Ten

TETRATOGEN TESTING

The Tragedy of Thalidomide: Misleading Animal Tests

The thalidomide tragedy focused the attention of the public on the inadequacy - prior to the 1960's - of the testing of drugs, food additives and environmental chemicals to see if these substances were "teratogens," that is, could cause malformations in the offspring of women who had been exposed to them. DCBL, the pharmaceutical branch of the large liquor company, Distillers, which promoted thalidomide in Britain, relied on assurances by the German firm, Chemie Gruenenthal, which developed the drug in the mid 1950's, that it could safely be used in obstetrical practice without harm to mother or child. However, although Gruenenthal had carried out tests on rats and mice to demonstrate the drug's sedative effects, none had been done on pregnant animals. The final toll of this failure to recognize that thalidomide was a teratogenic agent was thousands of deformed children, most of them born with rudimentary or missing limbs. Their mothers had received the medication to control nervousness and nausea during pregnancy.

These tragic occurrences stimulated belated investigations of the drug in 1961 and 1962. Tests were performed on pregnant mice, rats and guinea pigs. Although rats showed some reduction in litter size after massive or prolonged doses of thalidomide, there were no deformities in their offspring. It was only after a particular strain of rabbit had been tested between the 8th and 16th day of pregnancy that malformations showed up in the litters. This happened during the phase of "organogenesis," when the body organs begin their development a period of maximum sensitivity to teratogenic agents. (Page, B., 1976).

The significance of this, and of the clinical observation that in humans too thalidomide was teratogenic only if given during a limited period in pregnancy, was demonstrated by the 1971 experiment of Lash and Saxen. In tests on cultured human embryonic tissue it was found that in the presence of thalidomide there was a marked decrease in cartilage development of tissues of embryos five to seven weeks old, predicting the limb deformities which actually occurred in the children of women who had received the drug during the second month of pregnancy. (Lash, J., 1971).

If tests similar to this had been performed at the time the drug was first developed, the pitiful deformities it caused might have been avoided. But it is unlikely that tests on pregnant animals alone, even if they had been done, would have signalled danger, given the relative immunity of rats, mice and guinea pigs to the teratogenic effects of thalidomide. Only an understanding of the action of the drug in the bodies of human mothers and foetuses, based on laboratory and epidemiological investigations, might have saved the 8,000 children whose lives have been crippled by these deformities.


Many Variables Interfere with Animal-Human Comparisons

The factors which modify test procedures in animals, and hence the variables which can affect comparison (extrapolation) between these and the results in man, are virtually endless. Here are some of them. (Grice, H.,1975, p.135).

1. Anatomical differences (one placenta in humans, two in common test animals like rodents and rabbits).

2. Difference in metabolic patterns between animals and humans both on the adult and fetal level, affecting the concentration, distribution and excretion of the test substance.

3. Variations in response of animal species; e.g., rats are relatively insensitive to cortisone and thalidomide; the cat exhibits unique pharmacologic reactions. "Spontaneous" malformations unrelated to known teratogens appear in the offspring of all species, including man.

4. Short gestation period in most experimental animals compared to man. Thus tissue levels of chemicals under test may not reach levels comparable to that producing teratogenicity in human pregnancy.

5. Sensitivity of animals to many environmental factors which may cause variable results. For example, pesticide residues in bedding can affect metabolism; temperature, high or low barometric pressure and audiovisual stimulation can induce congenital malformations in some species; unbalanced diet, including vitamin excess or deficiency, can be teratogenic, while inconsiderate treatment, the number of animals per cage, the age of the mother, and the season of the year have all been shown to influence the test results.

6. Route of administration of the test substance; vehicle or solvent in which chemicals are given; dosage. For instance, large amounts of test material can affect palatability of food and water and produce nutritional imbalance. An experiment to test Coxsackievirus B3 produced severe fetal growth retardation. But this proved to be largely the result of destruction of the mother's pancreas and consequent secondary undernutrition of the fetus rather than a direct effect of the virus on the fetus. (Coid,C.,1978,p.675).

