWOCs Moss et al. (2002)
log Kp = 0.59 T + 0.637 E + 3.487 F + + 1.79Vx 5.05
H Alcohols, steroids Ghafourian and Fooladi (2001)
log Kp = 5.33 0.62 T + 0.387 E + 3.347 F + + 1.85Vx
Steric descriptors
E 1 H LMWVOCs; HMWOCs Moss et al. (2002)
Kp = (b1 + 0.0025/(b2 + b3 + 3RZ )) MWb5
Kp = (b1 + b2Po:w)e(b3MW) H LMWVOCs; HMWOCs Moss et al. (2002)
log Kp = 5.14 0.477Ca + 0.237Cd + 0.038Pol H Alcohols, steroids Raevsky and Schaper (1998)
log Kp = 6.14 0.427Ca + 0.237Cd + 0.21L 0.11W H Alcohols, steroids Raevsky and Schaper (1998)
log Kp = 7.29 + 0.15Pol H Alcohols Raevsky and Schaper (1998)
log Kp = b1 + b2 log Po:w + b3MW0.5 H LMWVOCs; HMWOCs Moss et al. (2002)
H Hydrocorticone esters Ghafourian and Fooladi (2001)
log Kp = 0.428H 4.80 ; 93& + 28.06
log Kp = 0.652 log Po:w 0.00603MW 0.623ABSQon 0.313SsssCH 2.3 H Dermal drugs; LMWVOCs; Patel et al. (2002)
HMWOCs
log Kp = 0.77 log Po:w 0.0103MW 2.33 H LMWVOCs; HMWOCs Moss et al. (2002)
2 H Alcohols, steroids Ghafourian and Fooladi (2001)
log Kp = 0.786OT + 0.252 O 1.617 T 5.767
V
log Kp = 0.82 log Po:w 0.0093Vm 0.039MPt 2.36 H Steroids Moss et al. (2002)
log Kp = 0.84 log Po:w 0.07(log Po:w)2 0.27Hb 1.84 log MW + 4.39 H LMWVOCs; HMWOCs Moss et al. (2002)
log Kp = 28.4q + 0.018Vm + 2.824 H Barbiturates; Isoquinoline; Ghafourian and Fooladi (2001)
Salicyclic acid
log Kp = 3.99 log TA + 4.53 T V 0.762OT 11.364 H Alcohols, Steroids Ghafourian and Fooladi (2001)
H Pharmaceuticals Moss et al. (2002)
Kp = 1.17 v 107 3RZ + 2.73 v 108
E E H HMWOCs Moss et al. (2002)
K p = b 1( 3 RZ /(b3 + 3 ))
RZ
H Steroids Seydel and Schaper (1982)
log Kp = 0.207 log 3RZ + 1.49 log Po:w 5.42
H Phenols Testa et al. (2000)
log Kp = 0.37 log 3RZ + 2.39 log Po:w 8.71
log Kp = 0.544 log Po:w 2.88 H Aliphatic alcohols Seydel and Schaper (1982)
log Kp = 0.80 log Po:w 8.883 H Hydrocorticone esters Ghafourian and Fooladi (2001)
log Kp = 1.46 (log Po:w + 0.29 log Po:w 3.75 H Alcohols, steroids Testa et al. (2000)
log Kp = 4.36 0.387Ca + 0.247Cd H Steroids Raevsky and Schaper (1998)
Mechanistically based equations

Kp = (Pvo:w
* 0.028Dl /0.0340) + (Pp:w
* 0.88D p/0.0018) H Acids; Alcohols; Hydrocarbons Poulin and Krishnan (2001)
a + + +

