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failure, and in some cases of prostate cancer. From 1940 to 1970, DES was used to help maintain pregnancy. In utero exposure to DES has been associated with abnormal development of the uterus. It has also been associated with certain types of tumors. Women who were exposed in utero often developed vaginal neoplasia, vaginal adenosis, and cervical erosion. Effects were not seen in offspring until they reached puberty. Clear cell carcinoma of the vagina is a type of adenocarcinoma found in young women who are exposed to diethylstilbestrol in utero. The reproductive organ of males can also be affected subsequent to in utero exposure. The outcomes include hypotrophic testes, poor semen volume and quality.
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13.5.4 Alcohol
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Fetal Alcohol Syndrome. Fetal alcohol syndrome (FAS) is a pattern of mental and physical defects that develops in some offspring when exposed to alcohol in utero. The rst trimester is the most susceptible period. Some babies with alcohol-related birth defects, such as lower birth weight and body size and neurological impairments, do not have all of the classic FAS symptoms. These outcomes are often referred to as fetal alcohol effects (FAE). Currently there is not total agreement among medical scientists concerning the precise differences between FAE and FAS. In addition to growth retardation, the most common outcomes of fetal alcohol syndrome include psychomotor dysfunction and craniofacial anomalies. The observed growth de ciencies are associated with an inability of the baby to catch up due to a slower than normal rate of development. Other infrequent outcomes include skeletal malformations such as deformed ribs and sternum, scoliosis, malformed digits, and microcephaly. Distinctive facial anomalies have been associated with a diagnosis of fetal alcohol syndrome: small eye openings, epicanthal folds, failure of eyes to move in the same direction, short upturned nose, at or absent groove between nose and upper lip, and thin upper lip. Visceral deformities may also be present: heart defects, genital malformations, kidney, and urinary defects. A common concurrent manifestation of FAS include central nervous system defects. These include irregular arrangement of neurons and connective tissue. Mental retardation may also be present and associated with learning disabilities as well as dif culties in controlling body coordination. 13.5.5 Non Chemical Teratogens
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Teratogens are not only xenobiotics. There may be other factors having the ability to cause malformations in the developing conceptus. Restraint stress in mice (12-hour restraint during early period of organogenesis) elicits axial skeletal defects (primarily supernumerary ribs). The Rubella virus ( rst reported in 1941, Austria) is associated with a number of fetal outcomes depending on the stage of development that the exposure occurs. Exposure during the rst and second month of pregnancy was associated with heart and eye defects. Exposure during the third month was associated with hearing defects (and mental retardation in some cases).
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13.6 TESTING PROTOCOLS
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Formal testing guidelines were established after thalidomide disaster. In 1966 guidelines were established by the FDA: Guidelines for Reproduction Studies for Safety Evaluation
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of Drugs for Human Use. Since then (1994) new streamlined testing protocols have been developed with international acceptance. This newer approach, ICH (13.6.2), relies on the investigator to determine the model to access reproductive/developmental toxicity. However, many scientist currently conduct and publish the FDA version of testing (i.e., segment studies). Therefore both (FDA & ICH) approaches will be discussed. Further practical details of testing protocols may be found in 21 as well as some discussion of the regulatory implications for different agencies.
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13.6.1 FDA Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use Multigenerational Study. This approach involves the continuous exposure of a rodent species (usually mice). The parental animals are exposed shortly after weaning (30 40 days of age). At reproductive maturity, the animals are mated. The rst generation is produced (F1 ). From these an F2 is produced and then subsequently an F3 generation. The effects of the test is monitored through each generation. The measured parameters include fertility, litter size, and neonatal viability. This is a time-consuming effort that usually takes about two years to complete. Single-Generation Studies. Single-generation studies are short-term studies conducted in three segments: Segment I: Evaluation of Fertility and Reproductive Performance. Male rodents are treated for 70 days (to expose for one spermatogenic cycle), and nonpregnant females for 14 days (to exposure for several estrous cycles). Treatment is continued in the females during mating, pregnancy, and lactation. Fifty percent of the females are killed and the fetuses are examined for presence of malformations. The other 50% are allowed to give birth. After weaning, these offspring are killed and necropsied. Segment II: Assessment of Developmental Toxicity. This involves the treatment of pregnant females only during the period covering implantation through organogenesis (typically from gestational days 6 to 15 in mice with 18-day gestational periods). One day prior to birth, the animals are killed and fetuses examined for viability, body weight, and presence of malformation. Segment II: Postnatal Evaluation. Pregnant animals are treated from the last trimester of pregnancy until weaning. Evaluated are parturition process, late fetal development, neonatal survival, and growth as well as presence of any malformations. 13.6.2 International Conference of Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) US FDA, 1994
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As previously mentioned, new streamlined testing protocols with international acceptance have been developed. Below is a description of these guidelines as it relates to similarity to a particular segment-type study.
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ICH 4.1.1: Fertility Assessment. This study duration is typically shorter than segment I studies. Males are exposed for four weeks before mating and females two weeks
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