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BPA and Plastic Lab Animal Cages

When Disaster Strikes:
Rethinking Caging Materials

Lab Animal v.32, n.4, Apr03

Kara E. Koehler, PhD, Robert C. Voigt, Sally Thomas, BS, Bruce Lamb, PhD, Cheryl Urban, LAT, Terry Hassold, PhD, and Patricia A. Hunt, PhD

Koehler, Lamb, Hassold, and Hunt are with the Department of Genetics, Case Western Reserve University, Cleveland, OH; Voigt was with the Animal Resource Center, Case Western Reserve University and is currently with Lab Products, Inc., Seaford, DE; Thomas is with Thoren Caging Systems, Inc., Hazelton, PA; and Urban is with the Animal Resource Center, Case Western Reserve University. Please send reprint requests to Hunt at Department of Genetics, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4955, or email: pah13@po.cwru.edu.

The value of research using lab animals hinges on the ability to carry out experiments in a tightly controlled environment. Diet, caging materials (e.g., cages and water bottles), and other environmental variables have the potential to create serious disruptions in animal studies. The authors describe the inadvertent damage of polycarbonate caging materials during the course of routine cagewashing, providing an instructive example and illustrating the importance of defined and controlled environmental conditions in biomedical research.

Since the wooden box was retired 50 years ago, a variety of plastic materials have come into use for rodent caging, including polystyrene, polypropylene, polycarbonate, high-heat polycarbonate, polyetherimide, polysulfone, and polyphenylsulfone (Table 1). The quest for better plastics continues, with the ‘holy grail’ being a caging material that is simultaneously strong, sufficiently resistant to heat and chemicals, inexpensive, durable, and transparent.

Polycarbonate, first introduced to caging in 1962, was the industry standard for 25 years and is still in wide use in animal facilities. The accidental exposure of high-temperature polycarbonate (polyphthalate carbonate) cages and water bottles in our facility to a harsh alkaline detergent intended for washing floors rather than cages resulted in degradation of the plastic polymer. The damaged caging materials (both cages and water bottles) leached high concentrations of the weakly estrogenic compound bisphenol A (4',4'-isopropylidenediphenol, hereafter referred to as BPA). Exposure to this compound had an immediate dramatic impact on the experimental results obtained by the Hunt and Hassold laboratories, both of which study meiosis1. Subsequently, exposure effects appeared in the Lamb laboratory, which studies animal models of Alzheimer’s disease, and control animals in the facility showed a substantial increase in both mortality rate and tumor susceptibility. Subsequent studies have shown the potential for even low-level BPA exposure (e.g., amounts comparable to animal exposures expected from the use of well-worn polycarbonate caging[2]) to influence substantially the outcome of experiments, especially those involving reproduction[1].

New high-temperature polycarbonate cages (Thoren Caging Systems, Hazelton, PA) were purchased when our facility opened in 1993. Normal cagewashing procedures involved the use of a phosphoric acid (pH 2.0) detergent, Lifegard 7000 (Rochester Midland, Rochester, NY), in a cage and rack washer, with subsequent autoclaving. A temporary worker’s confusion resulted in the inadvertent washing of several batches of polyphthalate carbonate cages and water bottles with the quaternary ammonium detergent A-33 (Airkem Professional, Ecolab, St. Paul, MN). With a pH of 9.2, this detergent qualifies as one of the strong bases that manufacturer instructions for polycarbonate caging generally warn users to avoid. Quaternary ammonium products, when added to household detergents, accelerate the breakdown of animal fats and other organic substances; furthermore, because quaternary ammonium binds tightly to organic material, residue may persist even after multiple water rinses.

Because BPA, the monomer used to make polycarbonate plastics, is one of a class of estrogen-like substances known as ‘estrogen mimics’, and it has been reported to leach from some composites of which it is a component[2–4], we suspected that BPA exposure was the source of the rapid changes in experimental results first noted in the Hunt laboratory. Subsequent studies of virgin polyphthalate carbonate cages and both polyphthalate carbonate and polycarbonate water bottles allowed us to determine that a one-time application of A-33 detergent is sufficient to cause polymer degradation1. Furthermore, the damage to exposed caging materials was progressive, in that during the course of several weeks, the surface of the normally yellow transparent high-heat polyphthalate carbonate cages became initially slightly crazed, then opaque, whitish, and rough, and finally sticky and bubbly (Fig. 1a). In contrast, damage to the polyphthalate carbonate water bottles was far less evident. Initially, an increase in the number of bottles with leaky seams was the only evidence of damage. However, as the degradation of the plastic polymer progressed, crazing on the sides of the bottles became evident (Fig. 1b). Because bottles, unlike cages, are in contact with water during the autoclaving procedure, differences in the visible manifestation of damage presumably reflect the protective effect of water. Nevertheless, as subsequent studies showed, the lack of visible damage was not an accurate indicator of the amount of BPA leaching from exposed bottles[1].

