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SPECIAL TOPIC OVERVIEW

Refinements in the Care and Use of Animals in Toxicology Studies—Regulation, Validation, and Progress

PATRICIA V. TURNER, DVM, DVSC, DIPLOMATE, ACLAM, 1* KATHLEEN L. SMILER, DVM, DIPLOMATE, ACLAM, 2 MAUREEN HARGADEN, VMD, MS, DIPLOMATE, ACLAM, 3 AND MICHAEL A. KOCH, DVM, MS, DIPLOMATE, ACLAM 4

During the past decade, important advances have been made in the humane care and use of laboratory animals used in toxicol- ogy studies, and there is strong international interest by the pharmaceutical sector in continuing with this progress. Because of the potential influence on human health, safety studies are highly regulated, and implementing refinements in laboratory animal care and use may require an initial validation study to demonstrate a lack of effect on study outcome. This paper provides an overview of issues surrounding implementation of animal care refinements in toxicology laboratory settings, including regulatory constraints, conducting validation studies, current progress in refinements, and areas for future consideration.

As our scientific understanding of the physical and social needs of laboratory animals and the importance of the effects of the interaction between these animals and their environment on study outcome grows, our care and use of research animals has become increasingly refined. Researchers have become aware that good science is equated with humane treatment of labora- tory animals, an idea first proposed by Russell and Burch in 1959 (1). This association has led to productive partnerships between scientists, laboratory animal veterinarians, and those caring for the research animals. Over the past decade alone, refinements in animal enrichment and husbandry have had a pronounced effect on how animals are housed and cared for in research fa- cilities. This effect includes areas such as animal socialization programs, provision of toys and food treats to research animals, permitting opportunities for exercise and social housing, envi- ronmental modifications into more naturalistic or complex settings, and establishing criteria to be used for humane end- points for experimental animals. There has been strong interest in extending these refinements and advances in animal care and use to animals used in regulated toxicology studies (2-6). Because of the highly regulated nature of toxicology studies and their direct application to human health and safety, safety testing is an area of research that has demanded a conservative study design coupled with a reasoned reluctance to change. Change in any aspect of toxicology study design can require ex- tensive validation to demonstrate that the proposed improvements will not significantly alter study outcome. Further, consideration must be given to comparability of new data with laboratory historical control databases if significant changes are instituted. This paper will review the regulatory rigidity that has hampered refinement innovation in toxicology studies and the validation process for implementing refinements, and it will ex- plore recent progress and future advances in refinements and enrichment techniques for animals used in toxicology studies. This article is not intended to be an overview of alternatives in testing, for which the reader is referred to other papers (7-10). We hope that this information can be used by laboratory animal

Pfizer Global Research and Development—Sheridan Park Laboratories, 2270 Speakman Dr., Mississauga, Ontario L5K 1B4, Canada 1 ; Wyeth Research, 641 Ridge Rd., Chazy, New York, 12921 2 ; Hoffmann–LaRoche, 340 Kingsland St., Nutley, New Jersey, 07110-1199 3 ; Covance Laboratories, 3301 Kinsman Blvd., Inc, Madison, Wisconsin, 53704-2595 4

* Corresponding author: Patricia V. Turner, DVM, DVSc, Diplomate, ACLAM, Depart- ment of Pathobiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

veterinarians and technicians to initiate innovations within their own institutions.

