The reproductive cycle encompasses a wide variety of complex interactions at the molecular, cellular and structural levels within a specific chronological sequence. The cycle starts with gametogenesis and includes all the differentiational and developmental processes occurring during the prenatal and postnatal periods. A broad spectrum of biological processes is represented, and includes cellular replication, tissue development and differentiation, neuroendocrine regulation, peptide and steroid hormone synthesis and action, secretory processes, and smooth muscle function, among others. Each of these processes is vulnerable to a multiplicity of toxic interferences. It is, therefore, extremely unlikely that a small number of 'simple' tests will ensure the identification of all possible adverse reproductive and developmental effects. In recent years, a great number of short-term tests for assessing reproductive toxicity has become available and can be performed in in vivo and in vitro systems. These have been recently reviewed in Vouk and Sheehan (1983). These tests have been developed as adjuncts to the existing array of primary test batteries and are designed to: (a) rank compounds for scheduling further testing; (b) generate more specific data on the basic biological processes which are involved in the reproductive cycle; and (c) further characterize the mode(s) of action of agents known to affect essential biological processes of the reproductive cycle. In vivo tests evaluate reproductive performance and perinatal toxicity. In vitro methods assess responses to test chemicals and other toxic agents (for example, ionizing and non-ionizing radiation), employing systems involving the growth and development of cells, tissues and organs of invertebrate and vertebrate origins, and whole-embryo culture~ of each. As our knowledge of basic mechanisms increases, it

The testes of marmosets (Callithrix jacchus), which had been treated with a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0.3 microgram to 10 micrograms/l kg body weight (BW)) were studied after 7 days using morphological and histochemical techniques. Light microscopic and electron microscopic examination revealed decreased intercellular contact in the germinal epithelium, as indicated first by enlarged intercellular spaces between the Sertoli's cells and between the Sertoli's cells and neighboring germ cells (i.e., spermatogonia and preleptotene spermatocytes), particularly in the basic compartment of the germinal epithelium. Second, decreased intercellular contact was indicated by the accumulation of premature spermatids and spermatocytes in the tubular lumen after TCDD treatment. The Sertoli's cells exhibited an increased amount of lipids, phagolysosomes, and vacuoles in their cytoplasm. Spermatids were frequently affected by TCDD, particularly during early spermiogenesis. These alterations included vacuolization of the cytoplasm and the development of additional germinal vesicles. This special effect on spermiogenesis became even more evident quantitatively by determination and counting of tubular stages in semithin sections. Tubular determination on the basis of the appearance of spermatids revealed that the ratio of tubular stages I to III became lower and that of stages V to VII became higher, dose dependently, indicating a maturation stop at the beginning of spermiogenesis caused by TCDD treatment. After TCDD treatment, Leydig's cells were morphologically unaffected, but histochemical investigations revealed decreased activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). The sensitivity of the applied methods was different in view of the level of unaffection. The effect of Leydig's cells, as indicated by the decreased activity of 3 beta-HSD, had already been found at a dose of 1 microgram/kg BW TCDD, whereas clear-cut morphological and morphometrical effects were seen at 3 micrograms/kg BW for the first time. Moreover, with the special effect on spermiogenesis in marmoset monkeys, the findings demonstrate that the toxicity of TCDD on testicular morphology is species specific.

Testes of rats, which had been injected with a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0.3 micrograms/kg-25 micrograms/kg body weight [BW]), were studied after 7 days using morphological and histochemical means. Light and electron microscopic examination revealed that TCDD affected testicular morphology in a dose-dependent manner. TCDD led to decreased intercellular contact, indicated by wide intercellular spaces between Sertoli cells between and Sertoli cells and neighbouring germ cells. Morphological alaterations in rat testes after TCDD administration included the sloughing off of premature spermatids into the tubular lumen and numerical increase of necrotic germ cells, in particular pachytene spermatocytes. Compared with control animals, Sertoli cells of treated rats exhibited an increased amount of lipid droplets and phagolysosomes. Vacuolization of the cytoplasm and fragmentation of the Sertoli cells occurred frequently. Examination of the different spermatogenic stages revealed that no stage was specifically susceptible to TCDD. In Leydig cells a decrease in enzyme activity of 3 beta- and 17 beta-hydroxysteroid dehydrogenases became evident by histochemical investigation. This effect on steroidogenesis was already found at a dose of 1 microgram/kg BW TCDD, whereas morphological effects were seen in the germinal epithelium for the first time at 3 micrograms/kg BW.

