Selected aspects of animal husbandry and good laboratory practices.

Clinical Toxicology Downloaded from informahealthcare.com by University of British Columbia on 12/09/14 For personal use only.

CLINICAL TOXICOLOGY, 15( 5), pp. 539-553 (1979)

Selected Aspects of Animal Husbandry and Good Laboratory Practices

JAMES FOX,D.V.M. Division of Laboratory Animal Medicine Massachusetts Institute of Technology Cambridge, Massachusetts 02139

Certainly, vast amounts of literature are available on laboratory animal science and animal husbandry. This paper will address only selected aspects of environmental and management p a r a m e t e r s that directly affect animal health and maintenance, particularly f o r rodents on long-term bioassay testing. In the last several decades the scientific community has continued to accumulate and assess knowledge and awareness of the complexities and potential problems that may arise in utilizing laboratory animals for toxicology and pharmacology bioassays. Loss of, and misinterpretation of, experimental data due to diseased animals which were housed in less than optimum animal care facilities, anim a l s with improper identification, and generally poor husbandry has posed a major problem to the research worker. Many examples can be found in the literature where various diseases of laboratory animals o r naturally occurring lesions have been confused with pathological data thought t o be caused by experimental dosing of a test compound. Fortunately, today the laboratory animal, in many experimental protocols, is critically assessed as to i t s health status before being placed on toxicology testing. This is especially important in rodents where the s t r e s s of dietary o r parenteral intake of toxic substances may trigger the clinical onset of latent diseases.

539 Copyright 0 1980 by Marcel Dekker, Inc.

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TABLE 1. Recommended Temperature and Relative Humidity for Common Rodents [ 11


Temperature Rodent

"C

"F

Relative humidity, %

Mouse Hamster Rat Guinea pig

20-24

68-75

50-60

20-24

68-75

40- 55

18-24

65-75

45- 55

18-24

65-75

45- 55

ANIMAL F A C I L I T I E S :

MACROENVIRONMENT

Animal husbandry is certainly one of the most decisive factors in a research project. In discussing laboratory animals on chronic toxicology studies, several key areas must be addressed. In each particular toxicology study involving animals, criteria f o r selection of facility design and managerial operation must be considered. Various factors such as species, strain, and quality of animal will influence the type of housing necessary for each particular experiment, All managerial assessments of laboratory animals must consider the immediate environment' s influence on the behavioral physiological and biochemical status of the animals on experimental toxicology studies. It is important to keep records on the various environmental parameters, temperature and humidity being among the most critical factors in the animal's environment. The temperature and humidity should be regulated within a specified range, monitored, and the results recorded daily (Table 1) [ 11. Variations above o r below the animal' s thermoneutral zone and changes in relative humidity can drastically alter food and water intake. The condition known as ringtail in neonate mice and r a t s occurs at a higher incidence when animals a r e housed in an environment of low humidity, especially if the animals a r e placed in a wire mesh cage [2]. A review of the effects of temperature on drug action has been recently published; the author s t r e s s e s the importance of defining the size of the animal population, the type of cages, and the ambient temperature used in drug toxicity trials [3], The ventilation, including gaseous and particulate matter, should also be regularly checked. The influence of aerosolized particulate matter in the incidence of benz (a)pyrene-induced pulmonary tumors was compared in hamsters housed in a conventional housing system versus those hamsters similarly treated but housed in a laminar-flow unit. The hamsters housed in laminar-flow units developed tumors more slowly than did hamsters housed in conventional type housing.


