Thirteen

TESTING SPECIFIC CHEMICALS

1. BACTERIA TEST OF WATER TOXICITY

The pollution of water is an ever-increasing concern, and has led to the development of biological testing methods using fish, water fleas and other aquatic organisms. Fish, for instance, are immersed in different concentrations of the effluent to be tested, and the percentage surviving after 96 hours is recorded. The concentration in which 50% are killed gives the LC/50. Beckman Instruments has introduced an alternative, the Microtox Acute Water Toxicity Monitor, using a strain of luminescent bacteria as the bioassay organism. The lightproducing metabolism of the bacteria is about six times more sensitive to toxicants than fish lethality, and the decrease in light emitted can be measured by the Microtox quantitatively in half an hour as against 96 hours for the fish test.

Since each testing population of fish is potentially unique, they are much more difficult to standardize than hacteria; thus tests using the microorganisms are more reproducible. Also, they are said to be two orders of magnitude less expensive than the fish test (once the approximately $10,000 outlay for the instruments and stock of reagents has been made), and the bacteria test is certainly more humane. (Beckman Instruments,1979).


2. TESTING THE TOXICITY OF FOOD DYES AND INSECTICIDES ON UNICELLULAR ANIMALS

A group of Japanese researchers led by F. Sake, at Hokkaido University, have shown that Paramecium caudatum , a unicellular animalcule, can be used to assess the toxicity of insecticides and food dyes. Comparing the use of small rodents and protozoa, they state:

"Although the LD/50 values for small animals have been generally accepted as indicators of the toxicity of food additivies, use of such animals seems both time consuming and uneconomical, judging from the fact that there has been a rapid increase in the numbers of various chemical compounds, including food additives, that require testing. Chlorella, Saccharomyces cerevisiae var. and Tetrahymena have [also] been used as indicators for these purposes." (Sako, F., 1977).

Dr. Oscar Frank at the New Jersey Medical School, East Orange, has also been working with protozoa, especially Tetrahymens, devising assays for vitamins and amino acids in foodstuffs. He has shown these tests to be faster and cheaper than rat tests, and more sensitive. The American Fund for Alternatives to Animal Research (AFAAR) awarded him a grant in 1979 for this research. (Frank,O., 1980).


3. CELL CULTURE TESTING OF MEDICAL DEVICES

A standard method of testing plastic and other "biomaterials" is by implantation either in a rabbit's muscle or skin. W. Guess and associates devised an alternative test using a cell culture of mouse fibroblasts. These were covered with agar and neutral red (dye) and the sample of test material was placed on top. In 24 hours samples which were toxic killed the surrounding cells: these had lost the dye and were now colorless. After testing thousands of biomaterials in succeeding years and comparing the tests with the rabbit implants, it turned out that the tissue culture was actually more sensitive than the implant.

The U.S. Pharmacopoeia is considering this test as an alternative for the rabbit muscle implant procedure. (Guess, W., 1965).

In 1973, Wilsnack and associates reported a similar test on biomaterials, but instead of mouse fibroblasts they used a human cell culture (WI-38 embryonic fibroblasts). The authors point out that the human cells are free from the contaminating leukemia viruses often found in mouse or chick embryo cell cultures. They also proved more sensitive than the rabbit implant test. (Wilsnack, R.,1973).

4. TEST FOR EYE IRRITANTS

The use of the rabbit's eye to test not only cosmetics but detergents, pesticides and any substance which might be hazardous to human eyes is routine. This is known as the Draize test, after the Food and Drug Administration doctor who standardized it 35 years ago. Since it is a test which must cause extreme suffering it ought to be closely scrutinized and a humane alternative should be developed.

The rabbit's cornea is particularly sensitive: it is only 0.36 mm. thick compared with 0.51 mm. for both man and monkey. Furthermore, the rabbit's eye is not supplied with effective tear glands; thus the irritating substance cannot be readily dissolved. (Coulston, F., 1969).

Fig.l. Chart showing reaction of rabbit's eye traumatized by test solutions.


The Consumer Product Safety Commission test for eye irritants can be found in the U.S. Code of Federal Regulations. (US Laws, Stats.,1979,CFR,Title 16, TI,1500.42). One eye of an albino rabbit is used, the other, untreated, is the control. The substance to be tested -liquid, flake, granule, powder - is "gently" placed in the eye, and the ocular reaction is recorded at 24, 48 and 72 hours. The positive reactions, increasing in severity, are: "Ulceration of the cornea, opacity of the cornea; inflammation of the iris, hemorrhage, gross destruction; or in the conjunctivae an obvious swelling with partial eversion of the lids or a diffuse crimson-red with individual vessels not easily discernible." The accompanying chart (original in color) depicts responses of varying intensity to specific test solutions (Fig.1). Of course the rabbits are in restraint during the one to three days they are undergoing this suffering (Fig.2).



The familiar row, of rabbits (USA)


When animals are forced to undergo such suffering, the very least one expects is that the tests will be reliable and the results meaningful. A comprehensive study of these eye and skin irritation tests, conducted in 25 cooperating laboratories (including Avon, Revlon and American Cyanamid-Lederle) by M. Weil and R. Scala of Mellon Institute, Pittsburgh, and the Medical Research Division of Esso, revealed "extreme variation" in the way the laboratories evaluated the rabbits' reactions to standard irritants. The investigators concluded that "the rabbit eye and skin procedure currently recommended by the Federal agencies... should not be recommended as standard procedures in any new regulations. Without careful reeducation these tests result in unreliable results." (Weil, M., 1971).

