E. Marshall Johnson

Application of the Hydra Assay for Rapid Detection of Developmental Hazards

Adult hydra and artificial “embryos” composed of randomly reaggregated cells of dissociated hydra were exposed to test chemicals. The lowest concentration killing adult hydra became the numerator, and the lowest concentration disrupting development of “embryos” became the denominator of a ratio. This adult toxic (A) to developmentally toxic (D) or A/D ratio from hydra was compared to the A/D ratio calculated from published reports employing standard laboratory animals. Data from rodents were considered if both adult and developmental toxicities were consistent for: species, route, and duration of treatment and had a direct dose-response relationship indicating that the toxic manifestations were near the low end of a dose-response curve. The minimal expression of adult toxicity was usually loss in body weight, but developmental toxicity varied from abnormal development or death to reduced fetal weight. The hydra assay proved as effective as standard safety evaluations in rodents at detecting developmental hazards, i.e., substances capable of disrupting the conceptus at a small fraction of the minimal dose toxic to adults. It also was as effective as rodents in establishing that the majority of substances are capable of disrupting development of the conceptus only at doses also toxic to adults.

Multiple chemical exposures: synergism vs. individual exposure levels.

Exposure to single chemicals is known to produce congenital malformations in both pregnant animals and humans exposed at sufficiently high intensity. However, real life involves multiple, simultaneous exposures. Using as a database the 43 multiple chemical exposure studies located by Nelson (Teratology 49:33-71; 1994) where synergism was reported, we explored the degree to which such concerns may be realistic from the viewpoint of the current standard developmental toxicity safety evaluation process. Focusing on the assessment of the lowest tested dose of a given agent participating in synergistic activity as compared to its threshold level for eliciting toxicity when administered alone, we found that while the availability of adequate data was limited, all cases, with the possible exception of one, demonstrated synergistic toxic expression only when at least one, and usually both, compounds were used at or above their individual threshold for toxicity. These findings suggest that in animals such phenomena of synergistic chemical interactions are likely to occur only when at least one and more likely both agents are administered at or above their individual threshold for toxicity. To the extent animal studies are predictive of human developmental hazards due to single chemical exposures, available data do not establish multiple chemical exposures as a major human developmental concern.

The developmental toxicity of xylene and xylene isomers in the hydra assay

Two laboratories tested multiple forms of xylene for their developmental toxicity hazard potential (A/D ratio) by means of the hydra assay. The three isomers, as well as a solution of mixed xylenes, all interfered with development (D) at or near to concentrations that also were toxic to adult (A) hydra. The A/D ratios ranged from 1 to 2 in hydra as they had in conventional tests made in pregnant laboratory animals. Each testing laboratory concluded that xylenes were not primary developmental hazards but coaffective agents capable of disrupting development only at or near to concentrations also toxic to adults. In each instance every xylene tested interfered with the same stage or developmental sequence and in a concentration-related manner. The hydra assay may be useful for establishing priorities to test agents in a more elaborate system, but substances less soluble than xylene may exceed the tests applicability.

Perspectives On Reproductive and Developmental Toxicity

Human reproduction and development is a cycle of interdependent events. Virtually all of its phases have been shown to be the primary target of one or more non-mutagenic exogenous agents (Table 1). Such agents interfere with certain of the countless epigenetic or ontogenic events essential for normal completion of the cycle. Mutagens disrupt this cycle at some points, but the overwhelming majority of reproduc tive and developmental toxins are not mutagenic. As in all aspects of toxicology, the reproductive and developmental effects of chemicals are determined by the intrinsic nature of the chemical, the quantity of the chemical exposure, the duration of exposure and the stage of the cycle at which it occurs. Signs of repro ductive toxicity range from reduced fertility to spontaneous abortion. Adverse effects on the conceptus are categorized as functional deficits, developmental retardation, structural abnormality and death. One or more of these is anticipated to occur as a result of excess exposure to most chemicals. Although the degree of hazard and risk potential can be calculated in each instance, chemicals differ markedly in their ability to interfere with reproduction (Amann, 1982) and/or develop ment (Johnson, 1984). Standardized methods for reproductive and developmental toxicity safety evaluation are available for detecting adverse effects upon any

On the Subject of Incorrect Terminology

To the Editor: A 1993 article published in the Journal of the American College of Toxicology (Christian MS. Problems in developmental toxicity caused by incorrectly used terminology. J Am Coll Toxicol 19!33;12:323-8) has elicited a rather vigorous Letter to the Editor. As I am the Developmental and Reproductive Toxicology Section Editor of the Journal and a person mentioned in the original article, the Editor-in-Chief has asked that I add an additional perspective on what to him seemed a valuable article. Dr. Christian’s article emanated from her platform presentation in an ACT symposium on the topic of terminology problems in developmental toxicology. Her writing highlights and briefly illustrates some difficulties generated by our general lack of agreed-upon definitions for the terminology we all use. She points out that (a) different languages can provide different connotations to some scientific terms andor lack of terms present in other tongues and (b) different regulatory agencies, courtrooms, and even individual pregnant women view agent safety differently based on the presence or absence of terata in a standard animal test and may do so to an extent that this finding overshadows other signs of perturbed in utero development or even exposure intensity. The above observations are followed by a brief historic and contemporary series of referenced practical situations she and others have encountered. The examples are well chosen and make interesting reading through illustrative examples most of us have encountered over the years. The last page of the article provides a historic background of EPA, MARTA, JTS, and ETS efforts at resolution of nomenclature problems across venues and languages and concludes with an aspiration for IFTS consideration of this topic. The letter to the editor submitted by Dr. De Sesso et al. considers that Dr. Christian lays the “blame” for incorrect terminology on Dr. James Wilson. Their letter opines that Dr. Christian is incorrect in doing so on three bases: (a) The FDA Segment I1 study really is a teratology study; (b) this terminology, i.e., “teratology study,” did not result in an exclusion of other forms of embryotoxicity; and (c) if calling the standard developmental toxicity study a “teratology study” was a misnomer of great impact, it would not have taken two decades for the problem to be raised. The first and last paragraphs extol some of Dr. Wilson’s many virtues and abilities, but seem to do so as though his reputation needs defense from anyone. They conclude that he provided inspiration and scientific insight and that a misperceived sin of incorrect terminology is not his legacy. My reading of the original article is that Dr. Christian also extolled the virtues of Dr. Wilson, but chose her words incautiously: i.e., in considering that others understood the axioms involved, he made a “communication error.”