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Review of Animal Studies
Relevant to Silicone Toxicity
Nancy I. Kerkvliet, MS, PhD
Professor of Toxicology and Extension Toxicology Specialist
Department of Environmental and Molecular Toxicology
Oregon State University
Table of Contents
I. What Is Silicone? I-1
II. Utility and Significance of Animal Studies for Human Toxicity Assessment I-1
III. Rationale for Analysis of Specific Animal Studies Relative to Silicone Toxicity I-3
IV. Animal Models for Atypical Connective Tissue Diseases I-4
V. Historical Perspectives on Silicone Toxicity I-5
VI. Silicone and “Adjuvant Disease” I-7
VII. Adjuvant Activity of Silicone I-8
VIII. Effects of Silicone in Animal Models of Autoimmune Disease I-9
Arthritis-prone DBA/1 Mice I-10
MRL lpr/lprModel of Lupus I-11
New Zealand Black (NZB) x New Zealand White (NZW) F1
Murine Model of SLE I-12
Tight Skin Mouse Model of Scleroderma I-12
Type II Collagen- induced Arthritis I-13
NZB Mouse Model of Autoimmune Hemolytic Anemia I-14
IX. Immunotoxicity of Silicone in Animals I-15
Evidence that Silicone Alters Immune Responsiveness of Animals I-16
Evidence for Antigenicity of Silicone I-17
Evidence that Silicone Induces Inflammation I-19
Evidence that Silicone Activates Macrophages I-20
X. Potential Contribution of Other Materials in SBIs to Toxicity I-23
Low Molecular Weight Cyclosiloxanes I-23
XI. Conclusions I-24
Review of Animal Studies Relevant to Silicone Toxicity
I. What Is Silicone?
Silicone is the name given to a family of synthetic polymers composed of a repeating Si-O backbone and carbon-linked side-groups. Si-C bonds do not exist in nature but can be formed under appropriate manufacturing conditions. The most common example of a silicone is poly(dimethylsiloxane)(PDMS), shown in Figure 1. The dimethylsiloxane units are the basic building blocks of silicones (Lane et al., 1996). Depending on the number of dimethylsiloxane units linked together as a linear polymer and degree of cross-linking between polymer chains, products of various textures and strengths are produced, including forms that mimic human body tissues. In general, straight chain polymers are liquids that increase in viscosity as the chain lengthens (liquid ÷ gel). Increased cross-linking of the chains leads to increasingly rigid silicone materials (gel ÷ elastomer). Substitution of methyl (-CH3) groups with other side chains produces silicone derivatives with varied physical characteristics and chemical reactivities.
Based on a 1950 review article, initial laboratory studies of silicone oils had shown that silicones were “remarkably stable in comparison with other fluids of similar viscosity . . . and more resistant to oxidation and more water repellent than other fluids” (Barondes et al., 1950). They were also shown to have “good resistance to chlorine, nitric acid and hydrochloric acid, sodium chloride, sulfur dioxide and sulfuric acid up to 30% concentration.” This early review also cautioned that silicone polymers “are not to be confused with the silicon (Si) compounds as sodium silicate, silica gel, and siliceous earth. Silica gel, for example, is a colloidal silica that absorbs water.”
II. Utility and Significance of Animal Studies for Human Toxicity Assessment
Experimental animal studies are used for safety assessment purposes prior to the introduction of a chemical or device for use in humans. These studies primarily use laboratory rats and mice, dogs, and rabbits, with additional animal species tested to address specific toxicology questions.
The data obtained from animal studies provide three main types of information. The first tests conducted in animals are generally referred to as “hazard identification.” These studies are carried out to determine the possible biological/toxicological effects the chemical is capable of causing, and often incorporate very high exposure levels or unnatural routes of exposure. These studies are not intended to address the likelihood of effects in humans, but allow scientists to understand the basic ways in which the particular chemical interacts with the cells and tissues in a living mammalian organism.
The second major purpose of animal studies is to establish the relationship between exposure and effects and to characterize the dose-response for those effects. Animal studies are carried out using nearly identical groups of animals that differ only in their exposure to the test substance of interest. By controlling for as many other variables as possible (for example, age, sex, genetic background, environment, diet, etc.), any differences in responses between the controls and treated groups can be causally linked to exposure to the test substance.
Furthermore, by testing different levels of exposure (doses), it is possible to see the relationship between severity of effect and dose; that is, how much chemical is necessary to cause specific effects. The results of this phase of testing are useful in predicting human effects to the extent that appropriate animal models are used and good scientific methods are employed. Applicability of results to humans is also enhanced when similar effects are reported by different laboratories and when consistent effects of exposure are seen in more than one animal species. The results of such studies often determine the fate of products prior to marketing. Once a product is marketed, if problems appear to arise from human exposure, the animal data are valuable to support or refute limited or conflicting evidence in humans.
