ANIMAL MODELS IN TERATOLOGY

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ANIMAL MODELS IN TERATOLOGY

• Bettina M. Francis
• Associate Professor
• Entomology Department

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Basic Principles of Teratology Testing

• Extrapolation between species is relatively
  unreliable
• 60 concordance for structural defects
• Statistically, each assay misses 40 of
  teratogens
• In vivo developmental assays are very expensive
• In vitro assays are useless for identifying
  teratogens

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Different Questions Require Different Models

• Regulating chemicals
• How do we prevent bad pregnancy outcomes without
  banning too many useful chemicals?
• Regulatory research
• Is the damage we see in animals important or
  relevant to humans?
• Basic research
• How does this chemical perturb development?

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Regulatory Developmental Toxicology

• Regulators have two goals
• To prevent the marketing of chemicals that are
  developmentally toxic to humans.
• To allow marketing of chemicals that are not
  developmentally toxic to humans.
• Tools include
• Laboratory testing
• Epidemiology
• Clinical experience

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Regulating Chemicals

• Basic problem
• preventing bad pregnancy outcomes
• infertility
• miscarriage
• stillbirth
• malformations
• functional deficits
• mental retardation
• problems of aging?

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Protocol for 2-Generation Assay
F0 parental animals
Initiate dosing at 30-40 days of age. Pair for
1st mating at 100-120 days of age
1st mating
Continue feeding chemical at each dosing level
Necropsy at weaning
F1A
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Protocol for 2-Generation Assay
F0 parental animals
2nd mating
F1B
1st mating
Necropsy at weaning
F1A
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Protocol for 2-Generation Assay
F0 parental animals
2nd mating
F1B
1st mating
Select F1 parental animals
Necropsy at weaning
F1A
2nd mating
1st mating
Necropsy at weaning complete histopathology
F2B
Necropsy at weaning
F2A
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2-Generation Assay Endpoints

• Fertility Index
• term pregnancies per mating
• Gestation Index
• liveborn per litter born
• Weaning Index
• weaned per litter born
• Growth index
• litter weights on selected days

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Advantages of the 2-Generation Assay

• A single assay identifies
• acute or cumulative toxicity leading to
• male or female infertility,
• pre- and post-natal mortality,
• pre- and post-natal growth retardation,
• functional deficits in offspring
• transplacental carcinogenesis
• infertility
• behavioral anomalies

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Disadvantages of the 2-Generation Assay

• Cost
• over 500,000 per species
• Labor-intensive
• Necropsies of all parents
• Necropsies of offspring of all litters
• Histopathology of offspring from 2nd litters
• Identifies the existence of a problem, but not
  always its nature

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FENARIMOL
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Fenarimol 2-Generation Assay
F0 parental animals
2nd mating
F1B
1st mating
Select F1 parental animals
Necropsy at weaning
F1A
???????
1st mating
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Developmental Toxicity of the Fungicide Fenarimol

• Fungicide
• Absence of 2nd generation offspring
• Mechanism
• Brains of newborn males dont imprint properly
• Exposure is through milk, not through placenta
• Imprinting in humans occurs prenatally
• Dose required is extremely high

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NITROFEN
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Toxicity of Nitrofen in Rats

• Adult toxicity
• LD50 gt 1 g/kg
• Adverse effect at LOAEL liver enlargement
• Fetal toxicity
• NOAEL lt 0.1 mg/kg/day
• Adverse effect at LOAEL diaphragmatic hernias
• Other heart, lung, kidney defects cleft palate.

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Brief History of Nitrofen

• 1966 First registered in U.S.
• 1971 Ambrose et al, Toxicol Appl Pharmacol
  Neonatal mortality at 100 ppm in maternal diet
• 1974 Kimbrough et al, Arch. Environ. Health
  Neonatal mortality confirmed
• 1981 Costlow and Manson Heart and lung defects
  identified
• 1981 Withdrawn from all U.S. uses

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Regulatory Research

• Is the damage we see in animals important or
  relevant to humans?
• Problem
• Are supernumerary ribs a malformation or a
  harmless variation?

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BROMOXYNIL
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Bromoxynil

• Major Commercial Use on genetically engineered
  cotton
• Acute lethal dose 160 mg/kg
• Developmental toxicant ??
• no effects seen in 2-generation assay
• causes extra ribs at maternally toxic doses

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Ossification Site or Rudimentary Rib
Branch et al 1998
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Supernumerary Ribs
Branch et al 1998
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Supernumerary Ribs SNR Harmless Variant?

• Kavlock et al In mice, 6 of 10 pesticides cause
  SNR at maternally toxic doses
• Chernoff et al Maternal stress on day 8 of
  gestation causes SNR in mice
• Kimmel et al SNR disappear after birth

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Isotretinoin 13-cis-Retinoic Acid
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Kessel, Development, 1992
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• Persistence of bromoxynil-induced SNR

Chernoff et al, 1991
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Persistence of bromoxynil-induced SNR
Chernoff et al, 1991
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Length of femurs and 13th ribs in fetuses with
and without SNR
Branch et al, 1998
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Summary SNR

• Two kinds of SNR
• ossification sites are transient
• not accompanied by altered size of last normal
  rib
• become part of tranverse process of vertebra
• longer SNR are permanent
• sign of altered pattern formation
• last normal rib is larger
• vertebra has more anterior pattern
• may be associated with ongoing problems in adult

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Brief History of Bromoxynil

• Major Commercial Use on genetically engineered
  cotton
• Acute lethal dose 160 mg/kg
• Suspect Carcinogen estimated to increase cancer
  incidence by 1.7 per million
• Developmental toxicant?
• 1998 Use on cotton banned by EPA

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Supernumerary Ribs Conclusions

• True supernumerary ribs (SNR) differ from
  rudimentary ribs or ossification sites (OS)
• OS disappear postnatally and are probably
  harmless
• SNR result from fundamental changes in embryonic
  patterning
• SNR are true malformations

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