ANIMAL HUSBANDRY AND GOOD LABORATORY PRACTICES

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The decreased incidence of pneumonia in hamsters housed in laminarflow units may have contributed to this effect. Separate studies in r a t s indicated that respiratory tract infections may be cocarcinogenic to pulmonary tissues [4]. Another intriguing study in C3H/He mice documented the effect of special housing and the reduction of s t r e s s in the incidence of viral-induced mammary tumors [ 51. Various groups of mice, each carrying the Bittner oncogenic virus, were subjected to different degrees of chronic stress. Results indicated that mammary tumor incidence a t 400 days was modified; incidences of tumors ranged from 92% in the group with sustained stress (handling and orbital bleeding) housed in open, conventional racks versus a 7% incidence of mammary tumors in those animals (with moderate intermittent stressful stimuli) housed in specially constructed, enclosed ventilated shelves. Continuous illumination of high-intensity light can cause retinal photoreceptor cell degeneration in the albino rat [6-81. However, if a dark-light cycle o r automatic timer is used, the effect of continuous high-intensity light a r e not likely to be a consequence in normal animal-holding areas. Environmental lighting certainly has a profound effect on the many metabolic and behavioral parameters which a r e known to have a 24-hr cycle, referred to as the circadian rhythm. ANIMAL CAGING:

MICROENVIRONMENT

Regardless of the use of laboratory animals on toxicology experimental research, three interests, not mutually exclusive but sometimes competing, must be considered when housing laboratory animals. These are: the animal’s welfare and health, the experimental results, and the worker’s safety and health. The toxicologist should attempt to keep his animals healthy, except for the possible effects of the test material, for the duration of the study. He hopes to achieve definitive results with respect to the pharmacologic o r toxicologic activity of the materials under test and he tries to minimize the expos u r e of a worker to a material that is potentially toxic. The manner in which animals a r e housed has a bearing on all three of the interests mentioned above. The stainless steel, wire mesh cage offers the following advantages: Animals can be housed individually with efficient utilization of space, thereby allowing frequent and reliable clinical observations and prevention of trauma by cage mates. Feces and urine are collected in a pan s o that the excreta do not come into contact with the animals. This is particularly important in dietary studies where coprophagy is a normal component of the rodent’s dietary habits. The solid-bottom caging system with a filter bonnet also has advantages. An enclosed microenvironment offers the animal protection from various disease-producing microorganisms, and the risk of exposing personnel working in the a r e a to toxic substances being admin-


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istered to the animal is therefore minimized. This barrier system is not available in the stainless steel wire mesh cage system, and decubitus ulcers on the ventrum of the foot a r e noted in rodents maintained for long periods of time on wire-bottom cages. The solid-bottom cage with a filter top does limit the free exchange of gases and vapors between micro- and macroenvironment. The degree of accumulation of gases such as carbon dioxide, ammonia, and volatile compounds from wood contact bedding and increased humidity and temperature depends on many factors such as the number of animals per cage, type of bedding, and frequency of changing [9, 101. It has been reported that levels of ammonia 25 ppm o r greater consistently increased the severity of rhinitis otitis media, tracheitis, and pneumonia characteristic of murine respiratory mycoplasmosis [ 111. Dirty bedding can impair hepatic microsomal enzyme levels; ammonia may be responsible for this [ 121. Regardless of the cause, the important thing to remember is that frequency of cleaning cages is a very important component of the animal maintenance protocol. Hardwood shavings (softwood shavings induce hepatic microsomal enzymes) a r e recommended for contact bedding for animals on carcinogenic bioassay, and certainly this is applicable for pharmacology o r toxicology studies utilizing animals. However, use of contact bedding may introduce another variable in animal studies. Wood workers exposed to fine, particulate dust of hardwoods o r to their constituents volitized during machine processing develop a high incidence of nasal tumors [13]. Sinapaldehyde (present mainly in angiosperms), whose p-o-methyl derivative was prepared synthetically, has been proved carcinogenic to rats, inducing a variety of tumors including nasal squamous cell carcinomas [ 141. The oxidation product of sinapaldehyde has induced sarcomas at the sight of the subcutaneous injections in mice and rats ~51. The use of cedar bedding has been implicated in the high incidence of liver and mammary tumors in CSH-AVY and CSH-AvYfb mice [16]. It was suggested, however, that the reduced incidence of these tumors observed when animals were bedded on sawdust was due to the lower weight gain in ectoparasitic infestation rather than to food o r bedding [ 171. However, when evaluating the incidence of spontaneous tumors in a given strain of mouse o r rat housed for long periods of time on hardwood bedding, the specific type of bedding used may be of importance. This is of particular concern with solid-bottom, filter-top cages where volitilization products can reach high concentrations. E X P E R I M E N T A L DOSING O F ANIMALS WITH CHEMICALS Because of concern for the safety of personnel working with 15 of the recognized chemical carcinogens, the Department of Labor, Oc-