More recently, investigators have attempted to recheck the findings of Weil and Scala. F. Marzulli and D. Ruggles with the cooperation of ten laboratories performed numerous Draize tests on rabbits and confirmed that there were striking differences in the way different technicians reported what they thought they saw in the rabbits. Nevertheless, Marzulli and Ruggles insisted that the test could still be valid for irritancy on the grounds that only one of the four observed criteria had to he positive - and the validity was still not discounted even if the observers differed on which of the four that was! (Marzulli, F., 1973).

Another pair of investigators, although they claimed to have found consistency in observations of irritancy, at the same time noted large variability in the reactions of individual rabbits. (Bayard, S., 1976).

Public Protests Against the Draize Test

The squirming in pharmaceutical circles to salvage an outworn, unreliable and inhumane test has not succeeded in silencing the growing public criticism of this procedure.

A group of several hundred local animal welfare groups, backed by the Humane Society of the United States and other national groups, has been organized by Henry Spira. Its purpose is apparent from its name, the Coalition to Stop Draize Rabbit Blinding Tests. A campaign of public information and protest has started, stressing the need for a humane alternative to the Draize test, and directed at the Cosmetic Toiletry and Fragrance Association, the federal agencies, involved, and the leading cosmetic companies. These companies, starting with Revlon, are being asked to allocate one-hundredth of one percent of gross annual sales to finding such an alternative. (Anon.,1980a).


Court Ruling: Test on Rabbits' Eyes Inapplicable to Humans

In 1974, the Northern District Court of Ohio handed down a decision which has strengthened the public protests against the Draize test. A girl using a shampoo suffered eye damage. Applying the Draize test, the Food and Drug Administration determined that the shampoo was an irritant and brought suit against the manufacturer. The ruling of the court, in favor of the manufacturer, was partly based on the finding that the FDA had failed to show that the results of tests on rabbits' eyes can be extrapolated to humans. (Ibid.).


5. SKIN TESTS FOR TOXIC OR IRRITANT SUBSTANCES

The Consumer Product Safety Commission's recommended skin tests call for acute (24 hours) or subacute (20 and 90 day) exposure. In the acute tests the animals, usually rabbits, have the skin of the trunk clipped free of hair and abraded. They are then anointed with the test substances, wrapped for 24 hours in a close-fitting "sleeve" of rubber, and immobilized. In the case of toxic materials, "dosage levels are adjusted... to be fatal to 50% of the animals." (US Laws,Stats.,1979,CFR,Title 16, II,1500.40-41).

Irritants are applied similarly, but in patches, and evaluated according to the following scale after 24 to 72 hours.

 Symptom

Description

Value
 
ERYTHEMA [redness]
 None
Very slight
Well-defined
Moderate to severe
Severe (beet redness) to slight eschar formation (injuries in depth)

0
1
2
3
4
 EDEMA [swelling]  Evaluation as above, from "none" to "severe" (4).  


Compounds which are only slightly toxic on acute exposure may be severely damaging an repeated application. Therefore, tests are recommended in which large doses of the substances are applied daily ("20-day subacute experiment") to areas of both intact and abraded skin. To abrade the skin, adhesive tape may be attached, then stripped off. Repeated applications of the tape allow the stripping of successive layers of skin down to the required level. (Lansdown, A., 1972).

Some experiments require the intact skin to be anointed daily for as long as 90 days. The skin is not covered with a rubber sleeve, as in the acute experiments, but the aninals have to be in restraint and the potential suffering can be gauged by the comment: "the agent may produce a mummification or a coagulative effect on the skin...or complete necrosis, or severe eschar formation with desquamation." (Assn of Food and Drug Officials, 1979).


"Pain and Distress": a Report from the
Laboratories on Rabbits

Rabbits, as we have seen, are a favorite subject for testing irritants on eyes and skin, and cited below are excerpts from Annual Reports to the Dept. of Agriculture under the Animal Welfare Act of companies which were among the largest users of these rabbits. The experiments described were all listed under Column 1) of the report form since they involved "pain or distress" for which pain-relieving drugs were not given.

Dow Chemical USA, Midland, Michigan. "1516 rabbits. The animals listed in the column headed 'Pain-No Drugs' were used in eye and skin irritation studies, such as those required by the Federal Hazardous Substances Act and by the Federal Insecticide, Fungicide and Rodenticide Act." (USDA/APHIS,1976, Dow Chemical Co.).

Procter and Gamble Co., Cincinnati, Ohio. "1561 rabbits. These are product safety tests required by the CPSC [Consumer Product Safety Commission] and FDA to evaluate the ocular irritancy and potential damaging effects of products after topical instillation in the cul- de-sac. It is necessary that no topical anesthetics or analgesics be used since their use may interfere in the proper evaluation of the test. Most products cause only transi,nt irritancy and very mild damage to the conjunctiva and/or cornea. If severe irreparable damage is induced, the animal is humanely euthanized." (USDA/APHIS,1976,Procter & Gamble Co.).

Hill Top Research, Inc., Miamiville, Ohio. "1604 rabbits. The rabbits in our laboratory are often involved in several studies, sequentially. These include eye irritation studies, primary skin irfitation and corrosivity studies, and finally a dermal LD/50 study. Generally, at least one of these studies involves a test substance that is painful to the rabbit. If a rabbit suffers ill effects as a result of a particular study, it is sacrificed and not used in any subsequent study." Pain relief omitted "because this would have interfered with protocol design and test results." (USDA/APHIS,1976,Hill Top Research, Inc.).

