Birth Control in Clinical Trials: Industry Survey of Current Use Practices, Governance, and Monitoring.

The Health and Environmental Sciences Institute (HESI) Developmental and Reproductive Toxicology Technical Committee sponsored a pharmaceutical industry survey on current industry practices for contraception use during clinical trials. The objectives of the survey were to improve our understanding of the current industry practices for contraception requirements in clinical trials, the governance processes set up to promote consistency and/or compliance with contraception requirements, and the effectiveness of current contraception practices in preventing pregnancies during clinical trials. Opportunities for improvements in current practices were also considered. The survey results from 12 pharmaceutical companies identified significant variability among companies with regard to contraception practices and governance during clinical trials. This variability was due primarily to differences in definitions, areas of scientific uncertainty or misunderstanding, and differences in company approaches to enrollment in clinical trials. The survey also revealed that few companies collected data in a manner that would allow a retrospective understanding of the reasons for failure of birth control during clinical trials. In this article, suggestions are made for topics where regulatory guidance or scientific publications could facilitate best practice. These include provisions for a pragmatic definition of women of childbearing potential, guidance on how animal data can influence the requirements for male and female birth control, evidence-based guidance on birth control and pregnancy testing regimes suitable for low- and high-risk situations, plus practical methods to ascertain the risk of drug-drug interactions with hormonal contraceptives.

Terminology of developmental abnormalities in common laboratory mammals (version 1).

This paper presents the first version of an internationally-developed glossary of terms for structural developmental abnormalities in common laboratory animals. The glossary is put forward by the International Federation of Teratology Societies (IFTS) Committee on International Harmonization of Nomenclature in Developmental Toxicology, and represents considerable progress toward harmonization of terminology in this area. The purpose of this effort is to provide a common vocabulary that will reduce confusion and ambiguity in the description of developmental effects, particularly in submissions to regulatory agencies worldwide. The glossary contains a primary term or phrase, a definition of the abnormality, and notes, where appropriate. Selected synonyms or related terms, which reflect a similar or closely related concept, are noted. Nonpreferred terms are indicated where their usage may be incorrect. Modifying terms used repeatedly in the glossary (e.g., absent, branched) are listed and defined separately, instead of repeating their definitions for each observation. Syndrome names are generally excluded from the glossary, but are listed separately in an appendix. The glossary is organized into broad sections for external, visceral, and skeletal observations, then subdivided into regions, structures, or organs in a general overall head to tail sequence. Numbering is sequential, and not in any regional or hierarchical order. Uses and misuses of the glossary are discussed. Comments, questions, suggestions, and additions from practitioners in the field of developmental toxicology are welcomed on the organization of the glossary as well as on the specific terms and definitions. Updates of the glossary are planned based on the comments received.

Two-generation reproduction toxicity study with isopropanol in rats.

A two-generation reproduction toxicity study was conducted in rats with isopropanol. Thirty rats of each sex per group (P1) were dosed once daily by oral gavage with 0, 100, 500 or 1000 mg isopropanol kg-1 for at least 10 weeks prior to mating. Parental animals were mated within groups for up to 3 weeks. Parental females were dosed during mating, gestation and lactation; parental males were dosed during mating through delivery of their last litter sired. The P2 adults were selected from the F1 litters and were dosed for 10-13 weeks before mating to produce a single litter. Findings in the parental animals included increased lactation body weight gain in the mid- and high-dose females, increased liver and kidney weights in the mid- and high-dose groups of both sexes and centrilobular hepatocyte hypertrophy in some P2 males. There was also accumulation of hyaline droplets and other microscopic findings in the kidneys from the mid- and high-dose P1 males and from all treated groups of the P2 males. Increased mortality was observed in the high-dose F1 offspring during the early postnatal period, although no other clinical signs of toxicity were observed in the offspring of either generation. In addition, offspring body weight was reduced during the early postnatal period in the high-dose F1 males and in the high-dose F2 pups of both sexes. Eighteen out of 70 F1 weanlings in the 1000 mg kg-1 group died or were euthanized prior to P2 selection. No treatment-related post-mortem findings were observed in the offspring from either generation.(ABSTRACT TRUNCATED AT 250 WORDS)

An abbreviated repeat dose and reproductive/developmental toxicity test for high production volume chemicals.