Regulatory Oversight of Toxicology Studies

Unlike many basic science experiments involving animals, toxi- cology studies often have a direct effect on human health, and for that reason, are highly regulated by government agencies. For example, the United States, Japan, Canada, and Europe all have legislated requirements for safety testing of pharmaceuti- cal and other products intended for human application or consumption. This regulation is detailed in the Federal Food, Drug, and Cosmetic Act, and the Public Health Service Act in the United States, and the responsibility for administering and enforcing these regulations lies with the Food and Drug Admin- istration (FDA) (11, 12). The purpose of requiring toxicity testing of novel pharmaceutical compounds in animals is to establish an initial safe dose to be tested in humans, to estimate and char- acterize potential adverse effects and toxicities associated with compounds, and to define the mechanism of action of any in- duced toxicities (13). Integral to designing and conducting safety studies are a set of regulations, Good Laboratory Practice (GLP) regulations, instituted by the FDA in 1978 (14). GLP regulations were intro- duced to ensure study reliability, that is, reproducibility of data, and to enhance the overall quality of safety studies. The regula- tions are mandatory for toxicology facilities and define requirements for all aspects of study conduct and documenta- tion, including animal care and use, and facility management. The net result of implementing GLP regulations in toxicology studies has been to decrease data variability and increase the precision of results (15). Responsibility for study conduct, docu- mentation, analysis, and reporting rests with the Study Director, and this person represents the single point of study control (GLP 58.33). Management is responsible for overseeing facility stan- dards, organization, and the resources necessary to conduct the study appropriately (GLP 58.31). It is important to understand the stringency of the process and the serious responsibilities of the Study Director, as there is zero tolerance for noncompli- ance with GLP regulations during FDA inspections and the specter of substantial penalties for facilities not in compliance. Given the grave consequence of an in-study error on human life, the potential negative financial and regulatory consequences of a poorly designed and executed study, the high degree of

precision required of study results, and the pressing timelines for compound development, it is understandable that incorpo- rating new or different procedures into toxicology studies is perceived to be fraught with risk. Contract research organiza- tions face additional pressures to comply with procedural requests of large pharmaceutical sponsors as an inevitable consequence of securing contracts with these organizations. In addition to specific toxicology regulatory oversight, toxi-

cology facilities in the United States, as any other research facility using animals, must comply with the animal care and use re- quirements of the United States Department of Agriculture (USDA) (16, 17). Those facilities conducting work for the United States Government must also comply with US Public Health Ser- vice (PHS) regulations (18). Many facilities are also accredited by the Association for the Assessment and Advancement of Labo- ratory Animal Care, International (AAALAC), an organization that strongly promotes the benefits of physical contact among social animals and solid-bottom caging for rodents (18, 19). To meet animal welfare and GLP requirements, enrichment pro- grams for colony and study animals must be detailed in Standard Operating Procedures (SOPs) or institutional animal care and use committee (IACUC)-approved policies and are often man- aged by enrichment coordinators with specialized training or background in animal behavior. Finally, to maintain high standards of ethical business con- duct, various industrial groups have joined ranks in an attempt to develop international benchmarks for animal care and use. With the support of organizations such as the International Coun- cil for Laboratory Animal Science (ICLAS), the Organization for Economic Cooperation and Development (OECD), and the Canadian Council on Animal Care (CCAC), sectors of regulated industry that use animals in testing have recently started to de- velop international “best practices” guidelines for animal welfare (20, 21). Pharmaceutical organizations form part of this coali- tion and are striving to meet or exceed these guidelines. Changing the interactions between animals and their envi- ronment or exposing animals to different environmental conditions through refinement in husbandry or environmental enrichment are potential sources of study variability that may increase the numbers of animals required for a study or decrease study precision. For example, pair housing of macaques may have profound effects on their immune system responsiveness (22). Facilities must endeavor to seek an ethical balance between pro- vision of husbandry conditions to enhance animal well-being and potential increases in biological variability necessitating increased numbers of animals or invalidation of results (15). The net re- sult of the regulatory dichotomy under which toxicology facilities operate is that while it is usually not difficult to incorporate re- finements and various types of enrichments for colony animals,

it is often much more difficult to do so for study animals without

clear evidence that the impact of the refinement on study out- come will be negligible. This evidence can be supplied by suitably designed and conducted validation studies.

Validation of Refinements in Animal Care and Use in Toxicology

Validation is the process by which reliability and relevance of

a particular refinement is assessed (23). Reliability refers to the

reproducibility of baseline results and the lack of interference with the toxicology study. Reliability must be assessed over time, as the effects of environmental or husbandry refinement may vary with time. Relevance refers to establishing the scientific meaningfulness and usefulness of a refinement. For changes in refinement of animal care and use, a validation study may be based on the results of published behavioral or preference stud- ies, but confirmation of relevance is important.

