1. Developmental Toxicology

2. Human Reproduction in the U.S.
Loss of conceptuses
50%-80% die before term
Most before implantation
Chromosomal abnormalities very common
Live births
Major malformations: 2% to 5%: 20 to 50 per 1000 live births
Genetic syndromes: 20% or ≤ 10 per 1000 live births
Environmental origins:
Maternal infections: 2%-3%: ≤ 1.5 per 1000 live births
Maternal metabolic imbalance 1%-2%: ≤ 1 per 1000 live births
Diabetes
Maternal infections 2%-3%: ≤ 1.5 per 1000 live births
Herpes, toxoplasmosis, rubella
Drugs and other chemicals: 4-6%: ≤ 3 per 1000 live births
Alcohol
Smoking
The causes of 60%-70% of birth defects are unknown

3. SCORE CARD Proven positive Rubella 1930s Radiation 1930s
Aminopterin 1952
Toxoplasmosis 1950s
Androgens
Thalidomide
Methylmercury 1960s
Warfarin s
Coumarin 1966
PCBs
Lithium 1970
Smoking s
DES
Alcohol
Diphenylhydantoin 1981
ACE inhibitors 1981
Isotretinoin (Accutane) 1983
Toluene (abuse) 1980s
Cocaine
Carbamazepine 1988
Hyperthermia s
Lead ongoing
Vit A derivatives ongoing
SCORE CARD
Probably negative
LSD
Sulfonamides
Adrenocortical steroids (cortisone)
Antihistamines (meclizin)
Bendectin
Hypoglycemics (insulin)
?????
Aspirin
Dioxin
Tranquilizers
Quinine
Marijuana
Barbiturates
Narcotics
Caffeine
Methadone s
Misoprostol 1991
Fluconazole (antifungal) 1992

4. Identifying Human Developmental Toxicants
Animal studies
65% concordance between species
Karnofsky’s law
Hundreds of animal teratogens known
~ 30 human agents
Alert clinicians
Rubella
Aminopterin
Thalidomide
Diphenylhydantoin
Warfarin
Alcohol
Valproic acid
DES
Epidemiology
X-rays
Smoking
Valproic acid
Dramatic discordance
Human/Rodent
Thalidomide
Cortisone
Rat/Mouse
Ethylene thiourea

5. Developmental Stages Birth to maturity Cognitive development
Brain continues to add neurons until ~ age 12
Connections continue to be made until early 20s
Growth
Adequate nutrition
Gene expression
Sexual development
Hormonal balance
Senescence
Long-term effects of early nutrition?
Environmental effects of childhood exposures?
PCBs?
Preimplantation Period
Totipotent cells
Damage is all-or-nothing
Possible exception: mutagens
Organogenesis
Pattern formation
Organ formation
Physical defects
Fetal Period
Maturation and growth
Functional deficits

6. Human Organogenesis
end of thalidomide sensitivity

7. W. His: Day 15 human embryo

8. W. His Day human embryo
(start of thalidomide-sensitive period)

9. W. His Day human embryo

10. W. His Day 31-34 human embryo:
(almost at the end of the thalidomide-sensitive period)

11. 6 week old W. His: human embryos 8.5 week old
~ 40 days old
~ 60 days

12. Principles of Developmental Toxicology
1. The developmental toxicant must have access to the target
Physical agents must penetrate maternal tissues
Chemicals must cross the placenta
2a. Dose-response relationships differ from other forms of toxicity
More malformations
More severe malformations
2b. There is a threshold dose below which there is no fetal damage
Most developmental toxicants are not mutagens
(But most mutagens are developmental toxicants.)
Remember Paracelsus!
3. Genetics interacts with the fetal environment
Most developmental toxicants affect only some exposed fetuses
~ 30% of offspring of alcoholic mothers have fetal alcohol syndrome
Thalidomide and possibly retinoic acid are exceptions to this rule
Anencephaly and spina bifida (ASB) illustrate this rule
Genetics acts
Across species
Examples: thalidomide, cortisone
Between strains or individuals within species
Example: cortisone

13. Interaction of Genetics and Environment: Anencephaly and Spina Bifida (ASB)

14. Principles of Developmental Toxicology
4. Time of exposure is very important in determining
Type of malformation
Incidence of malformation
Most birth defects must occur before a tissue or organ is fully formed
Exception: cocaine
The most sensitive period for malformations is during organogenesis
Well before the end of the 3rd month of pregnancy

15. Timetable of Thalidomide-Induced Malformations in Humans
(Timing is from the start of the last menstrual period)

16. Malformations Induced by a Hypothetical Teratogen

17. Principles of Developmental Toxicology
5. Manifestations of developmental toxicity include
Death
Malformation
Growth retardation
Functional deficit
A single chemical may cause several effects by different mechanisms

18. Functional Deficits Often occur in an already formed organ
Are not readily apparent at birth
Chemicals causing neurological deficits
Alcohol
Mercury
PCBs
Isotretinoin (AcutaneTM)
Diphenylhydantoin (DilantinTM)
All psychoactive drugs?