cupational Safety and Health Administration, adopted legislation re-



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garding the use of these particular compounds [ 181. Safety guidelines for research involving chemical carcinogens have been published by the National Cancer Institute. Many of these guidelines are designed to protect the laboratory worker and a r e directly applicable to the animal technician and to management of the animals under experimentation [19]. Testing procedures in which the investigator wishes to determine whether a chemical compound is carcinogenic, mutagenic, o r teratogenic in experimental animals have in the last several years become commonplace. The route of administration of the test compound, the frequency and dosage of exposure, the duration of experiment, and the species, strain, and s e x of the animal will influence the manner in which the animals are managed and housed, and the safety precautions instituted for each study. Feeding of a test compound in a diet is used commonly to mimic those compounds taken orally by intention o r as environmental contaminants in the food and water. In a recent study, 60 of 128 r a t s housed in wire-bottom cages in one room and 50 of 100 r a t s housed in soldi-bottom cages in another room were fed an agar-base gelled diet to which 3000 ppm sodium fluorescein was added [20]. The animals were housed in a conventional one-corridor animal facility. The trace r material , fluorescein , is detectable by spectrophotof luor imeter at a level of 0.5 ng/mL HzO, and more than 95% of the particle sizes of the material is respirable. Though data of this type must be interpreted with caution, results indicated that all operations performed by animal technicians and the activity of the animals produce contamination, which not only potentially exposes personnel within the work environment but also people outside the controlled environment. The test material also caused cross-contamination and exposure to control animals. The data presented in the report demonstrated that, from the pointof-view of contamination, solid-bottom cages are to be preferred to wire-bottom cages. The use of solid-bottom cages reduces the levels of floor contamination, the contamination of equipment associated with providing water to the animals, and also eliminated the handling of paper used to catch urine, feces, and particles of feed. The contaminated bedding that results from the use of solid-bottom cages can be dumped i n a ventilated enclosure [21]. This indicates that dosed and control animals should be housed separately, but also supports arguments to separate animals receiving different dose regimens. Small, separate cubicle systems with separate air handling systems [22, 231 o r the reverse flow laminal-flow cages systems that a r e equipped to exhaust air would afford the protection against this type of cross-contamination. Personnel were also contaminated with the fluorescein, including portions of the body covered by protective equipment. This illustrates the futility of relying too heavily on protective equipment. The preferred method of control of potential exposures is at the source. Comparison of the data using agar base diet with that of using