The purpose of the validation study is to collect objective in- formation about the proposed change to establish without a doubt for Toxicology Management, Study Directors, and indi- rectly, regulatory authorities, that the change can be introduced into safety studies without detrimental effects on results. Valida- tion studies frequently are used in-house to justify introduction of new equipment and techniques into toxicology laboratories but can also be used to introduce major changes in animal hus- bandry. How formalized the process is depends in part on the facility and the nature of the change to be introduced. An ex- ample of a commonly used refinement that has not been formally validated is the use of television in nonhuman primate holding rooms. Television has been shown to be a weak positive enhancer of the nonhuman primate environment (24), and many institu- tions have added these to colony and study rooms. For major changes, such as social housing of study animals, anecdotal or empirical data may not be acceptable, and scientific credibility and validation may have to be established before the changes can be incorporated. Examples of validated refinements in animal care will be dis- cussed later, but in general, as for any other experiment, validation studies require careful planning and should include collaboration and support of Toxicology Management and Study Directors. Objectives should be narrow in scope, safety issues must be carefully considered for both the animals and their hu- man caregivers, methods (preferably minimally invasive) to seek answers regarding the potential impact should be determined, and a thorough appraisal of the limitations of the proposed study should be conducted. Because the findings of a validation study may be shared with regulatory agencies in the future, it is impor- tant to conduct the study in compliance with GLP regulations. Depending upon the refinement to be introduced, this compli- ance may entail developing a protocol and seeking IACUC approval, consideration of appropriate group sizes for validity, use of control animals, assessment of variables over a sufficient period of time, strict adherence to facility SOPs including record keeping, and “blinded” data collection (25). After the study, the results should be analyzed to assess reli- ability and relevance to the study objectives. The information should be shared with Toxicology Management and Study Di- rectors to encourage support for the change, and facility SOPs should be developed or modified to incorporate the change, as appropriate. Initial implementation of a proposed and validated change may occur on an unregulated study as a final means of securing approval for the proposed refinement. Further, because implementing refinements in animal care may invalidate histori- cal data, additional consideration may need to be given to developing new reference ranges or standards. It would be naïve for us to suggest that the simple act of con- ducting and completing a validation study is enough to induce change, particularly for longstanding practices and when con- siderable expense will be incurred. After completion of the validation study, the most crucial step in the implementation process is acceptance by Toxicology Management and Study Directors of the results and the ethical imperative to change. This acceptance can only be accomplished through frequent, open dialogue between all parties, in concert with the IACUC, and prompt dissemination of results, preferably through publi- cation. Once the refinement is established as a standard practice, scientific justification should be provided to the IACUC when- ever exceptions from facility SOPs are requested.

Progress in Animal Refinement and Enrichment in Toxicology

Refining toxicology study design may substantively reduce animal use and thus the potential for stress and distress of addi-

Table 1. Summary of recommended animal care practices for industry a

Species

Housing

Social contact

Diet

Enrichment

Human interaction

Mice

solid-bottom caging with substrate or solid inserts

variable: strain and study-dependent

diet optimization

nesting material, plastic tubing or bottles for hiding, gnawing devices

not beneficial other than for acclimation

(strain-dependent)

Rats

solid-bottom caging with substrate or solid inserts

pair or group house

diet optimization

plastic box or devices for hiding, gnawing devices

beneficial

Rabbits

solid-bottom caging, oversized cages or floor pens with substrate

pair or group house juveniles and females when possible

diet optimization with occasional food treats (e.g., hay, fresh vegetables)

hiding place, shelf, toys, ball, gnawing devices

beneficial

Dogs

cages or pens with substrate

pair or group house

diet optimization with occasional food treats

platforms, chew toys, exercise

beneficial

Nonhuman

cages or pens

pair or group house

diet optimization supplemented with fresh fruit, vegetables, food treats, foraging opportunities

perches, mirrors, other toys rotated regularly, music/video

beneficial

primates

a Compiled from refs. 6, 26-28.