Possible effects on the next generation after long-term exposure (subcutaneous administration) of male rats to very high doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied. Two dose regimes were applied: TCDD-25 (initial dose: 25 micrograms/kg body wt; maintenance dose: 5 micrograms/kg body wt, once weekly) and TCDD-75 (initial dose: 75 micrograms/kg body wt; maintenance dose: 15 micrograms/kg body wt). Male rats were treated for 10 weeks before mating and then throughout the entire 12 week mating period. They were mated to unexposed virgin females. One group of pregnant females was used for teratological evaluations, and another group was allowed to deliver. No significant differences were observed in the number of implantations or fetuses per litter, and resorption rate, and fetal weight between the controls and TCDD-treated groups. No gross-structural anomalies occurred in any of the fetuses sired by TCDD-treated males. In the TCDD-25 group an increased frequency of two types of variations was observed which also occur in controls: incompletely ossified fingers (TCDD-25 = 5.1%, controls = 2.6%), and incompletely ossified ossa zygomatica (TCDD-25 = 1.8%, controls = 0.5%). In the TCDD-25 group a slight but statistically significant increase was observed in the rate of stillbirths (TCDD-25 = 1.3%, controls = 0.1%), apparently due to an unusually low frequency occurring in the controls (overall historical controls = 0.6%). There was no difference in postnatal mortality (TCDD-25 = 1.3%, controls = 1.3%). Taken together, despite the very high doses of TCDD used, the data do not provide evidence for biologically significant paternally-mediated developmental toxicity in the fetuses and newborn.

Mouse embryos were exposed to TCDD in vivo at various stages of organogenesis and with different dose regimes, calculated to give the same maternal tissue concentrations on day 12 of pregnancy. TCDD concentrations in maternal liver correlated well with corresponding concentrations in maternal adipose tissue. Similarly, the incidence of cleft palates (CP), as evaluated on day 18 of pregnancy, correlated well with maternal hepatic TCDD concentrations (on day 12 of pregnancy). These results may be taken as evidence for a direct action of TCDD on the palatal shelves during embryonic development.

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs and PCDFs) were given subcutaneously as a defined mixture (with a composition similar to that found in incinerator fly ash) to a marmoset couple (Callithrix jacchus).

Wistar rats were treated initially with very high single doses of 14C-2,3,7,8-TCDD (either 75 or 25 micrograms/kg body wt) followed by weekly maintenance doses of 15 and 5 micrograms/kg body wt, respectively. 14C-radioactivity was measured in various organs over a period of 22 weeks. 1) 75 micrograms TCDD/kg body wt (followed by the maintenance doses) was lethal for all the rats within a period of 9 weeks. While the concentration of 14C-TCDD equivalents in liver and thymus stayed reasonably constant during this period in the surviving rats, the concentration in adipose tissue and kidneys clearly increased in the same animals. 2) The dose of 25 micrograms TCDD/kg body wt (followed by weekly doses of 5 micrograms/kg body wt) proved to be a just tolerable dose over a period of 22 weeks for our strain of rats. 3) Within the individual variabilities the TCDD concentrations in the investigated organs showed no clear-cut decline, indicating that the animals were exposed to fairly constant levels of TCDD throughout the study. Thus, this dosing regime is suitable for maintaining constant TCDD exposure during long-term studies.

A defined mixture of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs and PCDFs) was subcutaneously administered to marmoset monkeys (Callithrix jacchus) and Wistar rats. Absorption and distribution in liver and adipose tissue were measured 7 days after treatment. High amounts of the applied dose could be detected only for the 2378-substituted congeners. The relative content in liver was found to be similar in both species for most of these compounds. The greatest difference was observed for 2378-T4CDF which may result from a shorter half-life in the rat. In adipose tissue higher concentrations of certain PCDD/PCDF-congeners were measured in monkeys than in rats. This may be due to a relatively low content of adipose tissue in marmoset monkeys.

Various chemicals or mixtures of chemicals were tested as vehicle for a reliable parenteral application of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to experimental animals. Great differences were observed in speed of absorption following subcutaneous or intramuscular absorption of 14C-TCDD. The best results were obtained with subcutaneous TCDD injection using a mixture of toluene/DMSO (1 + 2) as vehicle: 3 and 5 days after treatment the percentage of administered dose remaining at the injection site was about 10 and 2%, respectively.

The distribution of various polychlorinated dibenzo- p -dioxins and dibenzofurans (PCDDs and PCDFs) was measured in rat tissues following various routes of administration: subcutaneous or intraperitoneal (vehicle: mixture of toluene and DMSO). Subsequent to intraperitoneal injection the deposit within the liver was found to be almost identical with that found after subcutaneous injection. In contrast, much higher concentrations of the congeners were found in (abdominal) adipose tissue subsequent to an intraperitoneal injection. The extent of subcutaneous absorption was also measured using an oil-containing vehicle . Speed of absorption was found to be lower when compared with the vehicle without oil. This led to considerably lower concentrations in liver and adipose tissue for most PCDD/PCDF-congeners.