H = solubility parameter, T = dipolarity/polarizability, E = overall hydrogen-bond acidity, F = overall hydrogen-bond basicity, 7Ca = hydrogen bond
acceptor free energy in the molecule, 7Cd = hydrogen bond donor in the molecule, 2O = molecular shape index, ; 93& = connectivity indices, ABSQon
= sum of absolute charges on oxygen and nitrogen atoms, b1, b2, b3, b4, b5 = regression coefcients without any assigned role, BR = number of rotatable
bonds, Dl = coefcient for diffusion into the lipid fraction of stratum corneum, Dp = coefcient for diffusion into the protein fraction of stratum corneum,
Hb = number of hydrogen bonds formed by the substance, IB = Balaban index, L = molecular length, MPt = melting point, MW = molecular weight, OT
= number of hydrogen bonding heteroatoms, Po:w = n-octanol:water partition coefcient (or vegetable oil:water), Pol = describes bulk or volume related
effects, Pp:w = protein:water partition coefcient for stratum corneum, Pvo:w = vegetable oil:water partition coefcient, q = the most negative charge on
the hydrogen bond accepting heteroatoms, Q+/E = positive charge per unit volume, T V = sum of atomic charges on hydrogen bonding heteroatoms,
T V = sum of atomic charges on hydrogen bonding hydrogens, R2 = excess molar refraction, SsOH = sum of E-state indices for all hydroxy groups,
SsssCH = sum of E-state indices for all methyl groups, TA = total solvant accessible surface, Vm = molar volume, Vx = McGowan characteristic volume,
and W = molecular width.
c CFCs = chlorouorocarbons, HMWOCs = high molecular weight organic chemicals, LMWVOCs = low molecular weight volatile organic chemicals, and
VOCs = volatile organic chemicals.
515
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Table 40.9 In Silico Approaches for Estimating the Oral Absorption Constant (Ka) of Chemicals
Approacha Speciesb Chemical Class Reference
log Ka(absorption) = 0.58 log Po:w + 0.35pKa 1.77 F Barbiturates Seydel and Schaper (1982)
Ka(absorption) = km[(1/Rf) 1]n/(Q + [(1/Rf) 1]n) R Sulfonamides Testa et al. (2000)
log Ka(absorption) = 0.04 (log Po:w)2 + 0.22 log Po:w + 0.04 R Sulfonamides Seydel and Schaper (1982)
log Ka(absorption) = 0.067 + log Po:w log(1.4 + Po:w) R Pharmaceuticals Yamaguchi et al. (1996)
log Ka(absorption) = 0.082(log Po:w)2 + 0.268 log Po:w + 3.96 R Organic acids Seydel and Schaper (1982)
log Ka(absorption) = 0.09 (log Po:w)2 + 0.44 log Po:w 0.396 R Sulfonamides Seydel and Schaper (1982)
log Ka(absorption) = 0.09 log Po:w + 0.83 R Xanthenes Seydel and Schaper (1982)
log Ka(absorption) = 0.18 log Po:w + 0.23 R Carbamates Seydel and Schaper (1982)
log Ka(absorption) = 0.24 log Po:w 1.37 R Antihistamines Seydel and Schaper (1982)
log Ka(absorption) = 0.30 log Po:w 0.07(log Po:w)2 2.38 R Sulfonylureas Seydel and Schaper (1982)
log Ka(absorption) = 0.3 log Po:w 0.57 log (0.34 Po:w + 1) 0.15I 0.74 R Carbamates Seydel and Schaper (1982)
log Ka(absorption) = 0.46 log Po:w 0.36 log (0.60Po:w + 1) 0.23 R Sulfonylureas Seydel and Schaper (1982)
log Ka(absorption) = 0.5 log Po:w 0.61 log (0.07Po:w + 1) 0.39 R Sulfonamides Seydel and Schaper (1982)
log Ka(absorption) = 0.502 log Po:w log (0.053Po:w0.0862 + 1) 0.384 R Sulfonamides Seydel and Schaper (1982)
log Ka(absorption) = 0.56 log Po:w (0.04Po:w0.84 + 1) 0.63 R Phenols; Anilines; Esters Seydel and Schaper (1982)
log Ka(absorption) = 1.36 log Po:w + 0.36 R Organic anions Seydel and Schaper (1982)
(continued)
517
Table 40.9 (continued) In Silico Approaches for Estimating the Oral Absorption Constant (Ka) of Chemicals
518
Approacha Speciesb Chemical Class Reference
QSARs: FreeWilson type equations