tional animals and may not require extensive validation. Ex- amples of this include eliminating control animals from pilot or exploratory studies, using only one sex of a species in explor- atory studies, using the same subset of rodents for both toxicologic and toxicokinetic assessment in long-term studies, and reusing animals between minimally invasive studies. For this last example, there are obvious ethical, financial, and scientific imperatives for and against reusing animals between studies, and the IACUC may have to examine this practice on a case-by-case basis to determine ethical merit and reasonable limits on an animal’s use and confinement in a research facility. Best-prac- tice guidelines for industry have included further suggestions for refining animal care during studies, such as opportunities for social contact, housing, enrichment opportunities, and hu- man interaction (Table 1) (6, 26, 27). In particular, social housing of social species (e.g., dogs, nonhuman primates, and rats) is recommended whenever possible. In addition, solid-bot- tom caging is recommended for chronic rodent studies (26, 28). A marked disparity exists, however, between suggested indus- try guidelines and current practices. An informal survey of 21 veterinarians in the US and Canada working for pharmaceutical and contract research organizations suggested that only 33% of facilities surveyed currently paired or group-housed some large animals (e.g., dogs, pigs, rabbits, and nonhuman primates) on some regulated studies and that for most studies, rats and mice are housed on wire (Fig. 1) (2, 3). Although a recent study by Peace et al. demonstrated that rats weighing less than 500 g do not suffer any adverse physical effects from being housed on wire, it is common for animals on long-term studies to exceed this weight (29-31). Industry guidelines suggest that housing of rodents in solid-bottom cages with contact bedding increases animal comfort and provides for species-specific behavioral needs to burrow, regardless of the study length (28). There may be scientific reasons precluding housing rodents in solid-bottom cages for specific studies (e.g., compounds that are biotransformed into active metabolites in the feces of coproph- agic rodents), however these possible exceptions should be reviewed by the IACUC on a case-by-case basis. There are few published examples of validated refinements in animal care and use specific to toxicology studies, making it difficult for facilities to assess changes occurring across industry and removing peer-based incentives to implement change. Ar- eas in which validated refinement studies exist for toxicology animal care practices include provision of chew toys and nestlets to rodents on study (32), environmental enrichment programs for short-term housing of macaques (33), and pair or group hous- ing of study dogs (34). In this last example, the net impact of conducting a rigorous validation study for cohoused dogs was

that a subsequent study was deemed unnecessary to validate cohousing of nonhuman primates on study (35). Other examples of recently presented validation studies for refinement of ani- mal care include pair housing of cynomolgus and rhesus macaques on study (Fig. 2) and pair housing of study rats in solid-bottom cages (Fig. 3) (3). Various techniques that have been incorporated to refine ani- mal use on toxicology studies include jacket and tethering of animals for chronic continuous infusion studies to permit more freedom of movement (33-38), implementation of telemetry for noninvasive monitoring of physiologic variables (39), use of Holter monitoring devices for noninvasive, continuous cardio- vascular monitoring in macaques (40), use of alternative blood sampling sites for rodents (41), and use of portable chemistry analyzers that require minimal sample volumes for cageside monitoring of study animals (42).

Areas for Future Refinement of Animal Use in Toxicology Studies

Pronounced advances have been made in recent years in North America in refinement of animal care and use in regulated safety studies. Research animals are well cared for, however, there is always room for improvement. Consideration primarily should be based on our overriding goal of minimizing the stress and distress experienced by research animals and on professional and societal ethical concerns (43). Some may argue that it is neither convenient nor inexpensive to implement change even when validated studies exist. Organizations must juggle regula- tory compliance, budgets, ongoing study loads, and bureaucracy to move ahead. At times, a creative approach may be needed to make advancements. For example, switching from suspended wire caging to solid-bottom caging for all rodent studies is an expensive undertaking, with capital replacement costs estimated at over $16 million for a major US-based pharmaceutical com- pany (44). The provision of cage furniture (e.g., shelves, resting boards, tunnels) to rodents to permit a choice of flooring might be a good interim compromise. Much can be done to continue refinement of animal care and use in toxicology research. More consistent promotion of social housing for all species on chronic studies is a refinement that would have a major positive impact on animal well-being in toxi- cology studies. Now that validated studies exist for group or pair housing of the most commonly used species, it is hoped that facilities will be able to accept, renovate, and implement these changes in the near future. Another potential area for refinement that requires further validation is determination of toxicology study endpoints (4). A

Question 1: Do you pair or socially house large animals on study?