The morphogenetic differentiation of early postimplantation rat embryos (2 to 4 somite pairs) in culture was studied in the presence of 40 ng TCDD/ml (ppb) in the culture medium (bovine serum). The results suggest that the embryotoxic effects induced in rats in vivo are caused by an indirect action (presumably due to maternal toxicity).

The genetic and embryotoxic effects of bis(tri-n-butyltin)oxide (TBTO) were evaluated in multiple in vivo and in vitro short-term tests preparatory to its potential wide use as a molluscicide in control of schistosomiasis. When tested in the rec assay in Bacillus subtilis, TBTO was not mutagenic and it did not induce reverse mutations in Klebsiella pneumoniae. Neither in the presence nor in the absecne of rat liver activation system did TBTO produce point mutations in Salmonella typhimurium strains TA1530, TA1535, TA1538, TA97, TA98 or TA100. TBTO was matagenic in strain TA100 in a fluctuation test, but only in the presence of rat liver S9 (Aroclor-induced). TBTO did not induce gene mutations in the yeast Schizosaccharomyces pombe, mitotic gene conversions in the yeast Saccharomyces cerevisiae, nor sister-chromatid exchange in Chinese hamster ovary cells in the presence or absence of rat or mouse liver S9. In the latter cells, structural chromosomal aberrations, endoreduplicated and polyploid cells were induced. TBTO did not induce gene mutations in V79 Chinese hamster cells (to 8-azaguanine-, ouabain- or 6-thioguanine-resistance) in the presence of a rat liver postmitochondrial fraction or in cell (hamster embryo cells and human and mouse epidermal keratinocyte)-mediated assays. In mouse lymphoma cells, TBTO did not induce 6-thioguanine- or BUdR-resistant mutations. As many tumour promoters inhibit metabolic cooperation between V79 Chinese hamster 6-thioguanine-resistant/-sensitive cells, TBTO was tested but showed no such activity. TBTO was examined for the induction of recessive lethal mutations in adult Berlin K male Drosophila melanogaster, either by feeding or by injection. Doses of 0.37 or 0.74 mM did not increase the number of X-linked recessive lethal mutations. An increased number of micronuclei was observed in the polychromatic erythrocytes of male BALB/c mice 48 h after a single oral dose of TBTO (60 mg/kg bw), while a lower dose (30 mg/kg bw) was ineffective. Neither of the two doses had induced micronuclei 30 h after treatment. The reproductive toxicity of TBTO was studied in NMRI mice. In a 10-day toxicity study, the LD50 and LD10 were 74 and 34 mg/kg bw, respectively. An increased frequency of cleft palates was seen in the fetuses of mice (compared with controls, 0.7%) treated orally during pregnancy with 11.7 mg/kg TBTO (7%), 23.4 mg/kg (24%) or 35 mg/kg (48%).(ABSTRACT TRUNCATED AT 400 WORDS)

1.1. 14C-2,3,7,8-TCDD was administered s.c. to non-pregnant female and male marmoset monkeys. Radioactivity was determined between 3 h and 3 weeks in serum and 8 organs. There was no significant decrease in radioactivity in all organs within the 3 week period studied, except in the adipose tissue (37% decrease), indicating a very long half-life of TCDD in monkeys. Radioactivity in kidney and adrenals increased during the same period (approx. 40%).2.2. The tetra-, penta-, and hexa-CDDs tested, as well as 2,3,7,8-TCDF, induce cleft palates after s.c. administration in pregnant mice on days 9 – 11. The incidence of cleft palates was found to be: 25 nmoles TCDD/kg b.w. (34%); 100 nmoles penta-CDD/kg b.w. (20%); 200 nmoles hexaCDD/kg b.w. (15%) and 200 nmoles TCDF (40%).3.3. Studies on the teratogenic action of a mixture of TCDD with one of the other chlorinated compounds suggests a roughly additive effect with the dioxines and the furan tested.4.4. The radioactivity of 14C-2,3,7,8-TCDD was determined on day 18 in livers of pregnant and non-pregnant mice following a treatment on days 9 – 11 with TCDD alone or in combination with the pentaor hexa-CDD or with TCDF. The radioactive label found in livers of the non-pregnant mice were far higher (4 - 10-fold).5.5. Similar levels of TCDD were found in placenta, amniotic fluid and the fetus on day 16 of pregnancy after TCDD application on days 9 – 11. Compared with the trunk of the fetuses, the fetal head contained 50% less TCDD and the fetal liver contained 500% more.

Examples of a combined approach using in vivo as well as in vitro methods for the assessment of prenatal toxicity are presented. The topics discussed include the analysis of the possible embryotoxic potential of valproic acid (VPA), female sex hormones, bis(tri-n-butyltin) oxide (TBTO), and acyclovir and the problem of supplementing in vitro systems with drug-metabolizing activity.