FIGURE 1. Damaged cages and water bottles. (A) Polyphthalatecarbonate cages inadvertently exposed to a harsh alkaline detergent during routine cagewashing exhibited visible signs of damage: The cage on the left was subjected to 25 rounds of washing (without detergent) and autoclaving. The cage on the right exhibits the physical changes of haziness and bubbling that resulted from inadvertent exposure to the quaternary ammonium detergent A-33 (Airkem Professional, Ecolab). (B) Polyphthalate carbonate bottles exposed during the incident did not exhibit the same degree of visible damage as cages. However, crazing became evident with continued use, as seen on the sides of these two bottles.

In the two laboratories studying meiotic chromosome behavior, the impact of the chemical damage quickly manifested as a highly significant increase in chromosomal aneuploidy and other serious meiotic defects[1]. By comparison with values obtained before the cage-damaging event (‘Before’), there were an 8.3-fold increase in aneuploidy and a 20-fold increase in chromosome misalignment during the period of maximal exposure (‘During’). There was a gradual decrease in the level of chromosome misalignment during a period of several weeks as the most visibly damaged cages were removed (‘Partial cage replacement’). Surprisingly, following the removal of all polycarbonate plastics in the facility (‘After’), the level of aneuploidy remained slightly, but significantly, higher than preexposure levels (Fig. 2). However, when the colony was moved to a new facility, aneuploidy returned to pre-exposure levels (data not shown).

Furthermore, as shown in Fig. 3, a comparison of the mortality rate before and during the exposure revealed a significant increase in mortality, especially among young (one- to four-month-old) mice. A comparison of the five-month period preceding (February–June) and during (August–December) the incident suggests an increased frequency of deaths.

Correcting for cage inventory, the frequency of deaths per cage was 0.028 preceding the incident and 0.124 during the time of maximal exposure. Strikingly, 20/26 (76.9%) deaths during the exposure were of animals six months of age or younger; in the five-month period preceding the incident only 1/4 (25%) deaths involved animals in this age group. Anecdotally, during the months after the exposure we noted a number of animals with reproductive tract tumors, abnormalities that previously had been exceedingly rare in our colony. In two of the three laboratories, the effects led to a ten-month suspension of animal studies until the source of the problem was identified, the cages and water bottles replaced, and control experimental values returned to normal.

Such incidents are likely not as rare as might be hoped. Discussions with colleagues at other institutions provided numerous anecdotal reports of unexplained changes in experimental results and of rapid, visible changes in caging material, although there was no firm evidence of the striking correlation reported here. Surprisingly, a Comparative Medicine listserv inquiry by our veterinary staff at the time of the incident yielded numerous accounts of cage damage, with explanations ranging from amines in the autoclave steam to the action of various detergents.

BPA is a monomer that is polymerized to manufacture polycarbonate plastic products and resins, such as those used as dental sealants and to line food and beverage containers. Manufacturers widely recognize that polycarbonate is poorly resistant to chemical damage and incompatible with strong bases, among other substances. Further recent studies demonstrate that new cages leach low levels of BPA and that the level of leaching increases markedly under conditions of normal wear[2]. In fact, as a result of problems and experiences like ours, some manufacturers no longer mold cages using high-temperature polycarbonate. Other manufacturers warranty or recommend these polycarbonate cages for only 20 autoclave cycles, as opposed to 100–2,000 cycles for other types of thermoplastic (Table 1).

Even with extreme care, damaging events can occur, for example through a one-time accidental contact with a basic detergent as described here, through the continued use of cages beyond their recommended ‘shelf-life’, or through other inadvertent contact such as high concentrations of corrosion-controlling amines in autoclave steam. Importantly, a damaging incident like the one we experienced is not necessary for the release of the estrogenic substance BPA from polycarbonate plastics. Normal care and use of animal cages results in leaching of BPA at 10–135 ng/cm2 cage surface area[2]. Further, simply autoclaving polycarbonate flasks filled with water has resulted in detection of BPA at concentrations of 13 nM (3 p.p.b.)[5]. Similar lowdose concentrations (<50 p.p.b. range) of BPA reportedly affect numerous aspects of animal reproduction, including development of the reproductive tract[6,7], puberty onset[8–10], sperm count[9,11–13], sex-specific and maternal behaviors[14–17], abnormalities in meiotic chromosome behavior including aneuploidy[1], and disruption of estrous cycles[18]. Furthermore, BPA is only one of a large class of estrogen mimics and other hormone disruptors that have been suggested to be capable of causing such effects[19,20]. Although the reports of low-dose effects remain controversial, the sheer volume of reports of disrupted experiments, in addition to the results from experimental tests of substances, such as BPA, that may be released from caging or other environmental materials, warrants immediate attention by the animal research community.