2000

Response (n =17):

 
 

Yes

No

 

Pharma

CRO

Pharma

CRO

Dogs

2

2

11

3

Primates

1

2

10

4

Rabbits

0

0

13

4

Pigs

0

1

0

0

2001

Response (all nonrodent species; n =21):

 
 

Yes

No

 

Pharma

CRO

Pharma

CRO

4

3

10

4

Question 2: How do you handle uneven group sizes (e.g., n =3/dose/ sex)?

2001 Responses:

• house dogs in pens, use tunnels for primates

• group house 3 animals

• pair animals from different dose groups

• only design studies with even numbers/group

• use a naïve animal to create even numbers for pairing

Question 3: What clinical observations are not valid when animals are pair or social housed?

2001 Responses:

• food and water consumption

• emesis and diarrhea

• alopecia

• all are valid and attributed to both animals

Question 4: Do animals remain together for 24 h/day?

2001 Responses:

• separate during dosing

• separate only during feeding

• separate for up to 6 h during day (dosing, feeding, observation)

Question 5: How are socially housed animals randomized?

2001 Responses:

• on arrival, by body weight

• as single animals

• as pairs

Question 6: How are pair- or socially housed animals handled when an animal must be removed (e.g., due to toxicity, injury, death, or incom- patibility)?

2001 Responses:

• try to re-pair

• maintain separately for duration of study

Question 7: How is the social housing program assessed and documented?

2001 Responses:

• animals are observed by a combination of veterinary technicians, ani- mal behaviorists, and/or veterinarians; may be recorded in daily clinical observations or medical records

• formal, scheduled 15-min assessment after pairing

• records of compatible and incompatible animals

• meeting minutes

Question 8: What is your current use of solid-bottom caging for rodent studies?

2000 Responses (n =21):

Rats

Pharma

CRO

Mice

Pharma

CRO

80% to 100% on wire

9

3

6

3

6% to 79% on wire

3

3

5

0

0% to 5% on wire

0

0

1

2

2001 Responses (n =21):

Rats

Pharma

CRO

Mice

Pharma

CRO

80% to 100% on wire

9

1

9

2

6% to 79% on wire

3

1

2

0

0% to 5% on wire

1

3

1

3

Question 9: What are your facility and/or IACUC guidelines for weight ranges of rodents that can be housed on wire?

2001 Responses:

• no guidelines

• wire-bottom caging only used for studies < 1 month in duration

• all rats < 500 g

• caging inserts are given if foot lesions develop

Figure 1. Survey of enrichment and refinement of animal use in toxicology facilities (Pharma, pharmaceutical organization; CRO, contract re- search organization)(70, 71).

number of recently published papers are available concerning development of scoring systems to use as endpoints for different species (23, 45-47), but these are infrequently used in toxicol- ogy studies. More commonly, significant deterioration of clinical signs is used to justify withdrawal and euthanasia of animals on studies. Currently, because of regulatory requirements, the tox- icity of compounds must be clearly demonstrated, and this toxicity information, in turn, is used to estimate therapeutic safety of a compound. To provide more precise safety information for compounds under development, specific endpoints should be adopted that differentiate between direct pharmacologic or toxi- cologic effects and those occurring secondarily (e.g., dehydration secondary to emesis). Waiting for the moribund state to occur may result in an extended period of time in which an animal on study is experiencing unrelieved pain or distress (48). IACUCs may assist in study refinement by requiring clinical condition or endpoint scoresheets to be submitted prior to protocol approval. Little work has been done by industry to develop and validate expanded clinical observations and biomarkers that could be used as criteria for medical intervention or early removal of ani- mals from study prior to development of the moribund condition