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fluorescein-labeled meal diet [24] suggests that a gelled diet is to be preferred to the use of meal diet in carcinogen bioassay. It was stated that a gelled diet does not suffer from an intrinsic deficiency of a meal diet; meal diets consist of finely divided particulate matter and can easily be dispersed within the environment. This factor probably accounted for the lower levels of floor contamination and airborne contamination when using gel diet versus meal diet. A deficiency of the gelled diet used is its adhesiveness; it sticks well to almost any surface and is difficult to remove. The data also demonstrated that when equipment used for control and exposed animals is washed together, it is possible for the control equipment to emerge more contaminated than when it went into the washer. Such items should be washed separately. Washing often fails to clean completely, which suggests that attention be given to the reasons and that more efficient washing systems be designed. The alternative would be to employ disposable items. Another interesting study [25] using the techniques of phase mapping with microbial organisms and particle counts demonstrated that utilizing fundamental diurnal traits and physiological properties in laboratory animals [26, 271 can aid in establishing protocol for managing animals dosed with hazardous substances in the feed o r water. The study involved measurements of microorganisms and particles during the light and dark periods in animal rooms which housed rats, rabbits, cats, and monkeys. Correlations between both parameters were made, allowing a high precision of accuracy in ascertaining contamination from both sources. For rats, because of their nocturnal habits, the emission of particles and microorganisms was a peak activity during the night, while for cats and monkeys the emission increased only during the active feeding period. The activity of rabbits resulted in equal emission during day and night. In analyzing the data it was readily apparent that the floors in the rodent rooms were covered with a dense layer of the particules i n the morning, Therefore, not only is it important to wet mop the area o r use wet vacuum systems, but the floor should be washed first, before attending to the animals, thereby avoiding the creation of aerosols which disturb the settled dust. Cleaning the floor in the afternoon is not necessary if the personnel activity in the room is minimal during the day. Work by technicians should also be done in the early morning when the emission count is lowest, reducing the chance of aerosol exposure. Another important finding was that after changing cages on Mondays, the accumulation of soiled bedding during the week (samples taken at 7:30 p.m. before and 8:30 p.m. after the lights were turned off) did not appreciably elevate the microorganism count, and the particule count actually decreased. These results are opposite to those found in an earlier study where microorganism emissions were the highest on Monday, before cage changing [28]. The influence of bedding in the rat room was also studied by utilizing solid-bottom cages with "dust-free" sawdust (type not specified) compared to animals housed in suspended wire-bottom cages with


545

filter sheets placed 5 cm below the cages to hold feces, food, and urine. The actual values of both particle and microorganism emission were not significantly changed. With the bedding material used, the emissions seem to stem from the animal, feces, food, and urine


1251.

The hazards of experimental skin application of carcinogens were explored using spores of Bacillus globigii [29]. After applying the spores to clipped skin areas on the dorsum of 30 mice, it was noted that the spores remained on the skin and in the surrounding environment for 16 days. Changing bedding, sweeping floors in the animal room, and reclipping the hair of the mice resulted in elevated airborned spore counts during the 16 days posttreatment. Although the similarity of microbial spores to carcinogens differs in some respects, the fact that airborne particles can remain in the environment and be airborne dictates that skin application of carcinogens be treated with appropriate safety precautions. C H E M I C A L S IN T H E ANIMAL F A C I L I T Y In addition to purposeful dosing of chemicals to animals, animal husbandry and management must also recognize the animal facilities' chemical environment. The source of chemicals and toxin in the animal facility are, of course, numerous. Many of the substances occurring in our biosphere a r e potentially hazardous, and they may enter the animal facility via the air, the water supply, the animal food, o r the bedding. In each of our general work areas in the animal facility there is potential of contamination from a variety of chemicals. Chemicals and toxins can pose a threat, real o r potential, to the health and welfare of experimental animals. The safe use of such substances must receive primary consideration in the planning of the total laboratory animal environment. Not only may their misuse compromise the animals, but it may endanger animal c a r e personnel and investigators as well. Some dangerous chemicals may be necess a r y to the experimental environment such as those used to control pests and diseases o r to ascertain an agent's hazard when used in animals. If the laboratory staff strictly adheres to good laboratory practices and follows the recommendations on the chemicals used, then useful compounds necessary for animal experimentations are not likely to become hazardous. Managing the unintentional introduction of chemicals and toxins into the animal facility is, however, a more difficult problem. Whether contamination is the result of man's activities, such as lead in feeds, spilled insecticides, carcinogen feeding studies, o r a natural phenomenon such as microtoxins in feeds, the end result is the same-outright loss of animals or, more insidious, biased interpretation of experimental results. This is unlike potential biologic agents in animal diets which can be eliminated easily by steam autoclaving o r irradiation.