7. Potentiation - when a compound not teratogenic in a species may yet boost up another compound from inactivity to active teratogenicity. Or a physical state may be a potentiator: immobilization, normally not teratogenic in rats, potentiates Vitamin A teratogenicity. It is even conceivable that a hitherto unknown teratogen might potentiate a known one, or two unknowns might act together to produce teratogenicity and thus explain presently unaccountable human malformations. The Grice report of the Canadian Ministry of Health comments:

"The problem of the requirements for tests on combinations of chemicals has created concern in regulatory agencies. It is recognized that to test, in combination, the multiplicity of compounds to which man is exposed daily, is totally impractical." (Grice, H., 1975, p.153).

A few pages later, the report half admits the near-futility of the search for assurance against teratogenic risk with the statement:

"While predicting hazards it is necessary to make an allowance for the variability of species and individuals and other unknown factors which might adversely influence the teratogenic consequences and whose significance is difficult to evaluate. A subjective compensation or a 'safety factor' must, therefore, be applied." (Ibid., p.160).

A "subjective" approach to "variability" and "other unknown factors ... difficult to evaluate" may have been an honest description of teratogenicity research up to 1975, but today it is fair to ask, "Are these criteria reconcilable with the present 'state of the art'?"

In his book about alternatives to animal experiments D.H. Smyth says: "If there is one field of investigation where the use of animals is justified it is the avoidance of malformed babies." (Smyth,D., 1978). He couples this with considerable scepticism of in vitro methods in this area. Everyone would agree in principle that no efforts should be spared to avoid the terrible tragedy of such births, but conventional animal experimentation has not been strikingly effective in achieving this. The numerous variables which make teratogenicity experiments hard to interpret and to extrapolate to humans are probably responsible. Therefore, as happens so often, there are scientific as well as humanitarian reasons for exploring other than traditional methods of investigation.


Alternative: Epidemiological Studies

Since we are concerned primarily with effects on the human species, the first area to consider is that of studies on human beings. For example, it has been found that the nicotine and carbon monoxide which enter the system of women who smoke when pregnant can retard fetal growth, produce lower than normal birth weights, and increase still-births and infant mortality. (Am.Cancer Soc., 1978). Other epidemiological studies on the drug and eating or drinking habits of childbearing women can be correlated with teratogenic effects. Clinicians, especially obstetricians and pediatricians, should be encouraged to report suspected adverse reactions of drugs in confidence to appropriate regulatory bodies. (Inman, W., 1971). In Britain, such reports are made to the government's Committee on Safety of Medicines.


Alternative: Human Fetal Research

The above studies are valuable, but knowledge they bring may be difficult to relate to specific birth defects. An earlier line of defense lies in the use of fetal material or the fetus itself in research. The thalidomide study of Lash and Saxon using embryonic tissue cultures, cited above, is a case in point. This, of course, is a sensitive subject, but some useful guidelines have been proposed by an advisory group appointed in 1970 by the British Government "to consider the ethical, medical, social and legal implications of using fetuses and fetal material for research." (UK DHSS, 1972).

Their principal recommendations were as follows:

1. Any fetus less than 20 weeks of age (weight 400-500 grams) is pre-viable and has not reached the stage at which it can exist as a separate living entity. Only fetuses of less than 300 grams should be used for research. At this weight those parts of the brain on which consciousness depends are very poorly developed structurally and show no signs of electrical activity.

2. Where a fetus is viable after separation from its mother it is unethical to carry out any experiments on it which are inconsistent with treatment necessary to promote its life.

3. It is unethical to administer drugs or carry out any procedures during pregnancy with the deliberate intent of ascertaining the harm that they might do to the fetus.