(49). Molecular biology techniques are commonly used to iden- tify cellular damage in tissue samples (50). Biomarker panels for study endpoint determination might be developed using simi- lar biochemical or molecular screening assays to monitor early DNA damage, changes in mRNA and protein levels, cytokine release, and cellular toxicity in vivo using readily available samples (e.g., saliva, blood, and urine) (51). Other examples of technology that may be modified to screen for deterioration of animal condition and that also may provide important physi- ologic information during a study include capsule telemetry systems, bioimpedance cardiography, and total body electrical conductivity (52). Because of the large numbers of rodents used in many toxi- cology studies compared with other species and the burden that more intensive monitoring would place on the available workforce, less consideration is given to veterinary nursing sup- port of these animals when their clinical condition deteriorates and they cannot be immediately removed from study. This situ- ation is clearly inconsistent with industry best-practice guidelines and with professional ethical responsibilities for the well-being of all study animals. A number of inexpensive and readily imple-

Background: Although the USDA inspectorate had not issued any comments regarding social housing of nonhuman primates at the facility, AAALAC had strongly urged facility management to consider pair housing of macaques to enhance the enrichment program. Consultation be- tween facility management, Study Directors, and laboratory animal veterinarians and technicians led to provisional acceptance of a plan to develop pair housing of macaques.

Objectives of Covance–Madison Plan for Environmental Enhancement of Nonhuman Primates: To address the social needs of nonhuman pri- mates by pair housing, whenever possible, in addition to providing other environmental enrichment techniques (e.g., manipulanda, foraging opportunities, food treats, acclimation to study procedures).

Validation Plan: The approach was to practice and observe pair housing of colony animals over a 5-month period by using 16 adult (most were female) cynomolgus macaques. No formal study protocol was issued; however, weekly updates and discussions occurred between the technical staff, animal facility managers, veterinarians, and Study Directors. If the practice pairing proved successful, the long-term goal was to use the same method for pairing study animals.

Animal Pairing Protocol:

• Prior to pairing: disarm canine teeth, as appropriate; pair similar-sized animals; identify and record any stereotypic behaviors; ensure animals could be readily identified

• Introduction of animals (3-week period): 1 week of finger contact through cage dividers; 1 week of supervised pairing during the day, in which cage dividers were initially removed daily for a 1-h period under close observation; 1 week of extended pair housing culminating in overnight pairing

• Compatibility monitoring: close observation during contact periods for signs of compatibility including mutual play, grooming and/or hugging, fighting and/or food stealing

• Ongoing monitoring of pairs over 5-month period included: subjective evaluation of animal behavior and physical well-being; body weight; food consumption; fecal changes; observations recorded on comment log and reported to veterinarians and facility management

Results: No significant changes in body weight occurred. Reduced occurrence of stereotypic behaviors in paired animals were noted. No major physical trauma occurred during the 5-month observation interval of the paired animals; only minor lacerations and/or contusions of digits noted.

Implementation Plan:

• No pair housing occurs during quarantine, but colony animals are otherwise paired whenever possible.

• Animals are randomized individually based on body weights (pair randomization is also available).

• A graded plan for pair housing was introduced dependent upon study duration:

< 3 weeks: no pair housing used

3

to 5 weeks: at least 1 h/day, 3 times weekly

5

to 14 weeks: at least 1 h daily

> 15 weeks: at least 15 h daily (permits up to 9 h of study observation during which animals may be singly-housed).

• Study design issues: animals weighing < 4 kg may be housed as 3 per double-wide cage; and when groups of 3 are used, the extra animal is paired

with a naïve animal, or animals may be housed in groups of 3.

• Observation of food consumption and excreta: pair housing is suspended during urine collection; observations for abnormal feces and urine are made at the end of the single-housing periods; qualitative food consumption observations are determined at the end of the single-housing periods.

Impact:

• Study Directors and study sponsors report no impact on acceptance of regulatory submissions.

• More than 73 studies have been completed in a > 2-year period (to the end of 2001) using this approach.

• Fewer stress-induced problems (e.g., diarrhea, hair-plucking) are noted during studies.

• Improved animal technician morale has occurred.

• 2-year injury rate: 7.4% of pair-housed animals, compared with 4.9% of single-housed animals.