This last report gives a rare glimpse of repeated use of the same animals in painful experiments, which opens up a whole new dimension of suffering for these animals. How serious do the "ill effects" mentioned above have to be before the rabbit is put out if its misery?


"Professionally Acceptable"

By their own admission, these companies do not give pain-relieving drugs to the suffering animals. Why not? Because "it would interfere with the interpretation of the results." But the analgesic is to dull the sensation of pain; surely the results would still be plain to see on the blistering skin and in the hemorrhaging eye? No, the pain is declared "necessary," and the attending veterinarian, the laboratory director and the Animal Care Committee all certify that "professionally acceptable standards" have been maintained.

What these companies should be required to do is to produce scientific evidence that the use of analgesics would interfere with the results. If they cannot, then "unnecessary pain or distress" has been inflicted - and this is prohibited by Sec. 2.28(d) of USDA Regulations. (US Laws,Stats.,1979, CFR, Title 9,1).

To sum up: from the internal inconsistencies in these reports, and by comparing them with one another and with those of other companies, it appears that even the meager information requested under the Animal Welfare Act is not being supplied. These reports will continue to be useless until the regulations require that the number of animals used, the nature of each experiment, and the use or non-use of pain-relieving drugs (including postoperative analgesics) be specified.


Alternatives to Animal Tests for Eye Irritants

In 1975-76 Dr. P.B. Harper at Hazleton Laboratories Europe Ltd. investigated an alternative to the testing of irritants on the eyes of living rabbits. (Hazleton Labs. Europe Ltd.,1976 and 1977). The study was commissioned by the Dr. Hadwen Trust for Humane Research and involved the exposure of four different cell cultures (KB, HeLa, HEp2 and L929 cells) to shampoo-type products known respectively to be of low, intermediate and high irritancy.

These cell cultures were exposed to the shampoos for 72 hours, after which cells were counted to see how many had been destroyed and what had happened to the total protein content. It was found that the more irritating the shampoo, the greater the destruction of cells and the greater the decrease in total protein, the latter from interference with cell metabolism.

L929 (mouse fibroblast) cells showed great promise. These cells could distinguish between the different degrees of irritancy of all three shampoos. KB and HeLa, which are cultures of human cancer cells, were less sensitive, but could distinguish the low irritancy shampoo from those of intermediate and high irritancy (but could not distinguish between the latter two).

After this promising beginning, the investigation at Hazleton was discontinued, possibly because the Dr. Hadwen Trust could not continue financial support. However, an effort to develop an alternative test along these lines, without using animals, is currently being investigated at Johnson and Johnson pharmaceutical laboratories in New Jersey. Up to now, they have not been willing to divulge any details of their research, at least to this writer.

In addition to the above, a cited as alternatives: the use of human and animal corneas from eye banks, corneal tissue cultures, rabbit lenses prepared in organ cultures, and corneas, supplied by hospitals, which are frozen and may he kept indefinitely. (UAA,1973).

Alternatives to Animal Tests for Skin Irritants

When there has been exposure to toxic or irritating substances, the first question to ask is whether there is likely to be penetration of the skin and subsequent toxic effects in the body. if so, tests for general toxicity are necessary. If the substance is already being produced and is in use, these tests and epidemiological studies can be done on the workers in contact with the substance during the production and on those who are exposed to it after it has been distributed (e.g. farmers in the case of agrichemicals). (Gehring,P.,1973). However, if the substance, say as a pesticide spray, or detergent, or cosmetic, is in the phase of development, attention must first be focused on the skin itself and its relative impermeability or tolerance to the material.

Human volunteers are often used in testing cosmetics, detergents and the like, usually after screening on animals. However, animal tests can be misleading. Griffith and Buehler at the Procter and Gamble Company comment as follows on safety prediction for household products tested on humans, guinea pigs and rabbits:

"Though the guinea pig results were generally predictive of the human results, rabbit responses to the soap, the liquid detergent, and the cleaner would incorrectly place these products in the next higher, or moderate irritant category. Of the last five products, two (shampoo, pine oil cleaner) were weakly irritant, one (ammonia) was moderately irritating, and two (metasilicate detergent and bleach) were strongly irritating to human skin. Neither the guinea pig nor the rabbit skin would have predicted any of these responses correctly on the basis of absolute scores. Three of the rabbit results erred on the high side and two on the low side; the opposite was true for the guinea pig." (Griffith, J.,1977, p.158).

Griffith and Buehler used the "repeated-insult patch test" in their human subjects. A "scarification test," in which the test substance is placed on a small grid of scratches in the skin, under a 12mm chamber, is said to be faster and more accurate, especially for mild topical agents. (Frosch, P., 1977).