A novel protocol is described for obtaining preliminary data on repeated dose systemic effects and reproductive/developmental toxicity. The test protocol was developed by a group of experts at the request of the U.S. Environmental Protection Agency (EPA) for use as part of a Screening Information Data Set on high production volume chemicals. Interest in this protocol is shared by several regulatory agencies, including the Organization for Economic Cooperation, the European Community, and the EPA. To validate the study protocol, ethylene glycol monomethyl ether (EGME) was used. After a dosing period of approximately 6 weeks, EGME showed both systemic and reproductive/developmental effects similar to those previously reported using standard protocols. Thus, this test protocol may be used as a screening tool for high production volume chemicals.

Biotransformation, estrogenicity, and steroid structure as determinants of dysmorphogenic and generalized embryotoxic effects of steroidal and nonsteroidal estrogens.

A series of nine chemicals of varying structure and estrogenicity was investigated for biochemical determinants of their relative capacities to alter normal embryonic growth and developmental patterns during organogenesis in rats. In order to circumvent the potentially confounding influences of maternal factors, the direct effects of steroidal and nonsteroidal estrogens on cultured whole embryos were compared at concentrations producing readily measurable embryotoxicity but low embryolethality (2-20%). Nonsteroidal estrogens included were diethylstilbestrol (DES), hexestrol (HES), E,E-dienestrol (alpha-DIES), and tamoxifen (TAM). Steroidal estrogens were estradiol 17 beta (E2), estrone (E1), and 17 alpha-ethinylestradiol 17 beta (EE). For comparative purposes, the effects of two essentially nonestrogenic phenols, Z,Z-dienestrol (beta-DIES) and phenol, were also studied. TAM, a weak estrogen which also exhibits antiestrogenic properties, was studied for possible interactive effects with potent estrogens. Prosencephalic hypoplasia was the abnormality most consistently observed and was elicited by each of the chemicals investigated. Embryotoxicity was neither attenuated by TAM nor related to estrogenic potency or steroidal structure, but was strongly and unpredictably influenced by biotransformational determinants. Presence of a cytochrome P450-dependent oxidizing system in the culture medium resulted in marked increases in embryotoxicity of E1, E2, and phenol, only minor increases for beta-DIES and alpha-DIES, but in strikingly decreased effects of EE, TAM, and HES. It produced no statistically significant differences in effects of DES. The results obtained were compatible with the concept that effects of these agents on growth and development during the earlier stages of organogenesis are independent of steroid structure or estrogenic activity but strongly dependent upon pathways and rates of biotransformation of some (but not all) of the parent chemicals.

Contrasting effects of estradiol-17 beta and 17 alpha-ethinyl estradiol-17 beta on cultured whole embryos.

Estradiol-17 beta (E2) and 17 alpha-ethinyl estradiol-17 beta (EE) were compared in terms of their relative capacities to alter growth and developmental patterns of cultured whole embryos during the early stages of organogenesis. Embryos exhibited a notable differential susceptibility to the embryotoxic effects of parents E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically significant effects whereas EE elicited marked embryotoxicity. Inclusion of a P-450-dependent biotransformation system in the culture media resulted in a significant attenuation of the embryotoxic effects of parent E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically embryotoxicity by hepatic S9. The divergent results produced by the two steroids could not be attributed to differences in rates of catecholestrogen generation in the culture medium or by the conceptuses. The results demonstrate definitive dissimilarities between the effects of two steroidal estrogens on developmental parameters and document marked differences in the effects of biotransformation on their embryotoxic potential. The data strongly suggest that the embryotoxicity of these steroids is not mediated via interactions with estrogen receptors. Additionally, the data show that the differential capacity of these two steroids to produce embryotoxic effects is diametrically opposite to earlier reported patterns of their carcinogenic potential in the Syrian hamster kidney.