Figure 2. Validation of pair housing of cynomolgus and rhesus macaques in toxicology studies (72).

mented treatment options exist in this regard (e.g., cageside monitoring of electrolytes, fluid therapy, heat lamps or blankets, and food treats), which could be used to increase animal com- fort and the scientific quality of results (53). Use of handheld digital equipment programmed to interface with validated soft- ware for in-life databases that record changes in clinical condition and body weight at the cage level would provide a more conve- nient and immediate means to record and respond to alterations in animal condition (54). Other considerations for refinement that may require valida- tion prior to use in regulated studies include re-examining blood sample collection from rodents and nonrodents to minimize animal stress and its effect on data (55) and improving labora- tory analytical techniques such that required sample volumes are minimized. Some facilities continue to use retroorbital punc- ture for blood collection in rats and mice, whereas other well characterized, less-invasive techniques exist, such as lateral tail vein, jugular vein, sublingual vein, and saphenous vein punc- ture (42, 56, 57). Sensitivity of assays for gene expression, blood gas and serum chemistry determination, and detection of anti- body production in mice is such that a whole blood sample

volume of < 100 m l often suffices (58, 59). During toxicology studies, > 500 m l of plasma is often requested for clinical and ana- lytical chemistry sample protocols. Poor collection technique and sampling of large blood volumes adversely affects animal physiol- ogy by increasing animal distress and may alter overall blood composition if frequent sampling of large volumes is required over a short period of time (60). Requirement of large blood volumes for assays also necessitates large rodent group sizes. Continuing to explore and develop alternative dosing and re- straint systems for study animals is an important area of refinement (61). Involuntary emesis or regurgitation following naso- or orogastric gavage can result in aspiration and confound- ing pulmonary lesions with daily dosing (62). Mixing test substances with readily consumed, palatable ingredients (e.g., Prang for nonhuman primates or formulation in pediatric elix- irs or sprinkles as used in human juvenile clinical trials) would permit cageside dosing. This adaptation would require more prestudy animal training to ensure complete consumption, vali- dation to ensure a lack of interference with test article activity, and a different approach to concentration, homogeneity, and stability testing of the test article. However, this practice would

Background: At this facility, rats traditionally had been housed singly in suspended, wire-bottom cages. Solid-bottom caging and pair housing of rats were used at European facilities, and there was a strong desire by management to harmonize practices between sites. Further, the Guide recom- mends solid-bottom caging for rodents and this practice is encouraged by AAALAC at accredited facilities (18).

Objectives of Hoffman–LaRoche Plan for Environmental Enhancement of Rats: To address the social and environmental needs of rats by pair housing animals of the same sex and by housing in solid-bottom cages with substrate.

Validation Plan: The approach was to monitor changes in body weight, hematology, and serum chemistry over the course of the study for potential effects. In addition, different bedding types were evaluated for ease of observation of various clinical signs. A formal protocol was developed and approved by management and the IACUC. Toxicology personnel conducted the study.

Study Design:

• Wistar Hannover rats (40/sex ) were acclimated to pairing and solid-bottom caging for 1 week, followed by a 13-week study period

• 10 rats/sex were pair housed in solid-bottom caging on each of four substrates: hardwood chips, 1/8-in. corncob particles, 1/4-in. corncob particles, and paper chips

• Blood was collected on 20 animals/sex every 2 weeks for evaluation of hematology (WBC count and differential, hemoglobin, hematocrit, RBC, MCV, MCH, MCHC, RDW, reticulocyte count, platelet count, and MPV) and serum chemistry (glucose, ALT, AST, ALP, GGT, total bilirubin, cholesterol, triglycerides, total protein, albumin, A/G ratio, BUN, creatinine, calcium, potassium, sodium)

• Body weights were recorded weekly

• Urinalysis and coagulation parameters were assessed at study termination

Results: Body weights and blood parameters of paired study animals were compared with in-house reference values for Wistar Hannover rats housed individually in wire-bottom cages. There were no significant differences in body weights, blood parameters, or urinalysis parameters between the reference values and those obtained from male or female rats pair housed in solid-bottom caging. The results of the bedding evaluation are presented in following section. Increased exploratory behaviour and ease of handling was also noted for the pair housed animals.