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TABLE 2. Acceptable Levels of Feed Contaminants for Commercial Rations Manufactured for Rodent Use at the National Center for Toxicological Research, Jefferson, Arkansas Agents

Maximum concentration

Cadmium Selenium Polychlorinated biphenyls

0.05 pg/gm 0.50 pg/gm

Total DDT (DDE, DDT, TDE)

0.50 IJ.g/gm 0.05 pg/gm

Mercury Arsenic

0.05 pg/gm

Lead Dieldrin Lindane Heplac hlor Malathion Estrogenic activity

1.00 g / g m 0.01 g / g m 0.01 g / g m 0.01 pg/gm 0.50 pg/gm 2.00 g / k g

Total aflatoxins (Bl, Ba, GI,Gz 1

1-00IJ.g/kg

0.2 5 IJ.g/gm

Several reviews and articles have been published on the carcinogenic and teratogenic effects of insecticides in animals and the acute toxic manifestations of poisoning in man [30, 311. Insecticides commonly used in the animal facility, particularly chlorinated hydrocarbons, a r e potent inducers of heaptic microsomal enzymes of rodents [32, 331. Several reports recently have demonstrated the deleterious effect of insecticides on the immune system in laboratory rodents [34, 351. The evidence now indicates that the innocuous animal room deodorizers, occasionally seen in an animal room to keep down odors, contain volatile hydrocarbons [36], and the disinfecting sprays which contain oils and vinyl chloride are also capable of inducing or inhibiting the hepatic microsomal mixed function oxidase systems in the laboratory animal [37, 381. Unwanted variables ip the diet, i.e., the presence of chemicals and extraneous material o r variations in the concentrations of essential nutrients, can markedly influence the biological response of animals and thus alter the interpretation of experimental data. Analyses conducted over the past 15 years on standard, commercially prepared diets for rats have shown widely variable concentrations-not only of essential nutrients but also of biologically active contaminants (New-



547

berne, 1975) (Table 2). The NCTR has recently prescribed permissable levels of contaminants in the diet [39] (Table 2). It is advisable for investigators conducting chronic studies on animals to monitor pesticides, mycotoxins, and trace minerals in each batch of animal diet in view of the biologic effects they may have on the experimental regime [40-451. Many additional real o r potential factors a r e known to occur naturally in plants and plant products; in addition, substances may be incorporated into animal feeds [46]. These substances include toxic proteins and peptides, compounds in favism 471, vasoactive and psychoactive substances [48, 491, antivitamins 501, enzyme inhibitors [ 511, and estrogenic substances [ 521. Also of practical consideration is to routinely monitor milling dates of animal food and maintain fresh food in closed containers. Potential toxic contaminants also occur in tap water and should be considered when one performs long-term animal studies that utilize municipal grade water. Chloroform is commonly found in municipal water sources according to EPA reports. Other compounds with known or suspected carcinogenic ability and sometimes found in water a r e carbon tetrachloride, polychlorinated biphenyls, benzene, benzo(a)pyrene, trichloroethylene, bis ( 2-chloroethyl)ether, and diphenyl hydrazine. This list only partially covers the organic contaminants in drinking water; although studies have identified approximately 90% of the volatile organics that a r e present, these contaminants represent only 10% of the total organics [53].

t

PERSONNEL Any bioassay program must have a close working relationship among the personnel. This should include a toxicologist, clinical veterinarian, pathologist, toxicology technician, and animal technician, The success of an experimental animal testing protocol relies heavily on the training and dedication of the animal technician to provide high quality animal care. It is becoming increasingly important that the personnel caring for and monitoring the daily health of the laboratory animals on test approach the task with training in both theoretical and practical aspects. This training of animal technicians has been systematized in a national training program under the auspices of the American Association of Laboratory Animal Science. Currently, three levels of certificates of training (assistant animal technician, animal technician, and animal technologist) are granted to those persons successfully completing the required course work (American Association for Laboratory Animal Science, Manual for Laboratory Animal Technicians, 1970). Once these individuals have passed the didactic class work, each laboratory animal care program has the flexibility to train the individual in the pragmatic skills needed for individual experiments involving various laboratory species. The AALAS program offers a practical s e t of scientific information