The replacement of polycarbonate caging materials represents an expensive investment for some facilities. However, in comparison with the cost of potentially confounding years’ worth of experimental data with varying and uncontrolled levels of estrogenic substances, the cost of caging alternatives is minimal. Thus, we advocate an expeditious move away from polycarbonate caging and other environmental contact materials with poor performance records with respect to chemical damage. Furthermore, our experience underscores the importance of replacing worn caging on a regular basis. Currently there are no data on chemical damage and BPA release from polysulfone and polyetherimide, the two other common caging thermoplastics with a BPA component. Although these are considered stable polymers by comparison with polycarbonate, the recent finding of passive migration of small amounts of BPA from new polysulfone caging at room temperature in a neutral solution[2] suggests that further research is warranted.

Unfortunately, caging materials are not the only potential source of hormone-mediated environmental impacts on research animals. There are reports that soy-based or phytoestrogen-containing animal feeds can affect the timing of puberty21, susceptibility to cancer[21–26], body weight or organ development[27–29], thymic and immune changes[30], and memory and sex-specific behavior in rodents31. Although these effects remain controversial and may often be transient when exposure is restricted to adult animals, dietary composition nonetheless can potentially seriously alter experimental outcomes and requires more study. Additionally, facilities

Unfortunately, caging materials are not should carefully choose rodent bedding materials; for example, manufacturers of corncob bedding should provide contaminant analysis, because pesticide residues may be present[32]. There are recent reports that even very low levels of the common herbicide atrazine retard sexual development in frogs[33]. Furthermore, many breakdown products or even ‘inert’ ingredients of pesticides have the potential to function as hormone disruptors (including BPA and nonylphenol; a complete list of inert ingredients used in pesticides is available from www.epa.gov/opprd001/inerts/.

Our experience provides an instructive example of the importance of communication and cooperation among investigators, animal-care staff, and industry. Our ability to identify and confirm the source of the problem quickly was the result of just such cooperation. Increasingly sensitive methods of experimental analysis, coupled with an ever-expanding array of caging and husbandry options, have contributed substantially to the complexity of animal care. Thus, successfully meeting the needs of investigators and their research animals requires not only constant attention and vigilance but also recognition on all sides of the importance of communication.

FIGURE 2. Observed increase in meiotic errors with accidental exposure to an environmental source of BPA. Although the exact timing of the damaging event is unknown, a change in experimental results became evident at the end of August 1998. This figure shows the fold increase for two different types of meiotic disturbances, aneuploidy (red curve) and aberrations in chromosome alignment on the meiotic spindle (blue curve), in oocytes from exposed female mice. For further details, see Hunt et al1.

FIGURE 3. Unexplained deaths in the facility associated with inadvertent BPA exposure. This bar graph shows the frequency of unexplained deaths of mice past weaning age in Lamb’s colony.

TABLE 1. Properties of thermoplastics commonly used for rodent caging

		   Chemical 	   	       Maximum 	    Lifetime              			   Is BPA a
		   resistance to:  Impact      autoclave    autoclave  					   breakdown
		   Alkali  Acid    strength    temperature  cycles    Transparency  Color     	 Cost	   product?
Polycarbonate 	   Poor    Good    Excellent   250°F 	    NRa       Yes	    None 	 Low 	     Yes
Polyphthalate 	   Poor    Good    Good        270°F 	    ~50       Yes 	    Very light   Competitive Yes
  carbonate 									     amber
Polyetherimide 	   Good    Good    Fair        300°F 	    Limitedb  No 	    Dark amber   Very high   ?c
Polysulfone 	   Good    Good    Fair        300°F 	    ~200      Yes 	    Light amber  Competitive ?c
Polyphenyl-sulfone Good    Good	   Excellent   >300°F 	    >1,000    Yes/no 	    Amber 	 Very high   No

a NR, Not recommended.
b This resin can be autoclaved, but it becomes more brittle with increasing number of autoclave cycles.
c Although there currently are no published data concerning the release of BPA upon chemical damage to 
  these polymers, BPA is a component of both. In addition, recent data suggest that passive migration of 
  BPA from new polysulfone cages occurs at room temperature in a neutral solvent (ref. 2).

Acknowledgments

The authors gratefully acknowledge W. Thomas, W. Hallock and V. Percec for helpful discussions. The research studies affected by this incident were supported by National Institutes of Health grants HD31866 (P.H.), HD21341 (T.H.), and AG20202 (B.L.). In addition, subsequent studies and the writing of this manuscript were supported in part by a Culpepper Foundation Pilot Initiative grant (P.H.) and NIH grant ES11772 (P.H.).

Received 8/16/02; accepted 2/25/03.

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