log Ka(absorption) = BS(BEN-SO2-NHCONH)(2.272) + nH (0) + n2-CH3 (0.088) + n4- R Sulfonylureas Seydel and Schaper (1982)
CH3 (0.074) + n4-C2H5 (0.163) + n4-OCH3 (0.229) + n2-NO2 (0.324) +
n3-NO2 (0.207) + n4-NO2 (0.323) + n4-Cl (0.198) + n4-Br(0.122) + nn-
C4H9(0) + nCH3 (0.638) + nC2H5 (0.361) + nn-C3H7 (0.145) + ni-C3H7
(0.244) + ni-C4H9(0.035) + nt-C4H9 (0.149) + ncy-C6H11(0.135) + nallyl
(0.419) + nC6H5 (0.088)
a allyl = allyl group, Br = bromide, BS = basic structure, C2H5 = ethyl group, C6H5 = aromatic ring group, CH3 = methyl group, Cl = chloride group, cy-
C6H11 = cyclohexyl group, H = hydrogen group, I = family indicator variable, i-C3H7 = isopropyl group, i-C4H9 = isobutyl group, km = rate constant for
transfer out of the membrane, n = group occurrence in molecule, n = constant specic to the equation without any given role, n-C3H7 = n-propyl group,
n-C4H9 = n-butyl group, NO2 = nitrooxide group, OCH3 = methyl ether group, pKa = log dissociation constant of an acid in water, Po:w = n-octanol:water
partition coefcient (or vegetable oil:water), Q = constant specic to the equation without any given role, Rf = reverse-phase TLC lipophilicity parameter,
and t-C4H9 = tert-butyl group.
b F = sh and R = rats.
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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 0
1,1-dichloroethane 1 1 0 1 0
1,2-dichloroethane 1 0 2 0 0
1,1,1-trichloroethane 1 1 0 0 1
1,1,2-trichloroethane 1 0 1 1 0
1,1,1,2-tetrachloroethane 1 0 1 0 1
1,1,2,2-tetrachloroethane 1 0 0 2 0
Pentachloroethane 1 0 0 1 1
Hexachloroethane 1 0 0 0 2
a BS = basic structure (C-C).
1,1,1-trichloroethane 1,1,2,2-tetrachloroethane
H Cl Cl Cl
H Cl Cl Cl
H Cl H H
Basic structure:
(-C-C-)
Molecular fragments: Molecular fragments:

1 x (-C-C-) 1 x (-C-C-)
1 x H3 2 x Cl2H
1 x Cl3
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. Examples
of the description of 1,1,1 trichloroethane and 1,1,2,2 tetrachloroethane are pre-
sented.
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Table 40.11 Contributionsa of Chloroethane Structural Features to Rat Partition

Coefcientsb and Metabolic Constantsc
Fragments Pb Pl Ps Pf Vmaxc Km
BS 56.8 2.02 0.746 28.9 52.7 3.75
Cl2 42.7 0.319 0.0181 1.16 9.40 0.863
Cl3 7.00 1.60 0.233 14.1 15.3 0.0932
Cl 9.60 0.506 0.00710 7.22 7.22 0.234
H3 50.1 0.653 0.0770 8.56 12.9 1.65
r2 0.98 0.91 0.99 0.96 0.82 0.88
a Contributions were obtained by multiple linear regression from experimental data on
chloroethane, 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:blood
partition coefcients, respectively.
c Vmaxc (Qmol/hr/kg) and Km (QM) refer to maximal velocity of metabolism and afnity con-
stant, respectively.
Table 40.12 Contributionsa of Chloroethane Structural Features to Human Partition