Radioactive Isotopes Permit Systemic Tests in Humans

It is true that the very distressing studies described on p.220-222 frequently involve the absorption of toxic products into the system over a comparatively long period; thus animals are used rather than man because of the potential danger. However, with proper safeguards,- substances can be applied long enough in humans to allow systemic penetration and metabolic studies. R. Drotman tested a quaternary amonium,chloride widely used in laundry softener products , radiolabeled with 14 Carbon isotope, on the skin of several species - rat, rabbit, guinea pig and human - for 72 hours (144 hrs. for the human). He studied the systemic metabolic pathway of the product through the distribution of radioactivity in excreta (carbon dioxide, urine and feces). Since radiolabeled substances as small as one million millionth of a gram can be detected, the minute quantities of radioactive isotopes used were not dangerous. (Drotman, R.,1977, p.97)

When tissue damage is likely to be serious and put volunteers at risk, cultures can be exposed to the test material and cell injury can be observed microscopically. Time studies will show whether the trauma results in complete or partial cell destruction and, in the latter instances, with what degree of cell recovery. Non-specialized tissue may be adequate to assess the possibilities of cell survival, but if specific skin functions have to be studied, the human skin culture system used by B.A. Flaxman may be used. This culture includes proliferating epidermal cells and cells undergoing keratinization and desquamation. (Flaxuan, B.,1972). Pieces of skin taken from human cadavers have also been used in permeability studies, but these are not always accurate predictors of percutaneous absorption in living man. (Webster, R.,1977, p.120). One reason is the absence of desquaration in the excised skin.


6. TESTING BY COSMETIC COMPANIES

Avon, as befits a large cosmetics company, use - many animals in testing its products for "integumentary" (skin) irritation. In addition to their 3,800 rabbits, they had 4,900 rats and 1,600 guinea pigs in 1973. (USDA/APHIS,1973,Avon Prod.) Along with all approved laboratories in New York, they sign annually an agreement with the state in regard to the use of experimental animals, agreeing, among other things, to their "humane handling" and that "no greater number of animals will be used for scientific tests, experiments, or investigations than is actually necessary." Describing work that would involve the use of living animals during the year 1973, they stated:

"The work will involve the determination of acute toxicities of cosmetic products or ingredients. Rats, guinea pigs, and rabhits will be used. Interest will be primarily in the oral and percutaneous [through the skin] routes. In addition eye irritation and skin irriation tests as specified by the Food and Drug Administration will be performed on appropriate species. Sensiticing studies and inhalation toxicity studies will he done on guinea pigs and/or rabbits. The emphasis in this research will be on the integumentary system from a therapeutic, cosmetic or irritation aspect." (Avon Prod., 1973).

Although from the description of the tests it is plain that they must have caused much suffering, the company claimed in its 1973 Annual Report to the Dept. of Agriculture under the Animal Welfare Act that none of these experiments "involved pain or distress without the use of anesthetics, etc." In 19-76, they reuorted using 2,234 guinea pigs, 235 h3rsters and 2,248 rabbits, claiming that these too suffered no pain. None of these animals received pain- relieving drugs, but Avon admits that in the same year another 346 rabbits did suffer pain or distress, still without the use of anesthetics, analgesics or tranquilizers ("would interfere with test results"). The rabbits, they report, were part of the "Safety Testing (Draize Eye Irritancy Test) of all cosmetic products required by FDA." (USDA/APHIS,1976, Avon Prod.).

Revlon, another large New York-based cosmetics company, conducts similar tests, using 1,500 rabbits, 500 guinea pigs, 500 mice and 7,000 rats in 1972. A June 12, 1972 article in the Christian Science Monitor, poetically captioned "White Rabbits - Blue Eyes," described a visit to the five-story building containing Revlon's thirty laboratories and $2 million worth of equipment. The reporter wrote:

"Some experiences tattoo themselves upon the memory. Three white rabbits all in a row, encased in little boxes that look like reducing machines. Helpless, with their heads sticking out of holes in the top. Around the left eye of each what looks like blue eye shadow." (Loercher, D., 1972).

In 1972 Revlon stated that 150 experiments were performed on rabbits - "eye and skin irritancy tests on cosmetics" - and 140 on rats - "acute and subacute oral toxicity tests," and these involved "pain or distress without anesthetics, etc."1 (USDA/APHIS,1972, Revlon Res.Center). The number of animals per experiment was not given but could be quite large. In 1973 they again list numerous rabbits, guinea pigs and rats, and state that no anesthetic, etc., was used in performing the following tests: "Draize Eye Irritation, Acute Oral Toxicity, Primary Skin Irritation, Subacute Dermal Toxicity and Inhalation Toxicity," but now claim that none of these tests caused pain or distress. Yet some of them are the same ones which they reported as painful in the previous year. (USDA/APHIS,1973,Revlon Res. Center).

Finally, in 1976, Revlon reported a "total" of 1,600 guinea pigs and 2,000 rabbits used, but instead of a breakdown specifying, as the Dept. of Agriculture form requires, the number of these in the categories "no pain," "pain and (pain-relieving) drugs," or "pain no drugs," they simply wrote under the "no pain" and "pain no drugs" headings, "See Reverse." On the reverse is written:

"The following tests are run at this facility using the animals listed: a. Draize Eye Irritation; b. Acute Oral Toxicity; c. Primary Skin Irritation; d. Subacute Dermal Toxicity; e. Inhalation Toxicity; f. Guinea Pig Immersion Studies .... No anesthesia is administered in any of the above procedures. This is due to the nature of the studies involved." (USDA/APHIS,1976, Revlon Res. Center).

Product Safety Has to be "Adequately Substantiated"

The Food and Drug Administration denies that it "requires" testing of cosmetics (with the exception of colors in cosmetics, which have to be safety tested). However, it does require that the companies ensure that the safety of their products be adequately substantiated prior to marketing, otherwise they must be labeled: "Warning - the safety of this product has not been determined." (US Laws,Stats.,1978, CFR, Title 21, I,740.10).