Conversion of estradiol-17 beta to reactive embryotoxic intermediates by cytochrome P-450-dependent bioactivating systems.

P-450-dependent enzyme systems added to media of cultured rat embryos markedly increased the embryotoxicity of estradiol-17 beta. Increases were markedly attenuated by omission of NADPH, omission of enzyme, substitution of female for male rat liver as enzyme source, d) replacement of N2 with CO or replacement of estradiol-17 beta with diethylstilbestrol. Embryotoxicity correlated well (r = 0.84) with catecholestrogen generating activities. Addition of a catechol-methylating system failed to modify embryotoxicity even though large quantities of methoxyestrogens were formed. The results document that endogenous estrogen can be converted by P-450 to embryotoxic intermediates and suggest that reactive proximate metabolites are precatechols, perhaps epoxyenones.

Embryotoxicity induced by diethylstilbestrol in vitro.

The embryotoxic potential of diethylstilbestrol (DES) was examined in a whole embryo culture system containing a P-450-dependent bioactivating system. Sprague-Dawley rat embryos were explanted on day 10 and cultured for 24 hours. Concentration-dependent effects of DES on embryonic growth parameters, viability, and embryotoxicity were observed. Concentrations of DES greater than 0.26 mM (final concentration) produced 100% embryolethality, while those below 0.15 mM were without significant effects. At a final concentration of 0.19 mM, DES produced only a slight increase in embryolethality. The same concentration elicited a marked increase in observed embryotoxicity, including prosencephalic hypoplasia, incomplete axial rotation, and open neural tubes. In addition, reductions in embryonic length, somite number, and protein and DNA content were observed. An exogenous P-450-dependent hepatic biotransforming (catechol-generating) system failed to alter either the incidence of observed toxic effects or measured growth parameters. Likewise, exposure of cultured embryos to 20% carbon monoxide (CO) failed to reduce DES-induced embryotoxicity, indicating a lack of participation of an endogenous P-450-dependent embryonic bioactivating system. Arachidonic acid (0.20 mM) and/or indomethacin (0.50 mM) also had no observable effect on DES-induced embryotoxicity, suggesting that prostaglandin synthase was not involved in the embryotoxic activity of DES, as has been proposed to explain its carcinogenic effect. The antioxidants N-acetylcysteine (1.14 mM) and alpha-tocopherol (0.08 mM) failed to protect against DES-induced embryotoxicity, while the anti-estrogen tamoxifen (up to 0.85 mM) actually enhanced this effect of DES in culture. The DES analogs Z,Z-dienestrol (DIES, 0.10 mM) and hexestrol (HES, 0.48 mM) were both embryotoxic in vitro. The presence of an exogenous P-450-dependent hepatic biotransforming system appeared to protect against HES-induced embryolethality but had little effect upon DIES-induced embryotoxicity. The results were consistent with a direct effect of DES independent of either estrogenicity or exogenously generated metabolites.

The potential role of redox cycling as a mechanism for chemical teratogenesis.

A survey of the literature indicates that several chemicals whose reduced metabolites are capable of undergoing redox cycling in biological systems also possess significant teratogenic properties when tested in vivo. We have initiated investigations to determine whether the embryotoxic effects of such chemicals could result from their redox cycling properties and whether redox cycling could be an important mechanism in chemical teratogenesis. In order to obviate the potentially confounding influences of maternal factors, our initial studies have been performed with a whole embryo culture system with redox cycling agents added directly to the culture medium. Several representative redox cycling agents including doxorubicin, paraquat, a series of nitroheterocycles, nitrosofluorene, and diethylstilbestrol (converted metabolically to redox cycling quinone/semiquinone radicals) have been investigated thus far. The nitroheterocycles which bear nitro groups with comparatively high redox potentials produced a striking, asymmetric defect involving primarily the right half of the prosencephalic and mesencephalic regions. The effect was exacerbated under conditions of low O2 tension. Accumulated data to date strongly suggest that reduction of the nitro group is an essential feature in the embryotoxic mechanism. Quinones (doxorubicin, paraquat) and compounds metabolically converted to quinones (diethylstilbestrol) appeared to produce embryotoxic effects via mechanisms not associated with redox cycling. Nitrosofluorene embryotoxicity was markedly exacerbated by changes in both intra- and extracellular glutathione levels, but definitive dependence on a radical-mediated effect or redox cycling was not demonstrated.