Coefcientsb
Fragments Pb Pl Ps Pf
BS 37.4 2.72 1.099 38.9
Cl2 29.6 0.365 0.163 0.105
Cl3 7.53 2.16 0.166 12.2
Cl 8.92 0.446 0.0510 10.6
H3 39.3 0.699 0.0450 1.05
r2 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:blood
partition coefcients, respectively.
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1000
y = 1.0089x
R2 = 0.9159
Estimated parameter value
100
10
0.1
0.1 1 10 100 1000
Experimental parameter value
Figure 40.2 Comparison of rat experimental and predicted parameter values. Experimental
values from Gargas et al. (1988, 1989).
1000 y = 0.8984x
R2 = 0.8557
100
Estimated parameter value
10
0.1
0.1 1 10 100 1000
Experimental parameter value
Figure 40.3 Comparison of human experimental and predicted parameter values. Experimen-
tal values were derived from Gargas et al. (1988, 1989).
Table 40.13 Comparison of Experimentala (Exp) and

QSAR-Estimated (Est) Values of Rat Partition
Coefcientsb and Metabolic Constantsc for
1,1,1-Trichloroethane
Parameter Exp Est
Pb 5.67 13.7
Pl 1.52 2.97
Ps 0.56 0.90
Pf 46.4 34.5
Vmaxc 43.1 50.3
Km 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 coefcients, respectively.
c Vmaxc (Qmol/hr/kg) and Km (QM) refer to maximal velocity of
metabolism and afnity constant, respectively.
Table 40.14 Comparison of Experimentala (Exp) and QSAR-

Estimated (Est) Values of Human Partition
Coefcientsb for 1,1,1-Trichloroethane
Parameter Exp Est
Pb 2.53 5.56
Pl 3.40 4.18
Ps 1.25 1.31
Pf 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 coefcients, respectively.
Integrating FreeWilson QSARs into PBPK Models
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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 fragments
constituting the molecule. This information is fed to the program that contains the
model constants, the FreeWilson type SPR, the contribution values of each
molecular 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 prole as output. The example
of 1,1,1-trichloroethane is shown.
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Tissue concentrations obtained using conventional-type PBPK

10
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R2 = 0.9746
0.1
model (mg/L)
0.01
0.001
0.0001
0.0001 0.001 0.01 0.1 1 10
Tissue concentrations obtained using QSPR-PBPK type model (mg/L)
Figure 40.5 Comparison of steady-state blood and tissue concentrations of chloroethanes in

rats exposed to 1 ppm, as simulated by conventional and QSAR PBPK models.
Qg/L) of 1,1,1-
Table 40.15 Steady-State Tissue Concentrations (Q
Trichloroethane in Rat and Humans Estimated Using the
Conventional (PBPK) and QSAR-Based (QSAR) Physiologic
Model Following a Continuous Exposure to 1 ppm
Rat Human
Tissue QSAR PBPK QSAR PBPK
Blood 22.9 16.6 12.8 8.5
Liver 6.77 4.22 6.23 5.61
Slowly perfused 20.7 9.31 16.7 10.7
Fat 790 770 502 472
Richly 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 24.9
Human Ci (ppm)a 6342 4252
a Calculated using the QSAR-derived Cass (59.3 mg/L).
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CHAPTER 41
In Silico Application of Quantum Chemical