The FDA has stated that "adequate substantiation" can be achieved by carrying out the testing procedures described in a 1969 article by R. Giovacchini of Gillette Research Institute. (Giovacchini, R.,1969, p.13).


Cosmetics - the Alternatives

In addition to the animals sacrificed or severely distressed in the tests above, there are others who suffer just as much in the production of cosmetics and soaps. A fixative is used to prolong the fragrance of perfumet and the most popular is musk - the secretion from a gland near the genital organ of civet cats, beavers, Himalayan deer and rats. On a civet farm in Ethiopia, the 30 pound animals are kept for years in wooden cages, 10 to 18 inches wide, 11 to 18 inches high, 3 to 4 feet long, from which they are never released for exercise. It is denied that scraping the anal glands every ten days to collect the musk is painful, but confinement for seven to eight years in cramped quarters, in a dark, smoky building heated up to increase musk production, cannot be considered humane. Another fixative is ambergris - it is produced by sperm whales and is either found floating on the sea or is obtained from the intestines after death. But this is minor compared to the great quantities of whale oil required for soap, cosmetics, lubricants and margarine. For this the whales are mercilessly hunted down. They are shot by a harpoon carrying an explosive charge. Three seconds after they are hit the charge is detonated inside their body. They die agonizingly from massive internal injuries and hemorrhage.

These torments inflicted on the civet, the whale, the rabbit and the guinea pig are totally unnecessary. There are satisfactory alternatives.

Both soaps and every type of cosmetic can be made entirely from flower, herb and plant extracts. The few chemicals which have to be used in lipstick and eye makeup can be nontoxic synthetics. An organization in England, Beauty Without Cruelty, with an American affiliate, has produced all of these. Since the ingredients are harmless, it has been possible to test them on humans exclusively, and safely, and the Food and Drug administration has accepted them for importation into the U.S. There are also a few small American cosmetic manufacturers who make products, which, either in production or testing, have not involved animals.
2


Reducing the Number of Tests - and Animals

Since the methods of cosmetic manufacture advocated by Beauty Without Cruelty are not likely to be adopted overnight, it is important to encourage other efforts too, providing they lead at least to the reduction of animal testing. Andrew Rowan describes such an attempt:

"The Cosmetic, Toiletry and Fragrance Association is trying to reduce toxicity testing in the industry via their Cosmetic Ingredient Review program. This program involves collecting research of various substances from the files of cooperating companies, and making these results available to companies planning to use the same substance in a new product. In this way duplication of testing can be avoided and, consequently, many animals can be spared." (Rowan, A.,1979b, p.6).

A panel of experts has commenced its review of the safety of each ingredient in cosmetics (some 2,800 ingredients are known, of which 119 were under review by Dec. 1979). The experts will determine whether 1. the ingredient is safe as currently used, or 2. that the ingredient is unsafe, or 3. that there is insufficient information for the panel to make a determination. The tentative report is then made publicly available for a 90day comment period. (Elder,R.,1979). Of course, a decision of the third kind, above, may lead to renewed animal testing, but decisions of the first and second kind, if accepted bv regulatory agencies, should reduce pressure on test animals.

A Brighter Future for the Whales?

Aid for the whales may be coming from another quarter. Cetus Corporation, a biotechnology company in California, is now "working on modifying oil with microorganisms to make certain fine lubricants which could replace oil from the threatened sperm whale." (Wilkinson, J., 1980). And Witco, a British company, has produced a synthetic material, called Cyclol SPS, based on cetyl palmitate and other vegetable sources, which has the same properties as spermaceti, the main substance derived from while oil. (Spermaceti is used to give crean cosmetics a firm appearance and to make them flow easily). (Anon.,1980h).


7. TESTING ANALGESICS

The standard tests to determine the value of potential painrelieving drugs are skin-twitch and tail-flick (rats and mice) using radiant heat, the hot-plate, and the "writhing test." In the last-named, "mice are given phenylquinone, intraperitoneally, and after a few minutes begin to writhe and stretch their abdomen from injection of this irritant." (CJSDA/APHIS,1973,Warner-Lambert Res.Inst.). Since the movements are eliminated by low doses of narcotics, and by other pain-relieving drugs, it is a test of analgesia.

J. Cochin, reviewing methods for assessing analgesia, says: "The 'writhing test,' which is also euphemistically called the 'mouse peritoneal test,' has caused a great deal of furor, since writhing seems to offend the sensibilities of many investigators." (Cochin, J.,1973, p.704). Endo, in Garden City, New York, states: "minimal discomfort is experienced by unanesthetized mice and rats during exposure to phenylquinone writhing." (USDA/APHIS, 1976,Endo Labs.). Although the discomfort may be minimal in the opinion of the Endo spokesman, the Animal Welfare Institute comments: "This particular test has been under fire for many years .... The Universities Federation for Animal Welfare urged its abolition some ten years ago." (AWI,1973, p.2).

Pinching the skin, the end-point being a squeal, is also used, and an "ingenious variation" is described by J. Cochin (Cochin, J., 1973), whereby the sensitivity of the rat to the pressure stimulus is increased "by injecting a yeast suspension into the hind paw and applying pressure to the inflamed area produced by the yeast injection ... a struggle reaction being used as the end-point rather than vocalization." He also mentions "electrical methods, such as shock to the tooth pulp, or to the scrotal sac, or to the tail," but these give inconsistent results. More complicated methods have been devised to make the animal feel not only pain but fear - "conditioned anxiety, a concept that must raise some eyebrows of investigators who find it difficult to conceive of anxious rats."