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organized in a format easily assimilated by the technician to be utilized in a wide variety of laboratory animal settings. All animal care personnel in our program must have graduated from high school. The veterinary technician is another source of personnel which is helping to fill the need for persons with advanced university training in veterinary technical skills and knowledge. At latest count, some 40 colleges o r universities a r e offering 2-yr veterinary technician degrees. Persons trained in this role are invaluable assistants in treatment and daily follow-up care and surveillance after the initial diagnosis and treatment have been prescribed by the veterinarian. An indispensable member of any toxicology bioassay testing protocol is the toxicology technician. This person h a s the responsibility of administering the test material to the laboratory animals, mixing diets and test solutions, and assuring that these results a r e recorded in the proper manner. The individual must take and record clinical observations such as body weight, food consumption, presence of palpable abnormalities, o r grossly visible clinical signs in the test animals. He is also responsible in conjunction with the animal technologist to notify the clinical veterinarian of any clinical signs of disease. The technician also performs necropsy examinations on animals, records gross observations, and labels and collects specimens properly. The person at all times consults the pathologist in attendance regarding necropsies and the recording of pathological data, CLINICAL SURVEILLANCE A N D ASSESSMENT It is essential that the daily husbandry and care of the laboratory animals on test be handled by personnel familiar in general terms with the experimental protocol, thus enabling the animal technician and/or the toxicology technician to alert the clinical veterinarian and toxicologists if clinical signs of illness a r e noted. It is imperative that animals on long-term drug o r toxicology testing be observed on a daily basis. It is becoming increasingly important in toxicology studies to reduce the risk o r loss of animals due to intercurrent infection, and it is advisable that the test animals be monitored for specific pathogens [39]. When monitoring those animals which a r e on chemical testing or when assessing the reliability of the source of animals to be placed on subsequent experiments, special attention should be continually given to a wide variety of agents, including bacteria, viruses, fungi, protozoa, rickettsia, and mycoplasmas. Every effort must be made to prevent using animals infected with these microbial agents and also to prevent the introduction of these organisms into the animal enclosure. Two published documents offer invaluable information f o r those concerned with long-term testing and housing of laboratory animals. These a r e a guide for the care and use of laboratory animals and the recently published report of the long-term holding of laboratory rodents [39, 541. Several other booklets dealing with specific


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TABLE 3. ILAR Standards and Guidelines for the Breeding, Care, and Management of Laboratory Animalsa Syrian Hamsters (1960)

Laboratory Mice (1962)

Laboratory Rats (1962)

Guinea Pigs (1964)

Laboratory Cats (1964) Laboratory Rabbits (1965)

Chickens (1966)

Nonhuman Primates (1968)

Coturnix (1969)

Rodents (1969) (Revised 1976)

Gnotobiotes ( 1970)

Laboratory Dogs (1964).

Swine (1971) aCommittee on Standards, Institute of Laboratory Animal Resources, NAS-NRC, Washington, D.C. species of laboratory animals a r e also available. These are useful in alerting investigators concerning general items of c a r e and management, and they detail potential difficulties when dealing with a particular species of animal (Table 3). The clinical veterinarian who specializes in laboratory animal medicine must provide the major input into the design of an adequate quarantine, disease monitoring program, and clinical examination of animals on toxicological testing. It is this person's responsibility to aid the investigator in obtaining disease-free animals, provide proper diagnostic facilities to routinely monitor the health of the animals, and, if disease outbreak occurs, to advise the investigator whether the disease-causing microorganisms in the colony will compromise the interpretation of the experimental data. Often, in the past, poor husbandry techniques were not recognized o r animals with disease were not diagnosed clinically prior to large numbers of deaths occurring in a rodent colony on toxicology testing. However, with proper training in sound husbandry methods, successful research projects utilizing small laboratory animals can be made. Proper diagnosis and husbandry can significantly influence the final disposition of the animal; whether the animal can remain on test, is the disease o r husbandry practice affecting the animal adversely and altering experimental results, and is the presence of the disease jeopardizing the health of the remainder of the colony. These and other questions can often be answered with careful antemortem clinical evaluation and well-founded, modern animal husbandry. REFERENCES

[ 11 R. S. Runkle, Laboratory animal housing. Part 11, Am. Inst. Archit. J.. 41. 77-80 ( 19841.