Results of Bedding Evaluation: 1, excellent; 2, very good; 3, good; 4, not acceptable

Type of bedding

Hardwood

1 / 8 -in. Corncob

1 / 4 -in. Corncob

Paper chip

Cost/bag (USD) Visibility of food Visibility of urine Visibility of feces Dust control Cleanliness Odor

7.05

9.65

9.65

20.15

3

3

2

1

4

4

4

1

4

4

3

1

3

3

2

1

4

3

2

1

1

2

2

2

Implementation Plan:

• Rats are either cohoused or housed individually in solid-bottom cages, depending on study design (e.g., in a reproductive toxicology where breeding occurs over a 5-day period, animals would be individually housed).

• Choice of bedding is determined by study design and the types of clinical signs to be collected.

Impact:

• Technical personnel subjectively report that rodents are easier to handle and are more docile when paired. Animals conduct mutual grooming and play behavior and sleep in close proximity.

• Discussions have occurred with cage vendors to redesign cages and racks to meet space needs. This adaptation has included changing the feeder placement, altering rack heights, and grommet placement in shoebox cages for automatic waterers. This modification can be an expensive venture.

• The change in husbandry has not interfered with data collection from study animals.

Figure 3. Validation of solid-bottom caging and pair housing of rats in toxicology studies (73).

significantly reduce animal and technician stress. Incorporating a voluntary dosing technique also would have a direct and posi- tive effect on occupational health and safety concerns, as it would translate into less direct contact between technical support staff and animals during a study and reduce ergonomic issues induced by repetitive lifting and transporting of animals. In addition, tetherless methodologies incorporating infrared controls or al- ternative catheter instrumentation techniques have been developed for intravenous drug delivery and may be introduced into studies to increase animal mobility and comfort (39, 63, 64). The use of miniaturized and minimally invasive monitoring equipment to increase the information gained from each ani- mal and detect alterations in animal condition is another area of potential refinement in animal use that requires validation. Examples include in vivo scintigraphic imaging of areas of acute and chronic inflammation by using radiopharmaceuticals (65), laser Doppler flowmetry to examine tissue ischemia, and digital sonomicrometry to measure decreases in gastrointestinal func- tion and motility (66).

The recent explosion of knowledge and interest in compara- tive genomics and the widespread availability of databases containing comparative gene information should stimulate dis- cussion as to whether the most appropriate stocks and strains of each species are being used for testing and should guide prac- tices for refining use of these animals. This issue is particularly relevant when inbred strains of animals are used. For example, the benefits of caloric restriction may not be applicable to all mouse strains (67), and this difference may affect approaches to diet optimization for long-term studies. Further, numerous dif- ferences exist in chromosomal nucleotide sequences between inbred strains of mice (68). Some of these changes potentially alter gene transcription and translation, thereby accounting for known changes in strain phenotype, and some changes may in- duce as-yet uncharacterized alterations in strain-specific immune system function and responsiveness to xenobiotics. As informa- tion becomes available for various vertebrate and invertebrate species, a review of animal models used in toxicology studies should occur to ensure comparative relevance.

One final area of consideration for refinement in animal use and one that is immediately available to all institutions is to imple- ment consistent training of laboratory animals for routine procedures. Training of animals for various technical procedures has been shown to reduce stress for both animals and those who work with them (69). In summary, best-practices guidelines have been developed by industry to refine the care and use of animals in toxicology studies, and validation studies exist for several of these proposed changes. Within the pharmaceutical sector, there is widespread support for the idea that good science is equated with humane science. We hope that this support will increasingly translate into acceptance and implementation of validated refinements within regulated safety studies. A number of exciting opportunities ex- ist to partner new technologies and advances in molecular biology with laboratory animal science to further refine the care and use of animals in toxicology. Open discussion, including collabo- rative guidance from the FDA and USDA regarding animal welfare, and dissemination of advances in the refinement of ani- mal use must continue to encourage changes in practices during regulated studies.

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