This last comment is not a joke. Reflecting the attitude of this type of investigator, the Dept. of Agriculture actually issued a regulation stating that animals were not known to feel anxiety: "The word 'anxiety' is a psychiatric term that is only applicable to humans." (US Laws,stats.,1979, CFR, Title 9,I).


Tooth Pulp Stimulation

"Seventy-one rabbits had anesthetics for implantation of electrodes into tooth pulp of upper incisor teeth. Tooth pulp was subsequently stimulated electrically to test compounds for analgesic effects with no anesthesia." (USDA/APHIS, 1976, ICI-US). This statement appeared in the 1976 Annual Report to the Dept. of Agriculture of Imperial Chemical Industries. The nature of this implantation is described in an article in the journal Pain:

"Electrical stimulation of intradental nerves presents no special problem if, as in animal experiments, a cavity can be cut through the enamel and dentine to allow electrodes to be applied directly to the inner dentine or to the pulp surface, where the nerve endings are situated. In human experiments this is not usually possible and stimuli have to be applied through the enamel and outer dentine." (Matthews, B., 1976).

"No special problem" to the experimenter, but to the animals...?

During an Aug. 1978 visit to Huntingdon Research Center, Huntingdon, England, a subsidiary of Becton Dickinson, I saw a group of rhesus monkeys who had received tranquilizers and whose perception of pain was being tested by tooth pulp stimulation. Wires were led from a tooth, subcutaneously and through the cheek, to emerge just back of the ear, where they were connected with electrodes. The monkeys were all restrained in wooden collars and body stocks. The nearest one kept looking at me apprehensively and opening his mouth; every time the tooth was stimulated he twitched his lips. "It's not really painful," said the technician in a reassuring tone. Even if the sensations of the monkey as he was being shown to the visitor wore at that moment only mildly distressing, what may be only mildly distressing may become very disturbing if indefinitely repeated. I failed to inquire whether the monkey had anything to look forward to besides lifelong restraint and a perpetual toothache
.3

Tooth shock has become a fashionable experimental procedure only since the 1960's. In an article by K.A. Anderson and others, the following comment appears:

"The nature of the pain that resulted from electrical stimulation of human teeth was reported to be excruciating, poorly localizable pain that generally persisted for some time after the pulpal stimulus was terminated and can, therefore, be classified as diffuse rain." (Anderson, K., 1976).

It was concluded that in monkeys and cats, close to man on the nhylogenetic scale, the effects would be similar.

Alternatives in Analqesic Testing

The relatively harmless character of analgesics - a fact which could be confirmed by safety tests on human cell cultures should make them suitable for testing on human subjects. Barring veterinary use, this would also prove a shortcut to the ultimate beneficiary, and would eliminate confusion from interspecies extrapolation.

Cochin points out that although the "writhing test" is sensitive to the analgesic action of the narcotic antagonist drugs, one of its

"major drawbacks is its lack of specificity, since many nonanalgesic drugs also inhibit writhing. Also there appears to be a lack of correlation with potency of analgesics in man. It is of interest to point out that the analgesic activity of the narcotic antagonists is one property of this class of compounds that is relatively easy to demonstrate in man (Eckhardt, E-1958) and extremely difficult to show in almost any animal model that can be devised." (Cochin, J.,1973, p.704).

It is only fair to add that "human guinea pigs" have individual variations in sensitivity to pain, which can be affected by suggestibility, the environment and the motivation (cf.p.14). If animals have to be used, perhaps the hot-plate test is relatively benign. In this, a rat or mouse which has been injected with a drug to be tested for analgesic potency is placed on a plate which is heated to a constant temperature: 45º - 55ºC., for instance. The time to the end-point is a lifting of the hind leg or licking the front paws. If the animal is densensitized to pain by the analgesic, there is an automatic switch-off of the heat after so many seconds to prevent tissue damage. Since tissue damage would confuse the results - the animals are used repeatedly in what are usually long-term studies - the animals' feet must be protected.

However, there is also the possibility of using organ cultures, especially ones of the guinea pig ileum and the mouse vas deferens, without the disadvantage of involving the whole animal from which the drug, once administered, cannot be readily removed. Kosterlitz and associates found that the "correlation between the values obtained by the guinea pig ileum assay and those of the mouse hot-plate test was always very close." (Kosterlitz, H.,1975).

8. NEW DRUGS: DEVELOPMENT, AND TESTING FOR SAFETY AND POTENCY

Medicines are meant to alleviate or cure pathological conditions. To induce such conditions in test animals which are healthy to begin with it is necessary to make them ill by such procedures as the injection of pathogenic organisms or of cancer cells; the application of irritants to eyes, skin or mucous membranes; the inhalation of substances producing choking or bronchospasm; stressing by electric shock or other means to produce fear, depression and gastrointestinal ulceration; inducing pain; causing coronary occlusion and other cardiovascular pathology; producing burns; creating nutritional deficiencies, and performing other types of experiments all of which cause considerable distress or pain which may or may not be alleviated by the drugs being developed.

When procedures have caused pain in certain "reportable" animals, the Animal Welfare Act requires that these be specified in the research facility's Annual Report to the Dept. of Agriculture. If the pain or distress was not relieved by an anesthetic, analgesic or tranquilizer, the facility must explain why the appropriate drug was not given.