The third main value of animal toxicity studies is to determine the mechanisms by which a chemical interacts with living cells to produce its toxicity, an important factor in understanding how the chemical might induce or aggravate disease. Such mechanistic studies are particularly important if the benefits of the chemical (e.g., drug) outweigh the toxicity (e.g., side-effects) and the product will be marketed in spite of its recognized toxicity. By understanding the mechanisms for the toxicity, measures can be instituted to prevent or reduce the risk of toxicity. In this phase of testing, the approaches used are not dictated by government regulations and are limited only by the ingenuity of investigators and the amount of funding available for such studies. The relevance of such mechanistic studies in animals will depend on how well-defined
the toxicity is in humans and the ability to reproduce the same toxic effects in animals.
III. Rationale for Analysis of Specific Animal Studies Relative to Silicone Toxicity
When considering the whole data base of animal studies relating to silicone toxicity, several decision points were used during this analysis to determine the relevance of specific papers to SBI toxicity. The rationale for these decisions was as follows:
1. The term silicone has been used to represent many different types of materials that may have very different chemical characteristics when compared to PDMS. Therefore, toxicity studies of silicones that differ significantly from PDMS, are not present in SBIs, and are known to have different chemical reactivities than PDMS, were not included in this analysis. However, in many papers given to the Panel to review, the specific silicone materials tested were not described other than by code. In this case, it was assumed that a relevant form of PDMS was tested, and the data were reviewed and incorporated into this analysis.
2. Studies that examined the toxicity of silicone species that are found only as minor contaminants of SBIs were evaluated, but more critically in terms of their dose-response relationships. In general, minimal effects by minor species that were seen in animals only at high levels of exposure were considered not applicable to the SBI issue.
3. Studies in which relatively large doses of silicone were directly injected into tissues that would not be accessible by the silicone from SBIs in any significant concentration (eg., silicone injected into the brain) have been judged not relevant to the SBI issue.
4. Studies that are based on the oral route of exposure have been judged not applicable. Most silicones are poorly absorbed and do not result in appreciable systemic exposure from this route (Chenoweth et al., 1956). Thus, a lack of toxicity following dietary exposure cannot be used to infer lack of toxicity from SBIs. On the other hand, the possible hydrolysis of some small silicone molecules (e.g., D4) in the acid environment of the stomach also introduces a variable that would not be applicable to the SBI issue .
5. Based on the lack of any definitive evidence that silicone can be degraded to silicon or silica in the body, the toxicology of silicon or silica has not been reviewed for this report. Although very recent studies have provided evidence that D4 can be metabolized via oxidative demethylation (Varaprath et al., 1997), probably through the action of hepatic mixed function oxidase activity (McKim et al., 1988), there is no evidence that PDMS fluid, gel, or elastomer induce hepatic enzymes or are metabolized.
6. This review does not specifically address the issues of silicone leakage or metabolism since there is sufficient animal toxicity data available in which silicone fluids and gels were injected directly into tissue, modeling a worst-case scenario in which all of the silicone in the SBI, including minor species, had leaked through the elastomer and was free in the tissue. Furthermore, the results observed in long-term exposure studies of free fluid and gel would have reflected any migration or metabolism that might have occurred.
7. All literature forms provided to the Panel were reviewed, including peer-reviewed journal articles and non-peer- reviewed book chapters, abstracts, theses and reports. When only the abstract of a report was available, it was not used to provide the sole basis for any conclusions drawn. In judging the quality of individual studies, scientific credibility was strengthened by clearly written reports based on experiments that were hypothesis-driven, had adequate control groups (positive and negative), used well-documented and validated assays, evaluated the dose-response relationship, and were analyzed by accepted statistical tests. Credibility was also increased when conclusions drawn were biologically plausible.
IV. Animal Models for Atypical Connective Tissue Diseases
When considering the question of silicones and “atypical connective tissue diseases” (ACTD), the relevance of animal models to human disease becomes an issue. Because most of the symptoms of ACTD are subjective, the disease constellation cannot be modeled in animals unless a surrogate marker for the disease can be identified. However, since the biological basis for the subjective symptoms is not known, only hypothetical causes of ACTD can be examined in animal studies. These hypothetical causes have been articulated by the plaintiffs to the Science Panel and are also found in various publications provided to the Panel. The evidence
from animal studies to support or refute these hypotheses has been critically evaluated.
V. Historical Perspectives on Silicone Toxicity
The first review of silicone toxicology was published in 1950 wherein the results of standard testing of various PDMS fluids (DC200 series) in rats, rabbits, and mice were summarized (Barondes et al., 1950). Routes of exposure to silicone included oral intubation or intraperitoneal (ip) injection in rats; intradermal (id) or subcutaneous (sc) injection in mice and rabbits; intravenous (iv) injection in mice; and eye instillation or skin application in rabbits. The overall conclusions drawn from these studies was that the silicone fluids tested “are practically inert physiologically . . . . and nontoxic to the body tissues. When fed to laboratory animals in doses as high as 2%, no discernable ill effects were noted. There is little if any reaction when administered intradermally, subcutaneously or intramuscularly.”