Valproate hydroxylation by human fetal tissues and embryotoxicity of metabolites.

The oxidative biotransformation of sodium valproate was studied in liver, lung, brain, and adrenal homogenates from human conceptuses with gestational ages ranging from 50 to 77 days. Analyses of metabolites by GC/MS indicated the formation of 3-hydroxy-, 4-hydroxy-, and 5-hydroxyvalproic acid, with hydroxylation occurring preferentially at the 4- position. The adrenal homogenate was consistently the most active fetal tissue studied, with rates of hydroxylation similar to those in rat and macaque liver homogenates. Reaction rates in the fetal adrenal homogenate were approximately four times those in fetal liver and approximately 10 times the rates of the same reactions measured in fetal brain and lung. Although valproic acid itself (0.8 mmol/L) was highly embryotoxic to cultured whole rat embryos, none of the hydroxylated metabolites produced by human fetal tissues exhibited significant embryotoxicity at equimolar concentrations. This suggests that hydroxylation of valproic acid in human fetal tissues is a process of detoxification, and implies that valproic acid is a direct-acting teratogen.

Incidence and potentiation of external and internal fetal anomalies resulting from chlordiazepoxide and amitriptyline alone and in combination.

The teratogenic potential of a combination of chlordiazepoxide (Cdz) and amitriptyline (Amt) was examined with regard to both internal and external anomalies. Timed pregnant golden hamsters were given a single intraperitoneal injection on day 8 of gestation of one of the following: chlordiazepoxide hydrochloride (28.5 mg/kg), amitriptyline hydrochloride (70.3 mg/kg), Cdz-Amt combination (28.5 mg/kg Cdz + 70.3 mg/kg Amt, in order to retain the 1:2.5 dose ratio utilized in a clinically-used preparation of these agents), or saline vehicle (control). Fetuses were recovered on gestation day 15 following maternal sacrifice. Cranial malformations were analyzed in Bouin's-fixed fetuses by making 1-mm coronal sections through each head, whereas visceral anomalies were examined following general dissection of each body. Amt alone produced a significant (P less than 0.05) incidence of bent tail and encephalocele, whereas Cdz significantly (P less than 0.05) altered the male:female ratio of surviving fetuses when compared with saline-injected controls. The Cdz-Amt combination caused significant increases in cranial malformations, open eye, bent tail, abnormal lung, and urogenital anomalies. The teratogenic effects of potentiation between the components of this combination are discussed in terms of external and internal malformations.

Steroid-binding specificity of the progesterone receptor from rat placenta.

We have attempted to delineate some salient features of the progesterone binding site of the cytosol progesterone receptor (Rp) in rat placenta by studying the binding profile of various chemical modifications of the progesterone molecule (P). The relative competition ratio (RCR) was used to calculate the relative affinity of P and the modified ligand for receptor (Kaprog/Kainh). Cortisol exhibited no appreciable binding. Other corticoids (corticosterone, deoxycorticosterone, 11 beta-hydroxyprogesterone) had relative affinities 10-30-fold lower than P. Alterations in the structure of P which caused extensive declines in relative binding affinity (i.e. greater than or equal to 100-fold) include: reduction of A-ring to the 5 alpha-stereoisomere (A/B trans), introduction of a 17 alpha-hydroxyl group greater than removal of C17 side chain greater than reduction of C20 carbonyl. The binding profile of the rat placental Rp was similar to that described for uterine Rp from other species indicating a high degree of conservation of molecular structure for the progesterone receptor binding site from species to species.