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(a)
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Figure 42.1 Frame sequence showing the evolution of the action potential in an atrial tissue
using the Nygren model for the electrophysiology of the tissue. The times 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 Q%cm2 delivered to the leftmost
column of atrial cells. The stimulus lasted for 2 ms and VM in the legend is the
voltage in mV. (continued)
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Figure 42.1 (continued) Frame sequence showing the evolution of the action potential in an
atrial tissue using the Nygren model for the electrophysiology of the tissue. The
times 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 Q%cm2 delivered to
the leftmost column of atrial cells. The stimulus lasted for 2 ms and VM in the
legend is the voltage in mV.
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Figure 42.2 Action potential, entering from the left and encountering a region of high potassium
concentration region at t = 40 and 250 ms, respectively. The hyperkalemic region
is along the tissue central axis, in the center taking up 1 cm2 of the 3 cm v 3 cm
tissue.
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Figure 42.3 This gure 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 the
tissue 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 in
the right-hand panel. Modulation of the wave distortion and erratic behavior should
be noted.
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CHAPTER 43
Submillimeter-Wave Frequency
Studies of the Vibrational Modes
of Deoxyribonucleic Acid:
A Metric for Mutagenicity?
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Poly(dA)Poly(dT) Poly(dAdT)Poly(dTdA)
= 2 cm -1
Absorption
0 100 200 300 400 500 0 100 200 300 400 500
= 7 cm -1
0 100 200 300 400 500 0 100 200 300 400 500
Frequency (cm-1)
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.
Table 43.1 Comparison of Predicted Vibrational Band Positions (in

Wavenumbers) of Poly(dA)Poly(dT) with Values Observed
by Experiments in the Literature
Experiment Experiment
Calculations (Powell, 1987) Calculations (Powell, 1987)
20 Unknown 172 170
43 Unknown 199 200
57 62 211 214
71 80 238
94 95 270
106 106 348
131 136 400
160 459
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3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 250 0
-1
Wavenumber (cm )
Figure 43.2 Experimental absorbance spectra of herring DNA in the mid-infrared through the
very far infrared region.
0.60 Herring DNA

Salmon DNA
0.55
Transmission 0.50
0.45
0.40
0.35
0.30
0.25
24 22 20 18 16 14 12 10
Wavenumber (cm-1)
Figure 43.3 Fine structure in the absorbance spectra of herring and salmon DNA in the very
far infrared region.
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REFERENCES
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CHAPTER 44
Trophoblast Toxicity Assay (TTA):

A Gestational Toxicity Test Using
Human Placental Trophoblasts
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Cytotrophoblast
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Syncytiotrophoblast Trophoblasts Trophoblasts
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Figure 44.1 Pathways of trophoblast differentiation. Just as the basal layer of the skin gives
rise to keratinocytes, the cytotrophoblast the stem cell of the placenta gives
rise 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 studied
being hCG. cAMP, EGF, and even hCG itself have been implicated as stimulators
of this differentiation pathway. In addition to upregulating hCG secretion, cAMP
has also been shown to down-regulate trophouteronectin (TUN) synthesis. (Cen-
ter) At the point where chorionic villi make contact with external extracellular
matrix (decidual stromal ECM in the case of intrauterine pregnancies), a popu-
lation of trophoblasts proliferates from the cytotrophoblast layer to form the second
type of trophoblast the junctional trophoblast. These cells form the anchoring
cell columns that can be seen at the junction of the placenta and endometrium
throughout gestation. Similar trophoblasts can be seen at the junction of the
chorion layer of the external membranes and the decidua. The junctional tropho-
blasts make a unique bronectin trophouteronectin that appears to mediate
the attachment of the placenta to the uterus. TGFF and LIF have been shown to
induce cultured trophoblasts to secrete increased levels of trophouteronectin,
while down-regulating hCG secretion. (Right) Finally, a third type of trophoblast
differentiates toward an invasive phenotype and leaves the placenta entirely
the invasive intermediate trophoblast. In addition to making human placental
lactogen, these cells also make urokinase and plasminogen activator inhibitor-1
(PAI-1). Phorbol esters have been shown to increase trophoblast invasiveness in
in vitro model systems and to upregulate PAI-1 in cultured trophoblasts. The
general theme that comes from these observations is that specic factors are
capable of shifting the differentiation pathway of the cytotrophoblast toward one
of the above directions while turning off differentiation toward the other pathways.
See text for details.
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Figure 44.2 Purication of cytotrophoblasts from term placenta. A term placenta is minced and
digested with trypsin and DNAse. The supernatant is passed through calf serum
to inactivate the digestive enzymes; then these pellets are pooled and placed on
a Percoll gradient to separate out the cytotrophoblasts. (From Kliman et al. (1986)
Endocrinology, 118(4), 15671582. With permission.)
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Figure 44.3 In vitro morphologic differentiation of cytotrophoblasts. After purication, the