Based on the low toxicity of silicone fluids, and the development of a medical grade silicone rubber, the medical uses of silicone greatly expanded during the 1950s and early 1960s (Agnew et al., 1962; Andrews, 1966 ; Ballantyne et al., 1965; Braley, 1972; ). When certain adverse reactions to injected silicone fluid were reported, they tended to be attributed to the use of nonmedical grade or otherwise adulterated products. This position appears to have evolved from the fact that most clinical experience with silicone was very good, and most animal studies showed little reaction to pure silicone fluid (e.g., Dow Corning IND 2702, Informational Materials, 1968).
In the United States, the first SBI made of a silicone elastomer (rubber) envelope containing silicone gel was developed in 1960 and marketed in1963 (Braley, 1972). The silicone gel matrix was composed of high molecular weight linear PDMS polymers cross-linked via the presence of intermittent methyl vinyl groups within the linear chain (Lane and Burns, 1996). Based on the belief that the envelope protected the patient from exposure to the gel, clinical trials on SBIs were not conducted, and their use in humans was apparently allowed based on already- established successful clinical use of silicone rubber and other silicone prostheses. The major concern regarding silicone toxicity at this time appeared to be possible tumor development at the implant site, predicated on a mechanical theory of tumor induction. However, animal studies in the early 1960s described the tissue response to silicone rubber in rats as a fibrotic capsule formation that was accompanied by a mild chronic inflammatory response in some animals.
Histiocytes and giant cells were observed (Agnew et al., 1962). These findings were not considered serious detriments to the clinical use of silicone because of the focus on carcinogenesis and the fact that few tumors were observed (Agnew et al., 1962).
In 1966, the cellular response to silicone fluid was described by Andrews in a preliminary report. In this study, silicone fluid was injected directly into the subcutaneous tissue of mice. Tissue responses were compared to mice injected with saline. Tissue sections were reported to show macrophages that had phagocytosed silicone. Similarly, Rees et al.(1966) and Ben-Hur et al. (1967) reported that silicone fluid injected ip or sc appeared to be phagocytosed and distributed systemically, likely via the lymphatics. Other studies by Ballantyne et al. (1965) showed that massive injections of silicone fluid in guinea pigs, while accompanied by phagocytosis, were well-tolerated by the animals. Sparchu and Clashman (1970) also reported evidence of systemic distribution of ip or sc injected silicone fluid in rats, but noted that much of the silicone appeared to be in extracellular vacuoles and not associated with inflammatory cells.
As reviewed by Braley (1973), the use of silicone devices continued to expand in the 1960s, and by 1973, thousands of patients had received various forms of silicones in medical applications, including their growing use as mammary prostheses. Although complications from the clinical use of silicone fluid were recognized, few papers addressed complications from silicone gel implants and those were primarily related to local contracture. It is presumed that there was little concern over the safety of SBIs during this time. This viewpoint was supported by a report by Lilla and Vistnes (1975) who found little reaction to the long-term implantation of various types of SBIs in rabbits. Similarly, two-year dog and rat studies showed little reaction and no toxicity to multiple im, sc or id injections of silicone fluid (DC-360) (West and Jolly, 1976). Similar innocuous effects were seen in dogs that received various implant materials (presumably silicones) over a six-year period (Mastalski et al., 1977). In 1982, a conference at the National Institutes of Health on the safety of clinical applications of biomaterials noted the success of soft tissue augmentation of the breast in its Consensus Statement.
Additional toxicology studies continued to be carried out in the 1980s and into the 90s. The results of two independent two-year chronic toxicity studies of different silicone gels in rats indicated that tissue changes were observed locally at the site of the implant but no systemic toxicity was seen, based on the absence of changes in body weight and food consumption data, or clinical, gross or microscopic pathology results, including data from interim sacrifices
(Goodman et al., 1988; Lemen et al., 1992). Tumors were observed at the site of implantation but tumor development was related to the process known as solid-state tumorigenesis. This effect appears to be a process unique to rats injected with free gel since rats injected with liquid silicone (Agnew et al., 1966) or implanted with elastomer (King et al., 1989) did not develop tumors . Tumors were also not found in rabbits implanted with elastomer-covered gel for up to18 months (Lilla and Vistnes, 1976) or in mice implanted with silicone fluid, gel or elastomer for 180 days (Bradley et al., 1994). Long-term implantation of various synthetic (presumably silicone-based) materials in dogs for as long as six years did not result in tumor development (Mastalski et al., 1977).
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