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Federal Register, Occupation Safety and Health Administration, Dept. of Labor Carcinogens, 39(20), Pt. I1 (January 29, 1974). National Cancer Institute Safety Guidelines for Research Involving Chemical Carcinogens, D. H. E. W. Publication # NIH 76-900, 1975. E. B. Sansone and J. G. Fox, Potential chemical contamination in animal feeding studies: Evaluation of wire and solid bottom caging systems and gelled feed, Lab. Anim. Sci., 27, 457-465 (1977). C. L. Baldwin, F. L. Sabel, and C. B. Henke, Bedding disposal cabinet for containment of aerosols generated by animal cage cleaning procedures, Environ. Microbiol., 31, 322-324 ( 1976). C. M. Lang and G. T. Harell, A comprehenZve animal program for a college of medicine, J. Am. Inst. Archit., - 52, 57-61 (1969). S. M. Poiley, Housing requirements-General considerations, in Handbook of Laboratory Animal Medicine, Vol. 1 (E. C. Melby, Jr., and N. H. Altman, eds.), CRC Press, Cleveland, 1974. E. B. Sansone, A. M. Losikoff, and R. A Pendleton, Potential hazards for feeding test chemicals in carcinogen bioassay r e search, Toxicol. Appl. Pharmacol., 39, 435-450 ( 1977). W. H. Weihe, Phase maps for pa rt ic Es and microorganisms in animal quarters, Lab. Anim., 9, 353-365 (1975). J. L. Cloudsley-Thompson, Rhjthmic Activity in Animal Physiology and Behavior, Academic, New York, 1961. P. S. Siegel, Food intake in the r a t in relation to the dark-light cycle, J. Comp. Physiol. Psychol., 54, 294 (1961). K. Teelman and W. H. Weihe, Microorganism counts and distribution patterns in air-conditioned animal laboratories, Lab. Anim., 8, 109 (1974). D%rlow, D. J. C. Simmons, and F. J. C. Roe, Hazards from experimental skin painting of carcinogens, Arch. Environ. Health, - 18, 883 (1969). A. Hamilton and H. L. Hardy, Pesticides, in Industrial Toxicology, 3rd ed., Publishing Science Group, Acton, Massachusetts, 1974. F. D. Aldrich and J. F. Gooding, Pesticides in Trace Substances and Health (P. M. Newberne, ed. 1, Dekker, New York, 1976, pp. 159-243. B. D. Kolmodin, D. L. Azarnoff, and S. Siogvist, Effect of environmental factors on drug metabolism: Decreased plasma half-life of antipyrine in workers exposed to chlorinated hydrocarbon insecticides, Clin. Pharmacol. Ther., 9638 (1969). A. D. Poland, D. Smith, R. Knutzman, M. Jacobson, and A. H Conney, Effect of intensive occupational exposure to DDT on phenylbutazone and cortisol metabolism in human subjects, Clin. Pharmacol. Ther., 11, 724 (1970). M. Wasserman, D. Wasserman, Z. Gershon, and L. Zellermayer, Effects of organochlorine insecticides body defense systems, Ann. N. Y. Acad. Sci., 160,393 (1969). D. Keast and M. F. Coales, Lymphocytopenia induced in a

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Q U E S T I O N AND A N S W E R S E S S I O N

Q: You said that the animal technician performs the necropsy, does

he at that time have any findings? Does the pathologist have to be present in the autopsy room during necropsy o r can he be anywhere throughout the laboratory? Fox: A clarification. I did not state that the animal technologist performs the necropsy, but rather the toxicology technician who, presumably, is much more involved in experimental protocol on a day-to-day basis than would be the animal technician. The second part of your question is, in most instances, the pathologist is on call if he indeed is not present in the necropsy room. So he is readily available for a consultation if the need arises. Q: You mention various types of litter. You did not mention ground corn cob litter. Would you please? Fox: Yes, that is another type of commonly used contact bedding used primarily for rodents and the thing that we would like to specify about that particular type of bedding is that it is specified to be mycotoxinfree.

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