cytotrophoblasts are dispersed as individual cells (left). When plated in culture
media containing serum, these cells atten out and begin to move toward each
other. After 24 hr in culture, aggregates begin to appear, with some evidence of
cell fusion (center). After 72 hr in culture, most of the trophoblasts have fused and
formed large, multinucleated syncytiotrophoblasts. (From Kliman et al. (1986)
Endocrinology, 118(4), 15671582. With permission.)
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Figure 44.4 hCG secretion by trophoblasts in culture. Percoll-gradient puried cytotrophoblasts

were cultured in DMEM media for four days. Media was changed daily and assayed
for hCG by radioimmunoassay. hCG was not detectable at the time of initial plating.
(From Kliman et al. (1986) Endocrinology, 118(4), 15671582. With permission.)
Table 44.1 Regulation of Trophoblast hCG Secretion

Trophoblasts Effect on hCG
Factor (Trimester) 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)
F-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 (Petraglia et al., 1991)
stimulation of hCG
secretion
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 Term Inhibits (Beckmann et al., 1992)
hormone
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)
Figure 44.5 Effects of 8-bromo-cAMP on hCG and progesterone secretion by cultured cytotro-
phoblasts. Percoll-gradient puried cytotrophoblasts were cultured for 48 hr in the
absence () 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-treated
cultures secreted signicantly 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.)
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ALTERNATIVE

Progress in the Validation and Regulatory

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Historical Developments in the Humane

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RUSSELL AND BURCH

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A History of Interagency Approaches

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The Interagency Coordinating Committee

BACKGROUND AND HISTORY OF THE ICCVAM

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Table 3.1 Member Agencies

Table 3.2 Test Method Validation and Acceptance Criteriaa

Establishment of the ICCVAM

ICCVAM Authorization Act of 2000

Table 3.3 The Purposes of the ICCVAMa

Table 3.4 The Duties of the ICCVAMa

THE NATIONAL TOXICOLOGY PROGRAM INTERAGENCY

The ICCVAM Scientic Advisory Committee

THE ICCVAM TEST METHOD EVALUATION PROCESS

Test Method Validation

Test Method Submissions

Test Recommendations Agency

Figure 3.1 ICCVAM test method evaluation process.

ICCVAM Interagency Working Groups

Independent Scientic Peer-Review Panels

Expert Panels and Workshops

ICCVAM Test Recommendations

REGULATORY AGENCY CONSIDERATION OF ICCVAM

ICCVAM TEST METHOD EVALUATIONS

The Local Lymph Node Assay

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Frog Embryo Teratogenesis Assay in Xenopus (FETAX)

Up-and-Down Procedure for Acute Oral Toxicity

In Vitro Methods for Assessing Acute Systemic Toxicity

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Validation and Regulatory Acceptance

VALIDATION AND REGULATORY ACCEPTANCE

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prediction of systemic in vivo systemic

Figure 5.1 Building blocks of the ECITTS scheme.

Compounds and Test Battery

Table 5.2 Effects of the Test Compounds, Determined in Differentiated Human

CONCLUSIONS AND FUTURE PERSPECTIVES

Renement of the Neurotoxicity Test Battery

Estimated doses (mg/kg) or (mg/kg/day) 100.0

Lindane (CNC: inhibition of VOCC vs. LOED: learning; s.c.)

Different Exposure Times

Summary of the OECDs New Guidance

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