Within these bounds, fetal research can be a valuable alternative to animal investigations. The British advisory group includes an Appendix of 51 suggested research projects; here are a few which could relate to teratogenicity:

1. Fetal size in relation to maternal smoking habits in and before pregnancy.

2. Carbohydrate metabolism in hypoxic oxygen deficient] fetuses and the effects of maternal dextrose infusions [intravenous feeding].

3. Vitamin A content and activity of liver (and brain). (See Kochhar experiment below, p.181, for teratogenicity of Vitamin A).

4. Culture of renal tissues to elucidate the development of fetal renal malignancies.

5. Alterations in trace metal metabolism in relation to protein and electrolyte levels in amniotic fluid.

6. Placental metabolism: studies of the transfer of drugs, bacteria, and biochemical substances.

7. Chromosome studies, including abnormalities found in therapeutic as well as spontaneous abortions.

The advisory group adds this comment:

"There is a particular need to determine the ability or otherwise of the fetus to deal with substances, including drugs given therapeutically to benefit the 'mother, which may cross the placenta. Observations on the pre- viable fetus are necessarily limited to a period of two or three hours. They have, however, already contributed significantly to our understanding of vital physiological and biochemical processes before birth on which the development of a fetus into a normal child essentially depends ... and promise to be the most hopeful approach to understanding certain failures of the human brain to develop properly and the influence such factors as variants in sexual differentiation in utero may have on inherent behavioral patterns after birth."


Human Organ Cultures

Since such a fetus in its entirety is only viable, after removal from the mother, for two or three hours, it is fortunate that its parts can be kept alive in culture. These include cells, tissues (e.g. renal tissue, as in 4 above) and even organs. The value of organ culture is illustrated by an experiment performed by Karkinen-Jaaskelainen and Saxon at the University of Helsinki. These investigators wished to discover why and how rubella infection in the mother, when it occurs during the first trimester of pregnancy, often results in the occurrence of congenital cataract in the child. "To study the problem more closely," they state, "we looked for a model and immediately ran into the first difficulty, not uncommon in research on teratology: there was no good animal model for the study." However they found that they could produce cataracts in the lens fibres of chick embryo by infecting them through the eggshell with a viral disease similar to rubella, namely mumps. They observed that the chick lens, which forms from an invagination of the surrounding cells which then becomes a sealed vesicle, can only be invaded by the virus as long as it remains in open communication with the exterior. They continue:

"We now returned to the original problem of human congenital cataract and collected young embryos from therapeutic abortions. one of the eyes of each human embryo was infected with rubella virus in vitro and its pair, the uninfected control, was cultured in otherwise identical conditions. Twelve embryos obtained when less than 5-6 weeks old, had open-stage lens vesicles, and some hundred embryos were at the closed stage."

However, 50 of the latter had their lens vesicles opened surgically before infection and these, as well as the 12 whose vesicles were naturally open, developed cataractous changes, whereas the other with closed, unoperated vesicles, did not. (Karkinen-Jaaskelainen,M., 1976, p.275).


Advantages and Disadvantages of Organ Culture

The above paper is entitled "Advantages of organ culture techniques in teratology." The authors discuss some of the advantages and disadvantages of organ culture. Among the advantages are the following: it can be studied free of maternal or placental confounding factors; its stage of development can be exactly timed by observation; exact dosage and exposure time to the teratogen is known and can be simply controlled by changing the culture medium. Disadvantages are the necessity of working with very small organ fragments because of nutritional limitations - and even these fragments cannot survive for long; the laboriousness and delicacy of the technique as compared with simpler cell culturing; and the absence of the natural conditions which are found in whole animals experiments: for instance in situations where maternal metabolites may be more toxic to the fetus than the substance acting directly on it without maternal mediation.