Experiments at Warner-Lambert Research Institute

Reportable animals are dogs, cats, hamsters, guinea pigs, monkeys, rabbits, "wild animals" and marine marmals. Since rats and mice need not be reported, and since the vast majority of experiments are performed on them, the reports are very limited in scope. However, for some unknown reason, the Warner- Lambert Research Institute, which is connected with the large Pharmaceutical company in Morris Plains, New Jersey, included rats and mice in their Annual Report for 1973. They also gave unusually explicit details of the experiments and the pain caused, and explained why pain-relieving drugs were not used. These clear descriptions of experiments typical of those performed in all large pharmaceutical laboratories are exactly the way these organizations should report procedures associated with unrelieved pain, but judging by my inspection of hundreds of these Annual Reports forwarded to the Dept. of Agriculture, the agency practically never gets anything as straightforward as this, especially with the gratuitious but most informative mention of rats and mice. (USDA/APHIS,1973,Warner-Lambert Res. Inst.).

"Warner-Lambert Research Institute
170 Tabor Road, Morris Plains, N.J. 07950

Annual Report for 1973 to USDA/APHIS of Animals Used in Research or Experimentation.

 B. Names of Animals  C. Approx. No. Used  D. No. of Experiments* - Involving Pain or Distress without Use of appropriate Anesthetic, etc.
Mice
Rats
Hamsters
Guinea Pigs
Rabbits
Cats
Dogs
Monkeys

 120,000
65,000
5,000
27,000
1,000
300
2,000
15

181

275
4
188
12

197

*Unlike the new form issued in 1974 - cf.p.240 - this form does not elicit number of animals used per experiment.

"E. Reason for not using appropriate Anesthetic, etc.:

1. Experiments which are performed by the Toxicology Dept. involve a search for untoward reactions at high dose levels. The concurrent use of anesthetics, etc. would mask the reactions we are attempting to note. In general the number of animals showing distress from such studies amount to relatively few as compared to the numbers of animals used.

2. Approximately 100 experiments using 5,000 mice are conducted yearly in evaluating the analgesic properties of potential agents. Mice are given phenylquinone, intraperitoneally, and after a few minutes begin to writhe and stretch their abdomen from injection of this irritant. Other techniques required to induce pain in rodents ti.e. hot plate, tail flick) are used as secondary development.

3. Approximately 50 experiments are done yearly using mice as objects for mouse-killinq rats. This test is quite sensitive for anti-depressant agents and is considered one of the more reliable and predictive animal models. Usually mice previously used for some other experiment are employed.

4. mice are used in approximately 25 experiments a year in which aggressive behavior is elicited by means of isolation or painful electric foot shock. Drugs are studied in attempts to prevent this display. Such procedures are important in the development of tranquilizing agents.

5. Two hundred experiments a year, comprising thousands of rats, are conducted in conscious subjects given a phlogistic agent into a rear paw. This treatment induces pain and swelling at the site of injection (carrageenin) or at a distal site as well (adjuvant arthritis). Such experiments are essential in the screening and development of potential anti-inflammatory agents.

6. Rats are used in approximately 25 experiments a year in which aggressive behavior is elicited by means of isolation or painful electric foot shock. Drugs are studied in attempts to prevent this display. Such procedures are important in the development of tranquilizing agents.

7. Approximately 10 experiments a year using a total of 200 rats are conducted in evaluating the anti-ulcer properties of potential agents in a Stress Ulcer Model. Rats are restrained in wire mesh and suspended in a water bath (chest deep) for a period of time. Vocalization and gastric hemorrhage occur and drugs are studied in attempts to prevent these gastric lesions. Since stress is thought to be an important mechanism for causing human gastrointestinal ulceration, we attempt to duplicate this etiology in animals.

8. Our Chemotherapy Section uses approximately 300 guinea pigs annually in experiments involving the abrading of shaved skin to produce dermatophytic infections. These procedures are necessary in order to discover and evaluate potentially useful agents for the treatment of fungal infections.

9. For bronchodilator screening approximately 1,800 guinea pigs are subjected to an aerosol of histamine or methacholine. If animals are not protected by a bronchodilator thev develop intense bronchospasm and will ultimately expire of asphyxia. These studies must be done in the unanesthetized state in order to determine the end-point.

10. We use approximately 180 dogs that previously have had one coronary artery ligated under surgical anesthesia. They develop a ventricular arrhythmia and are used for screening anti-arrhythmic agents. On some occasions the drug may produce an effect which causes distress, e.g. convulsions. Under these circumstances the animal is usually given a barbiturate.

Edward Schwartz, VMD, PhD, Director, Dept. of Toxicology
Henry H. Freedman, PhD, Director, Biological Research
Richard C. Brogle, PhD, Director, Clinical and Regulatory Services"

The first paragraph above refers to studies of "untoward reactions at high dose levels" of drugs. This is a kind of toxicity testing - essentially the overdosage approach - well illustrated in an experiment at the University of Rochester, New York, by B. Weiss and others, reported under the title "Movement disorders induced in monkeys by chronic haloperidol treatment." (Weiss, B.,1977). Haloperidol is a major antipsychotic drug, marketed by McNeil Laboratories, and in common with many "neuroleptics" may produce movement disorders as side effects. In this experiment the monkeys, after several months of treatment, began to exhibit "violent, uncontrolled movements that flung the animals about the cage." Some exhibitod writhing and bizarre postures. These symptoms, in a milder form, have been observed in humans treated with antipsychotic drugs. In some cases they become persistent and irreversible, even when the drugs are discontinued.