19. Growth Retardation Temporary Permanent Catch-up growth
Fetal Alcohol Syndrome
Small until puberty

20. Current Test Requirements
2 species
1 must be non-rodent
2 generation assay:
Continuous dosing from before mating of parents (F0) to weaning of F2.
Look at
Survival
Weight
Pathology
Highest dose must affect dam adversely
Additional studies to examine effects on
Fertility and mating
Organogenesis
Parturition and lactation

21. In Germany:
Contergan
In Britain:
Distaval

23. Reproductive Toxicology
Fertilization
Early cleavage
Gastrulation
[Implantation & organogenesis
role of placenta in fetal development]
Growth and maturation
Birth
Childhood: growth and maturation
Puberty & sexual maturity [or metamorphosis]
Mating

24. Reproductive Cycle: Egg Layers
All necessary nutrients must be in the egg when it is laid.
All wastes must be processed or excreted by the embryo.
The egg and its embryo are not buffered by the maternal organism.
Metamorphosis adds another layer of complexity - even more than puberty.

25. Requirements for Successful Reproduction
Female
Hormones
Estrogen
Progesterone
Prolactin
Ovary
Ovulation
Fallopian tubes
Transport of ova
Uterus
Receptivity
Maintenance of pregnancy
Ova
Offspring
Growth
Maturation
Puberty
Sexual development
Male
Hormones
Testosterone
Estrogen
Testes
Sperm maturation
Accessory organs
Epididymis
Prostate
Sperm
Number
Motility
Behaviour
Fenarimol in rats

26. Reproductive Toxicity Observed
In ecosystems:
Species-wide epidemiology
There are no juveniles … because …?
Male or female does not produce gametes OR
Gametes are not functional OR
Male or female does not (cannot) mate OR
Female cannot sustain embryos to independent life OR
Offspring do not mature sexually
In humans:
Epidemiology or clinical observation
Followed by medical documentation
Males provide most examples because
System is simpler
Count sperm, check motility
More occupational exposures

27. Assorted Reproductive Toxicants
Wildlife
DDT in some species of birds
Greater sensitivity + greater exposure
Eggshell thinning + normal embryos
Pelicans, peregrine falcons
TCDD equivalents in Great Lakes
PCBs, dioxins
Fish: lake trout
Death of early life stages
Birds: gulls, eagles
Malformed chicks, poor parenting
Pulp mill effluents
Fish
Temperature?
Alligators, turtles in Lake Apopka
Sewage outflow (phthalates, hormones)
Feminizing fish
Human Male
DBCP
Chlordecone (KeponeTM)
Phthalates and other “endocrine disruptors”?
Human Female
DES
Major focus on developmental tox
diethylphthalate

28. Protocol for 2-Generation Assay NB: No known reproductive or developmental toxicants are missed by this assay.
F0: parental animals
2nd mating
F1B
1st mating
Necropsy parents [F0 and F1] aftertheir 2nd litter is weaned.
Select F1 parental animals
Necropsy at weaning
F1A
Necropsy F2B at weaning; including complete histopathology
Necropsy at weaning
F2B
F2A
Continue feeding chemical to each group at the appropriate dosing level throughout the study (progeny, too!)

29. Current Test Requirements
2 species
1 must be non-rodent
2 generation assay:
Continuous dosing from before mating of parents (F0) to weaning of F2.
Look at
Survival
Weight
Pathology
Highest dose must affect dam adversely
Additional studies to examine effects on
Fertility and mating
Organogenesis
Parturition and lactation
Behaviour

30. Cholesterol
DES

31. Environmental Endocrine Disruptors: Hormones
Simplified diagram outlining points of entry of estrogenic chemicals into the water supply, adapted from Velicu et al., 2009 (12).
Published in: Amber Wise; Kacie O’Brien; Tracey Woodruff; Environ. Sci. Technol.  2011, 45,
DOI: /es
Copyright © 2010 American Chemical Society

32. Estimated contribution of total natural estrogens (E1+E2+E3) and EE2 excretion to total estrogen excretion in the Dutch population. OCs are estimated to be 1% of total. Figure adapted from ref 51. This figure does not account for the higher potency of EE2 compared to natural estrogens.
Published in: Amber Wise; Kacie O’Brien; Tracey Woodruff; Environ. Sci. Technol.  2011, 45,
DOI: /es
Copyright © 2010 American Chemical Society

34. Estimates of fate and excretion of EE2 in the body, adapted from Johnson and Williams, 2004(57). All numbers are approximate and final numbers do not add to 100% of the original ingested EE2, as a complete mass-balance analysis has yet to be performed.
Published in: Amber Wise; Kacie O’Brien; Tracey Woodruff; Environ. Sci. Technol.  2011, 45,
DOI: /es
Copyright © 2010 American Chemical Society

35. “Environmental Endocrine Disruptors”:Phthalates
DEHP:
diethylhexylphthalate
General Structure

36. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY OF PHTHALATES Jan L
REPRODUCTIVE AND DEVELOPMENTAL TOXICITY OF PHTHALATES Jan L. Lyche et al, Journal of Toxicology and Environmental Health, Part B, 12:225–249, 2009
“The present human toxicity data are not sufficient for evaluating the occurrence of reproductive effects following phthalate exposure in humans, based on existing relevant animal data. This is especially the case for data on female reproductive toxicity, which are scarce. Therefore, future research needs to focus on developmental and reproductive endpoints in humans. It should be noted that phthalates occur in mixtures but most toxicological information is based on single compounds. Thus, it is concluded that it is important to improve the knowledge of toxic interactions among the different chemicals and to develop measures for com- bined exposure to various groups of phthalates.”

37. Current Concerns: Environmental Endocrine Disruptors
Phthalates
and other plasticizers
Agricultural and pharmaceutical hormone use
Steroids (estrogens)
Recombinant Bovine Growth Hormone
Pesticides
Atrazine
Vinclozolin