Four New Alternatives in Teratogen Testing

1. Enzyme Activation

That this last disadvantage can be overcome to some extent is illustrated by recent work of Manson and Simons who tested the effect of the teratogen cyclophosphamide on an organ culture of mouse embryonic limb buds. It is known that the teratogenic effect of this drug is not demonstrable unless it is first activated by an enzyme system known as "mixed function oxygenases" (MFO). Since hamster embryonic cells have high MFO activity, they were co-incubated with the limb buds and cyclophosphamide, whereupon the metabolites of the drug induced abnormal limb development (a similar effect occurs in humans). (Manson, J., 1979).


2. A Battery of Tests to Assess Drug Action

Since the dire consequences of thalidomide have become known, the testing of new drugs for teratogenicity on pregnant animals has vastly increased. Desirable as it would be to reduce this often irrelevant sacrifice of animals in favor of techniques using human materials, it is not going to be possible in the near future to substitute the latter for much of the animal work. However, it might be possible greatly to reduce the numbers of adult animals sacrificed, and the duplication of tests in different species, if attention is focused on the actual mechanism of drug action, and the principles of abnormal development, through organ culture techniques and microscopic observation of the behavior of cells in culture, instead of merely noting gross pathology (or its absence) in the fetus.

D.M. Kochhar has attempted this and has described it in a paper entitled, "Elucidation of mechanisms underlying experimental mammalian teratogenesis through a combination of whole embryo, organ culture, and cell culture methods." (Kochhar, D., 1976, p.485). He studied the effect of two substances, retinoic acid (Vitamin A) and 5-bromodeoxyuridine, on cartilage development in the limbs of mice embryos. Through the methods referred to in his title he was able to demonstrate various teratogenic effects, including a progressive decrease in cartilage development and disturbances in cell migratory rates as recorded by time-lapse microphotography of mouse limb bud cell cultures. He suggests that no single system but rather a combination such as he has employed may be required to assess the teratogenicity of a new drug before it can be confidently released on the market.


3. Biosynthetic Patterns of Cells

Since the number of potential teratogens has vastly outstripped the possibility of testing them by laborious methods using the intact animal, rapid in vitro assay systems must be found. One such has been developed by Ruth Clayton of the Institute on Animal Genetics at the University of Edinburgh. She first described it in a paper published in 1976 (Clayton, R., 1976, p.473), and discussed recent developments in a FRAME symposium at the Royal Society in 1978. FRAME's abstract follows:


"The system developed in their laboratory is based on the premise that there is a relationship between the synthetic capacities of cells and abnormal development. All the major protein products of the cell are analysed in,microgels where abnormal synthetic patterns can be readily identified by autoradiographic and densitometric techniques. The method has the added advantage that it can be adapted for routine use. Samples of amniotic fluid taken from treated and untreated animals are applied to foetal cells (e.g. lens, neural retina, kidney & limb fibroblasts) in culture and then the effects on biosynthetic patterns are recorded. Using this system, they were able not only to predict correctly which of the two unknown substances was teratogenic, but also to identify the effects of the teratogen." (Anon., 1978b).


4. Inhibition of Cell Attachment to Lectin-treated Surfaces

A still more rapid screening system for teratogens has been described by Braun and Nichinson. Tumor cells attach rapidly and irreversibly to plastic surfaces treated with plant lectins. Teratogens inhibit this attachment. The investigators tested a series of agents. Thirteen did not inhibit cell attachment and were probably non-teratogens. Another nine were also noninhibitors but were considered to be false negatives; i.e., they were thought to require metabolic activation for their teratogenicity to be expressed. Twenty-five other known teratogens inhibited cell attachment. (Braun, A., 1979).

Although some of the work described above is exploratory and requires further confirmation, it is obvious that there are now many systems being developed in teratogenicity testing which are approaching closer to the use of human material and are reducing reliance on the old-fashioned intact animal experiment. The pessimistic attitude to in vitro work in this area expressed both by D.H. Smyth (Smyth, D.,1978) and by H. Grice and his collaborators (Grice, H.,1975) is no longer justified.


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