Paragraph 2 refers to the testing of analgesics. For discussion of this, cf.p.232 ff.

Prey-killing experiments (para. 3) have bee my section on "aggressive" behavior, p.32 ff., also aggression resulting from painful electric shock or isolation (pares. 4 and 6) was discussed there an p.37 ff.

For gastric ulcers caused by stress (para. 7), see J. Weiss's use of shock, p.64, and S. Boyd and associates' use of shock and cold restraint, p.65.

The bronchodilator experiments of para. 9 were discussed in the chapter "Inhalation of Toxic Substances," p.104 ff.


Changes in the Annual Report Form under the Animal Welfare Act

Since the year of Warner-Lambert's report, above, the Dept. of Agriculture has introduced (1974) a revised Annual Report form under the Animal Welfare Act. It requires, in addition to the number of animals suffering pain or distress who received no pain-relieving drugs, a list of the reportable animals suffering pain or distress who did receive drugs, and a list of the animals who suffered no pain or distress. As before, the form asks for an explanation of why pain-relieving drugs were omitted in the case of animals suffering pain or distress. While the old form merely asked that the number of experiments causing unrelieved pain be given, the new (1974) form asks for the actual number of animals used.

Listing the total number of reportable animals rather than merely the number of experiments is an improvement on the old form, but since the research facility is not required to specify the nature of the experiments, it is still impossible to evaluate statements about the presence or absence of pain or distress and the consequent giving or withholding of painrelieving drugs. What, for example, is one to make of the A.H. Robins Co., which like other large drug houses manufactures antibiotics, antihistamines, analgesics and tranquilizers? Robins reports the use (including rats and mice) of 34,026 animals in 1976, but gave no pain-relieving drugs, and states that none of these animals suffered any pain or distress. (USDA/APHIS, 1976, Robins,(A.H.) Research Labs.). How did they do it? When one compares their statements with the Warner-Lambert Research Institute's 1976 Annual Report, Table 1, (USDA/APHIS,1976,Warner-Lambert Res. Inst.), one finds that the latter lists some thousands of animals (this time emitting rats and mice) requiring pain-relieving drugs or suffering pain and distress (Column D). 1976 explanations of why pain relief was withheld (keyed by index numbers to Column D) are much briefer than they were in the 1973 report, but they are still explicit:

"l. Experiments which are performed by the Toxicology Department involve a search for untoward reactions at high dose levels. The concurrent use of anesthetics, etc. would mask the reactions we are attempting to note. In general the number of animals showing distress from such studies amount to relatively few as compared to the numbers of animals used.

2. Safety studies of drugs on gross behavior or on electrical activity of the brain, in which distress may have been induced; required by FDA for potential side effects.

3. Aggression produced by electrical stimulation of brain; recognized technique for developing new antidepressants.

4. Animals subjected to bronchoconstriction due to histamine spray for screening and development of new drugs.

5. Avoidable and escapable shock schedule (Sidman) for testing tranquilizers." (Ibid.).


Table 1.
Warner-Lambert's Report to USDA/APHIS for 1976


The Effect of Drugs on Behavior

The first paragraph, above, repeats that in the 1973 report, and paragraph 4 is a repeat of the bronchodilator drug testing. The other tests are similar to the 1973 ones using behavioral evaluation of drug effects for which references in this book were civen on p.240. These references were mainly to basic studies in behavioral psychology in which an animal is first trained to behave in a certain manner - to act aggresively under the stimulus of painful electric shocks, or to develop a stereotyped pattern of food avoidance when subjected to a conflict situation in which food freely offered alternates with food accompanied by electric shock. Once a characteristic behavior pattern has emerged it can be used in a pharmacological investigation of a new drug. If the drug is a tranquilizer, the animal may become relatively indifferent to the shock, with a decrease in the food avoidance rate. If the drug is an antidepressive stimulant, the previously induced aggressivity may increase. This gives a rough approximation of how these drugs affect certain behaviors.

That there are disadvantages of drug testing based on behavior shaped by painful stimuli was pointed out on p.74 ff. The many variables inevitable in the behavioristic approach and the disturbing effect of fear and pain often introduce confusion into the results. Why are the tests done in this fashion, then? The answer may be in a phrase quoted in paragraph 2 of WarnerLambert's 1976 report: "Required by Food and Drug Administration." Have both the experimenters and the regulatory bodies lost sight of the inevitable variability of animal subjects in their preoccupation with ingenious technology and impressive statistical "verification"?

There may be better ways of arriving at the desired information. The science of drug design, as described in the next chapter, predicts the effects of therapeutic substances with precision sufficient to greatly reduce, if not eliminate, much of this painful and often hit-or-miss work with animals.

There may be better ways of arriving at the desired information. The science of drug design, as described in the next chapter, predicts the effects of therapeutic substances with precision sufficient to greatly reduce, if not eliminate, much of this painful and often hit-or-miss work with animals.


NOTES

1. For the nature of some cosmetic oral toxicity tests, see p.207.
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2. A current list of these can be obtained from Beauty Without Cruelty, 175 West 12th Street, New York, N.Y. 10011.
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3. I was unaware that during the year of my visit to Huntingdon, the laboratory had killed 1342 primates in acute toxicity experiments. The Int'l. Primate Protection League has drawn attention to this, and to the painful death there of 10 crabeating macaques, poisoned in an experiment with the weed-killer diquat. (Anon., 1980c).
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Chapter Fourteen | Back to Table of Contents